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545 Cards in this Set

  • Front
  • Back
zoonotic infections
viral infections that can cross species barriers
chicken flu
avian pandemic, huge cost to world economies
viruses with little impact on health
most viruses don't greatly damage health because of defense systems in host (immune and physical defenses)
virus in human genome
5-10% of genome is from integrated retrovirus DNA
humans as virus resevoir
viruses exist in lungs, GI tract; include adenoviruses, coronaviruses, rhinoviruses; guts have many plant viruses and bacterial species with their own viruses
viruses other functions besides disease
1. facilitators of genetic information transfer
2. major evolutionary forces in their hosts
viruses three-part strategy
1. nucleic acid genome packaged in protein particle
2. viral genome with information to start/complete infectious cycle within susceptible/permissive cell
3. viral genomes can establish themselves in host population to ensure survival
infectious cycle steps
attachment/entry of particle, decoding of genome information, host ribosome translation of viral mRNA, genome replication, assembly/release of full particles
survival of viral particles
occurs because of enormous number of particles released, many unselected mutants (natural selection removes these and keeps the "fit" ones), each selections creates new founders that can start cycle over and often give new results (evolution)
diversity of viruses
size/nature/topology of genomes, particle type, coding strategies, tissue/cell tropism (response to virus), degree of pathogenesis
underlying simplicity of viruses
viral genomes are obligate molecular parasites that function only after cellular replication, must make mRNA to be translated by host ribosomes
degree of harm done by viruses to host
completely killing host will also kill virus, if virus is too passive then host defenses block viral spread (faces elimination), middle ground is good
rates of microorganism discovery
fungi, protozoa, and many bacteria were found before the first viruses (TMV); technology to study cell/molecular biology was discovered to speed up virus discovery
filterable agents
not bacteria, go through small pores and still can infect
viruses and cancer
cell-free filtrates with viruses have the ablity to cause cancer
experiments that tested on humans viral vaccination, crucial in establishing vaccines as major health tool
viruses are usually 10-300 nm, but this is an 800 nm amoebe virus with a huge genome (not submicroscopic like most viruses)
viral actions in host cell
uses genome to synthesize by cellular systems components needed for viral genome replication and viral particle transmission, move to new celll and disassemble
sizes (nm) of: hydrogen atom, globular protein, ribosome, poliovirus, herpesvirus, e. coli, sperm, red blood cell (human), somatic cells, amoeba, human egg, frog egg
0.1, 4, 20, 30, 200, 1000x2000, 1000x5000, 9000, 10000-30000, 90000, 100000, 2000000
crystalization and viruses
some particles can be crystalized and the structure can be obtained (TMV), helps that viruses are identical and symmetrical
the study of serum/blood and antigen/antibody reactions, viruses are called serotypes
virus classification
by disease symptoms, city or area of classification, molecular themes (nucleic acid, protein coat)
classical system of virus nomenclature
phyla, class, order, family, subfamily, genus, species; grouped by shared properties (not disease types) like genetic material
4 characteristics of virus classification
1. genetic material (DNA, RNA)
2. capsid symmetry (helical, isometric)
3. naked or enveloped nature of nucleocapsid
4. dimensions of virion and capsid
nomenclature endings (order, family, subfamily, genus, species)
virales, viridae, virinae, virus, ? (characterization of viral species is problematic, similar characteristics that allow clustering separation from rest of viruses?)
virus strain
different lines of isolates of the same virus
virus type
different serotypes (different antigenic specificity)
virus group
sub-category of spcies, division based on genomic sequence similarities or origin
virus variant
virus whose phenotype differs from the original wild type strain but genetic difference for change is unknown
subviral agents
satellites, viroids, prions
viruses and RNA
only thing with RNA genomes, possibly connected to early RNA world, most viruses have RNA genomes (compared to DNA)
difficulties of in vivo infection (as opposed to ease of in vitro)
diffusion-limited (encounter host randomly), avoid harsh environment, break through physical barriers (dead skin, low pH of skin, mucous layers, ECM), immune defenses
epithelial cell infections
viruses infect exposed apical surfaces and can move through to basal side to get into body
simple squamous cells
thin cells, blood vessels
simple columnar cells
thick, mucous secreting, GI tract
transitional cells
distinct layers that are expandable
stratified squamous cells
epidermal layer of skin, mouth, genitals
cell membrane and viral entry
different lipid associations make certain microdomains for entry/exit (lipid rafts are dense patches of membrane with cholesterol that are resistant to this)
susceptible cell
has functional receptor for a given virus
resistant cell
no receptor for that virus
permissive cell
has capacity to replicate the virus
susceptible and permissive
has the receptor to take in the virus and can replicate it because it is permissive
plaque formation
monolayer of cells in dish, vius infects and speads locally to surrounding cells
logarithmic cell growth (compared to viral growth)
bacteria increase linearly (the log) and level off, viruses remain constant then burst then level off (the burst is steepest if the infection was highly synchronous)
old wrong ideas about viral growth
some thought they were host cell "lytic" enzymes out of control, some thought they were just very tiny cells
Delbruck and viruses to understand the gene
they self-replicate, mutations are inherited, small and easily manipulated with rapid growth
infected cell and number of viral progeny
either makes a burst or doesn't, most cells make the same number on average (each cell has a finite capacity to produce virus), number of particles per cell varies because of cell characteristics (bigger cells make more), virus type, and conditions
limiting factors for number of particles produced per cell
resources, sites for replication, timing systems, host defenses, others
cytopathic viruses
viruses that cuase the cell to burst (killing cell)
non-bursting viruses
don't kill cell, make particles as long as cell is alive and continuously release particles
Ellis and Delbruck experiment
virus particles added to e. coli, adsorbed to cells, diluted culture (reduces any further binding of virus to cells and synchronizes infection), samples of diluted cultures were taken over time to measure bacterial colonies versus number of plaques
viral growth curve when all cells infected
one burst with an eclipse period in the beginning and leveling at end
viral growth curve when a few cells infected
eclipse, first burst, eclipse, second burst, level off (uninfected cells from first burst become infected), slope of second burst is shallower (less synchronity), steps represent viral replication phases
intracellular infectious virus
lysed cells as time went on before time of usual burst (extracellular measurement), found increase to be linear from a certain (non-zero) time and up until the usual burst phase
eclipse phase of viral infection
after dilution of cells with virus, a complete loss of infectious virus occurs for some time because input infectious virions disappeared and no new phage particles were detected (uncoating and decoding of nucleic acid), no infectivity for this period before new virions assemble
latent period
the time it takes to replicate and release new virus
burst size/yield
number of infectious virus particles made per cell (total number of infective units divided by number of infected cells)
dificulties with animal cells in culture
cells cannot be suspended like in bacterial culture (monolayer of cells in culture with medium), infections cannot be synchronized by dilution
provide synchronous infections for animal cells
use high MOI (every cell will be infected with at least one particle), adsorb virus to cells on ice (block entry, and warm to 37 C so all enter at same time)
identical latent and eclipse period
when viruses bud from membrane (they become formed in cell and are released as they are formed)
host DNA replication and viral infection
normally cellular DNA synthesis (by radioactivity tests) increases over time, in infected cells it levels off at a lower level and viral DNA synthesis simltaneously increases
superinfection exclusion
virus in a cell will not allow same type of viral particles to infect cell (only one), maybe other kinds of viruses can
irrevesible actions during replication
no other virus at all can replicate after that virus gets in and begins its own replication
co-infections results
viral recombination, rapid evolution, helper functions by complementation
efficiency of plating
fraction of infectious centers (loci with one or more phage particles) that produces plaques
multiplicity of infection (MOI)
number of virions added per cell
circular zone of infected cells, distinguishable from the rest of the monolayer
single step growth curve
infection/growth (latent), burst (release)
burst size
number of virions released per cell after burst (average, varies from cell to cell)
eclipse period
phase of viral infection when viral nucleic acid is uncoated from its protective shell and no infectious virus can be detected inside the cell
latent period
phase of viral infection where no extracellular virus can be detected
synchronous infection
cells are infected at the same time so no "blurring" of growth curves occurs
Foot and Mouth Disease Virus particle
size of a ribosome, genome has just one ORF, not a human pathogen, rarely kills the animals it infects
Germ Theory
formulated by Robert Koch in 1879, viruses as units of infection were not known at this time (virology is not a very old discipline), Koch was a bacteriologist
1939, 1957 in virology
1939- Ellis&Delbruck described the one-step grwoth curve for phages
1957- Fraenkel-Conrat&Williams showed in vitro assembly of TMV
Dimitri Ivanovsky, Loeffler and Frosch, Walter Reed
recognized that TMV, foot and mouth disease, and yellow fever (respectively) is caused by a filterable agent
V Ellerman and O Bang, P Rous
found that cancer can be caused by cell free transmissions
F Twort and F D'Herelle
described bacteriophages (bacterial lysis)
light microscope can see
plant cells (10-100 um), animal cells (10-20 um), bacteria (1 um)
electron microscope can see
plant cells, animal cells, viruses (100 nm), ribosomes (20 nm), proteins (5-10 nm)
X ray can show
viruses, ribosomes, proteins, small molecules, atoms
NMR can show
proteins, small molecules, atoms
virus subtype
different protein composition as in H1N1 and H5N1 (differrent specificity (combo) of hemagglutinin and neuraminidase)
myristoyl group
lipid group that binds the proteins of certain viruses to the cell membrane during assembly
macroscopic effect
cell-cell spread, visible plaque instead of single bacterial cell infected (not visible)
"viral generation"
a burst of new viruses from one cell
tailed bacterial viruses
the most abundant viruses on the planet, bacteriophage T4
dilution step in burst experiment
reduces any further binding of virus to cells after particle adsorption to synchronize the infection
all cells versus a few cells infected
single burst for all cells, multiple bursts for a few (bursts occur, then more occur later to the originally unifected cells)
assay for intracellular infectious virus
zero up to end of eclipse phase, then increases up until the time of burst to the same amount of infectious particles that are released to the extracellular medium after burst (same y value on graph as burst)
methods used to lyse cells prematurely
freeze/thaw cycles, sonication, osmotic shock
problems with synchronous infection with animal cells
can't suspend cells (monolayer in dish), dilution cannot synchronize (instead use high MOI or adsorb virus onto cells on ice and then warm to 37)
cell with too many original virus particles
can lyse or die immediately (without any replication)
viral structural biology
X-ray crystallography and cryo-electron microscopy (freeze, structure not distorted by solvent)
TMV firsts
first shown to be deconstructed and have the ability to reconstitute an infectious particle, first discovered, first crystalized, first shown to have RNA as genetic material
common features/general principles of molecular architecture
symmetry (cubic, or helical), finite number of ways to put proteins together symmetrically, symmetry allows small genomes to encode machinery to build diverse structures
functions of virion proteins
protection of genome, delivery of genome, other interactions with host
protection of genome
assembly of a stable, protective protein shell, specific recognition and packaging of the nucleic acid genome, in many particles there is an interaction with host cell membranes to form the envelope
delivery of the genome
specific binding to external receptors of the host cell, transmission of specific signals that induce uncoating of the genome, induction of fusion with host cell membranes, interaction with internal components of the infected cell to direct transport of the genome to the appropriate site
other interactions of virion proteins with host
with cell host components to ensure efficient infectious cycle, with cellular components for transport to intracellular sites of assembly, with the host immune system
subunit (protein subunit)
single, folded polypeptide chain
structural unit (protomer, asymmetric unit)
unit from which capsid or nucleocapsids are built; may comprise one protein subunit or multiple, different protein subunits
morphological unit (capsomere)
surface structures (knobs, projections, clusters) seen by electron microscopy
capsid (coat)
the protein shell surrounding the nucleic acid genome
nucleocapsid (core)
the nucleic acid-protein assembly packaged within the virion; used when this compllex is a discrete substructure of a complex particle
envelope (viral membrane)
the host cell-derived lipid bilayer carrying viral glycoproteins
the infectious viral particle
virion metastability
protects the genome (stable), but must come apart quickly upon infection (unstable) if and only if cell gives proper signals (potential energy in assembled particle allows for disassembly)
virion molecular machine
has moving parts and does work but is not alive
virions as active containers
the particles just open up (not completely apart) and replication and transcription occur inside particle (cell can't detect viral nucleic acid, retroviruses convert RNA to DNA and reoviruses replicate and do mRNA synthesis inside virion)
virion structure
symmetrical arrangement of identical proteins, maximal non-covalent contacts (highly conserved structural motifs)
virion function
genome delivery made possible because the structure can be taken apart after infection
rules for virion self-assembly
each subunit has "identical" bonding contacts with its neighbors (chemically complementary surfaces), contacts are usually non-covalent (important for taking apart, error-free assembly, and minimization of free energy)
regular vs. irregular structures and bonding
regular structures form from interactions between identical subunits with identical bonds and aggregates, clumps, and disordered complexes (precipitates, inclusion bodies) form from non-identical bonds between identical subunits
virus-like particles (self-assembled from subunits), vaccines for HBV and HPV made like this (HPV VLPs are empty capsids with just one of its proteins)
solution to self-assembly problem
capsid proteins have highly conserved structures that enable symmetric interactions
pitch of helical capsid
angle between layers of the nucleocapsid (equals the number of units per turn times the axial rise per turn between adjacent units)
rigidity of helical capsids
either rigid or flexible, depends on capsid protein interactions
TMV, helical nucleocapsid
RNA lines the coat protein subunits, coat proteins engage in identical equivalent interactions with each other and the RNA, length of capsid determined by length of the RNA strand
virion families with (-) ssRNA genomes and helical capsids
paramyxoviridae, rhabdoviridae, orthomyxoviridae, filoviridae; capsids are always called nucleocapsids for these and the infectious particle has an envelope
when nucleocapsid term is used
when the nucleic acid-protein complex is a discrete substructure of the complex particle (not the virion itself)
solid with 20 triangular faces, 12 vertices related by 2/3/5 fold axes of rotational symmetry
number of proteins in icosohedral virus capsid
multiples of 60 usually, no virus with less than 60 subunits, spherical viruses can be many different sizes but usually have similar capsid protein subunits sizes (20-60 kDa), simplest has a homotrimer on each face
protein interactions in icosahedron
usually at least three proteins per equalateral triangle (60 or more subunits on 20 equalateral triangles), must be able to make 3 different types of interactions (at the 2,3,5-fold axes); theoretically a single protein unit could work but it needs to have three axes of symmetry and none have been found to have this
changing size of capsid
size of protein doesn't need to increse, just need to add more units to make a larger unit that can be put on the icosahedron as if it was just like the smaller unit, the face must be larger
T=4 compared to T=1
4 faces instead of 1 and 12 proteins instead of 3 (for trimers), proteins become only quasi-equivalent from new 6-fold axis
penton protein
for larger faces, may be a different protein to form the 5-fold axis
hexons vs. pentons
hexons are flat, pentons are puckered (12 corners of icosahedron)
larger T numbers
more asymmetric units have to be formed in the 20 faces (larger faces), T numbers are the only possible ways to assembly the asymmetric units in the 20 triangular faces
T number
number of asymmetric units per face of an icosahedron, assumes each asymmetric unit contains a capsid protein multimer
face versus structural (asymmetric unit)
capsid subunits = T x 60, number of subunits/face = 3 x T, protomer/asymmetric unit/structural unit = 3 (usually, can be on one or two different faces of the icosahedron)
T number equation
T = h^2 + hk + k^2 (h and k and non-negative integers), shows the allowed T numbers T=1,3,4,7,13... (area of icosahedron face increases directly with T number)
capsid protein motifs
must have certain motifs that define the 2,3,5 fold interfaces (most proteins do not fill this criterion), beta-barrel jelly roll is common
nano-fabrication of viral proteins
a plant virion protein is mutated to have cysteines at key symmetry centers, it assembles and silver or gold can be added to the cysteines and once virions crystallize to form lattices they can conduct electricity (nanowires)
open vs. closed viral structures
helices are open (unless something added to end), icosahedrons are closed
tailed viruses
majority of bacteriophages use tails for recognition and attachment to the host cell, penetration of the envelope, for DNA transfer from capsid to cell cytoplasm (attached at only one of the 12 penton vertices), elements in horizontal gene transfer
tail shape
helical symmetry in many places, some are contractile
spikes on icosahedron
can be at each 5-fold site, site of modification
opening for viral DNA in icosahedron
only at a single penton point out of 12, often a different complex protein present at the tail point
enveloped viruses and hosts
common for those that infect animals, less common for plants
derivation of envelope
lipid bilayer from host cell (viral genome doesn't code lipid synthesis machinery), occurs through budding of nucleocapsid from a cellular compartment or PM, either type of symmetry of nucleocapsids may have envelope
viral envelope components
lipid membranes, integral membrane proteins encoded by the virus (short alpha helices act as transmembrane domain), exterior domain has sugar groups, glycoproteins (hard to form antibody against sugars)
integral membrane glycoproteins
binding sites for cell surface viral receptors, major antigenic determinants, sequences that mediate cell fusion during entry
patches of viral membrane proteins
formed by lateral interactions of viral membrane proteins and by interaction with nucleocapsids or matrix proteins aligning along the inner surface of the modified membrane (different membrane components like lipid rafts can influence viral membrane protein assembly)
matrix protein binding
binds to cytoplasmic terminal of viral envelope proteins at the membrane and interacts with nucleocapsid proteins (proteins and nucleic acid), binding scaffold
structure of viral envelopes
most are unstructured, no physical connections between viral membrane proteins to underlying nucleocapside (only to matrix)
fatty acid modifications of matrix proteins
some use this modification to interact with membranes (HIV, myristoylation) to bring it into proximity to the membrane for viral exit (HIV uses protease cleavage of its matrix protein to activate it)
HIV MA protein
Interacts with the membrane through its N-terminal myristate chain (projects into membrane)
host membrane proteins and viruses
Not just viral proteins in membrane, also host proteins (not as abundant), passively or actively incorporated is the question, in low copy (not random incorporation, functional heterogeneity)
virus host membrane functions
inhibition of host defenses (functions used by the virus), polymerized actin for budding
viral envelope heterogeneity
unstructured virion envelopes have their copy number vary from particle to particle (try to figure out if this is functional or random)
functions of viral membrane proteins
receptor binding, membrane fusion, uncoating, assembly/egress, signaling (engages signaling pathway of cell), infected cell membrane interactions
membrane fusion by viral membrane proteins
fusion of virus envelope with cell plasma membrane for entry, fusion of infected cell plasma membrane with uninfected cell (syncytium formation)
syncytium formation
allows for cell-cell spread without release into medium (viral spread without release, avoidance of antibodies outside of cells)
viral membrane proteins on cell membranes
independent roles in cell membranes compared to roles on viral envelopes
gC proteins of herpes virus
bind complement proteins to block innate immunity from cell membrane
gE proteins of herpes virus
bind antibodies to block acquired immunity from cell membrane
M2 protein of influenza A virus
a tetramer with an aqueous pore to pump protons, it adjusts the pH inside the virus particle and inside membrane vesicles
influenza A virus structure (nucleic acid)
8 distinct pieces of RNA, one piece has a nucleation center for the other 7
herpes virus features
virus structure arose early in evolution, unusual architecture, 200 nm diameter, enveloped, three distinct layers, all types look alike under electron microscope, more than 12 membrane proteins
herpes virus structure
at least 35 different proteins in either capsid, tegument, or envelope (at least 8000 proteins per particle), capsid is T=16 (960 VP5 proteins), tegument has 10-20 different viral proteins (binds tails of envelope proteins plus capsid proteins), 12-20 different membrane proteins
poxvirus structure
200-400nm long, more than 100 different proteins, at least 10 enzymes per particle (mainly concerned with viral nucleic acid metabolism/genome transcription/replication)
vaccinia virus and membranes
four forms, number of membranes around core, IMV/IEV/CEV/EEV
intracellular mature virus, a single membrane
intracellular enveloped virus, same IMV membrane plus 2 more golgi-derived membranes (3 total)
cell-associated (stays outside cell, bound to plasma membrane), 2 membranes (IMV membrane plus inner IEV membrane), supported by microvilli formed by actin bundles (propel virion away)
extracellular enveloped virus, 2 membranes (same as CEV), not associated with cell membrane (unlike CEV)
results from fusion of IEV that loses outer golgi membrane
3 pathways of vaccinia virus
single membrane (no golgli) and lytic release, double golgi one lost on exit (extracellular), double golgi one lost on exit (stays connected to cell and pushes with actin)
HIV shape
cone shaped, can self-assmeble into cones and cylinders (facilitated by RNA presence but not essential)
Fullerene cones
not covered by quasi-equivalence theory, not normal symmetry
complex capsids with 2 icosahedral protein layers with no envelopes, concentric shells (inner is forbidden T=2 from dimers, rare; outer is unusual T=13 and uses a different capsid protein), 12 hollow turrets (pass through both shells) out of each point where RNA is synthesized and released
chorella virus
T=147, icosahedral with many structural twists
huge with T=1179
virion assembly procedure
individual protein molecules form structural units, coat assembly by structural unit interactions, nucleic acid genome incorporated, possible lipid envelope addition by budding out of cell or into a cell compartment, release of newly formed particle (possible protein processing to become infectious)
virus reaction conditions
requires specificity and coordination of multiple reactions, reasonable efficiency of assembly, overall irreversible
virus particle structure determines...
determines the nature of reactions by which the particle is formed, the mechanism by which it enters the cell (lipid based or naked), mechanism by which it replicates
picornaviruses (polivirus vs. rhinovirus)
poliovirus virions survive through stomach to replicate in gut (receptors are in the intestine, not lungs) so it is an acid resistant capsule, the rhinovirus receptors are in the respiratory tract and cannot survive low pH of stomach (acid sensitive)
metastability of virions
stable enough to survive in the wild, unstable enough to come apart during infection (irreversible formation in cell that produces it and reversible in cell that it infects)
host cell functions for viral assembly
cellular proteins catalyze folding of individual viral proteins, cell transport systems move viral proteins/nucleic acids to/from sites of assembly, secretory pathway moves viral proteins, nuclear import/export proteins for nucleic acid
concentration of viral components
faster reactions in less dilute solutions, concentrated into small inclusions, naked viruses often use internal membranes to concentrate proteins, viral replication/translation in a new compartment (localized site) to enable formation of independent subassemblies, lateral interactions between membrane associated proteins cause local membrane protein concentration
viral subassemblies
assembly from individual protein molecules (pentons (hole proteins), trimers...), polyprotein precursor (already in proximity/held together), chaperones to assist assembly
assembly line virion formation
orderly formation of viral particles and virion subunits, form discrete intermediates first (quality control, only go on once last step worked)
self-assembling viral structures
HIV capsid forms empty shells, VSV membrane protein can bud and form round lipid vesicles, HBV surface antigen can assemble into virus-like particles
viral particle assisted assembly
particles can't assemble on their own, proteins and nucleic acid genomes needed as scaffolds or chaperones to form structure
viral scaffolding proteins (herpes simplex virus type 1)
a transient intermediate structure using a scaffolding protein forms so the capsid proteins can form around it, then a protease breaks up the scaffolding so the genomic DNA can enter
localization of virion components
increase concentration to increase reaction rate, establish microenvironments to restrict side reactions, moving far in a cell requires a transport system like microtubules, and IFs (use dyneins, kinesins, myosins)
viruses and built in "address"
allows host machinery to localize it to correct place, membrane proteins go to appropriate membrane (signal sequences and fatty acid modification cause this), membrane proteins remain where they are (retention signals), nuclear localization signals or export signals
localization of viral proteins to nucleus
nuclear viral protein is translated on host cytoplasmic ribosome and has a signal for transport into the nucleus (nuclear import protein) through pore
localization of viral proteins to the plasma membrane
viral protein has ER signal and is translated into ER membrane, vesicle to golgi, sent to PM in vesicle membrane and remains in PM (then the internal proteins and nucleic acid are directed to these PM sites for particle assembly)
viral protein signal sequences
some hydrophobic regions for lipid binding (matrix proteins), localization signals, nucleic acid binding sites
concerted assembly of influenza A virus
concerted assembly is when capsid proteins only form in association with the genomic nucleic acid, for influenza A (-)RNA is replicated in nucleus, packed in helical nucleocapsid, a matrix protein enters nucleus with a nuclear export viral protein and the NEP binds to the matrix, some proteins go through secretory pathway and buds off
sequential assembly of adenovirus
in sequential the protein shell is preformed and then the genome is inserted, pentons and hexons are formed in the cytoplasm and transported into the nucleus and form into capsid and DNA enters, protease converts immature virion to full mature virion
issues with viral nucleic acid packaging
discrimination of viral genome from large population of cellular nucleic acid, energetic problem of condensing, collapsing, excluding water/ions
viral genome packaging
must be inside of capsid no matter what shape, interacts specifically and non-specifically with viral proteins
recognition of nucleic acid genomes (HIV)
packaging signals, HIV virus has a spot called psi that is the packaging site, stems and loops of RNA genome bind the capsid proteins
recognition of nucleic acid genomes (adenoviruses)
numerous DNA sequences important for packaging the genome, set of repeating sequences that overlap with enhancers that stimulate their late transcription, the structure is recognized by the viral protein IV2a (a transcription activator and packaging initiator)
implications of capsids with helical symmetry with respect to nucleic acid binding
capsid proteins can bind to any nucleic acid sequence (no specificity except for the first interaction of capsid protein initiation complex with the nucleic acid), in theory any length nucleic acid can be put into a helical capsid
initiation sequence
binds specifically at one end of the nucleic acid (usually), ensures viral genome is packaged, allows for other capsid proteins to add on
Traveling Loop model (helical capsids)
nucleic acid (RNA in TMV) keeps jumping forward as more and more capsids are added from the initiation complex
Influenza virus particle genome packaging (random packaging)
8 genome segments of (-)ssRNA required to be infectious, random packaging is likely as only a small portion of virus particles are actually infectious and particles with more than 8 segments have been observed
bacteriophage genome packaging (selective packaging)
ratio of infectious particles to total particles is around 1, 3 dsRNA segments S, M, L are packaged in sequence (each step changes packaging recognition)
connection between site of assembly, site of replication of genomes, and mechanism of exit
replication and assembly in cytoplasm associated with membrane compartments (most enveloped RNA viruses, poxviruses, reoviruses), nuclear replication/assembly in specific compartments (adenoviruses, papillomaviruses, polyomaviruses, parvoviruses, circoviruses, herpesviruses), replication in nucleus/move nucleocapsids out of nucleus and final assembly occurs as virions bud from plasma membrane (influenza viruses, bornaviruses)
budding definition
active process of pushing nucleocapsid structure into a lipid bilayer to acquire an envelope
budding from cell process
capsid formation is separate from envelope acquisition, capsid interacts with matrix proteins, membrane containing viral membrane proteins dictates assembly and release by binding matrix
reverse of endocytosis; used by viruses that assemble within vesicular compartments of the ER or golgi (herepesvirus assembly and egress)
viral maturation
final stages of producing an infectious particle (some can be immediately infectious, most take some time and some can take a long time maybe after encountering host (low pH may activate))
4 budding strategies
1. envelope glycoproteins and internal capsid important (interactions between envelope heterodimers with each other and capsid proteins)
2. only internal matrix and capsid proteins are required
3. only envelope proteins needed
4. internal matrix proteins alone can drive budding but is inefficient and creates deformed particles (so does need envelope glycoproteins)
viral particle activation and energy input
often by specific proteases which cleave capsid proteins or by conformational changes in the protein during assembly
HIV protease
a maturation enzyme required to make an infectious HIV particle (drugs are protease inhibitors)
retrovirus maturation
viral protease cleaves GAG polyprotein to release new C and N termini for interactions that define the final structures in the virion
2 mechanisms to leave an infected cell
release into external environment (either budding or lysis from cell), move directly into another cell by cell-cell spread
apical egress
virus goes out of apical side of cells (places in outside world, sneezing)
basal egress
virus moves into blood, lymph, and nerves (systemic spread)
egress at sites of cell contact
lateral spread (infection in lungs or gut tissue itself), syncytia can allow for cell-cell spread, lesions can occur in certain cells (many viral particles in one cell)
virological synapse (HIV infection)
HIV infected T cell releases particles onto epithelial cell for uptake into the blood
alpha herpesvirus spread of infection
polarized spread where the particles from cell body in neuron are made, sent down axons and released from axon terminals onto epithelial cells
direction/mode of egress and pathogenesis (neural spread)
local virus spread, systemic spread, or spread into brain (down axon terminals)
Viral needs covered by gene products and regulatory signals
replication of genome, assembly/packaging of genome, reulgation of timing of replication cycle, modulation of host defenses, spread to other cells/hosts
information not in viral genome
no complete protein synthesis machinery set (no rRNA, ribosomal/translational proteins), some have tRNA, no genes involved in energy products and membrane biosynthesis, no centromeres/telomeres
7 viral genome classes
dsDNA, gapped circular dsDNA, ssDNA, dsRNA, ss(+)RNA, ss(-)RNA, ss(+)RNA with DNA intermediate
different viruses connection and mRNA
all viral genomes must make mRNA that can be read by host ribosomes
+ or - strand conventions
mRNA is always + strand, DNA of equivalent sequence is also + strand, RNA/DNA complements are - strand (not all +RNA is mRNA)
simplification by Baltimore system
only a few ways to replicate nucleic acid and fewer to produce mRNA from them
common and rare types of genomes
all RNA genomes are linear (circular is very rare, except hep D satellite virus), linear ssDNA is rare (parvovirus), circular ssDNA is pretty rare (circovirus), ds circular DNA can ONLY be papillomavirus or polyomavirus, linear dsDNA is quite common
viruses with covalently attached proteins
probably a protein primer for replication (very few viruses use this strategy)
viruses with a stretch of RNA at the end of a DNA genome
probably a hepadnavirus (hep B), RNA is used as a primer for replication
viruses with cross linked ends of a DNA genome
probably a poxvirus (when denatured, you get a circular piece of DNA as ends are covalently attached)
viruses with stems & loops at ends of linear ssDNA genome
probably used as internal primers for replication
viruses with stems & loops at ends of linear ssRNA genome
probably used to initiated replication, translation, or even packaging into virions
human uses for viral genome sequencing
determine relationships between viral genomes, niche sequencing (viral ecology), origin/movements of viral agents from populations (animals, humans), compare sequences present in diseased versus normal tissues
virochip DNA array
all known viral genome sequences, hybridize to find related viruses to one that may be unknown
why both RNA and DNA
no clear answers, RNA probably from RNA world, DNA possibly replaced RNA as a result of reverse transcriptases
smallest, non-defective animal viral genome (1.7-2.3 kB); hepadnavirus is the second smallest
1.2 Mbp, giant amoebae virus, no envelope, 400 nm diameter (other large ones are iridoviridae, polydnaviridae, poxviridae)
RNA vs, DNA in genome size
RNA genomes never as large as DNA genomes (coronaviridae is the biggest with 31 kB of (+)ssRNA)
reason for big DNA genomes
usually associated with large capsids to hold the large genome (more genes needed), have more complicated lifestyles(viral replication in quiescent cells, nucleic acid metabolism, modulation of host defense systems), less dependent on cell functions (restrictions on host biochemistry)
virus definition
no genes that encode proteins to make functional ribosomes, membranes, or machinery to produce energy
big viruses compared to cells
some seem like they could have been derived from a primordial cell (large, diverse collection of genes), root of the tree of life, big DNA viruses often replicate in the cytoplasm (possibly around before nuclei were invented), cells have larger length DNA, RNA, mRNA
possible limitations on viral genome length
length of time needed to replicate (probably mainly only true for RNA viruses as DNA have multiple origins of replication), capsid size capacity
building a bigger capsid and genome size
takes more proteins to build, more machinery needed (proteins not incorporated in capsid, enzymes), high percentage of ORFs used for capsid building proteins
limits to RNA genome sizes
largest RNA genomes are in the coronaviridae (max ~31 kB), shear force may break long RNA, RNases may be important in limiting size, error elimination only for DNA poly (longer have more chance of more mutation 1/10^4 or 5) and no RNA repair enzymes known
helical capsids and RNA genome size
theoretically have no limit, but max is usually 19 kb (filoviruses, plant closteroviruses), shear force may break long RNAs
tricks for larger RNA genomes
segmented RNA genomes (orthomyxoviridae, reoviridae; doesn't work)
Dengue virus/HIV and defectiveness
more than 90% of all genomes in a person finishing a bout with the virus are defective (have mutations, demonstrating that RNA genomes do mutate and this is problematic), mutated viruses have replication problems, error catastrophe
3 viral DNA genomes
double-stranded (linear or circular), single-stranded (usually linear), gapped
mRNA production from dsDNA virus
copies (-) strand to mRNA, can only copy from dsDNA, ssDNA of the dsDNA doesn't work for transcription
viral family genomes that encode viral DNA poly
adenoviridae, herpesviridae, poxviridae
viral family genomes that use host DNA poly
polyomaviridae, papillomaviridae
viral family genomes that don't use DNA poly (non-RNA viruses)
retroviridae, hepadnaviridae
ssDNA virus genomes that infect mammals
circoviridae and parvoviridae
problem for ssDNA genomes
as RNA can only be transcribed from dsDNA, the ssDNA must have its second strand synthesized to form dsDNA before mRNA is produced, these genomes need cellular DNA polymerases as they don't encode their own
gapped DNA genomes
hepadnaviruses, protein covalently attached to 5' end of one strand, short RNA on other strand 5' end, one strand is complete and the other is not
hepadnaviral genome repair
can't make mRNA from its gapped DNA genome, must be repaired and converted to dsDNA (need perfect duplex DNA to make mRNA), makke ccc dsDNA
unusual hepadnaviral replication
no DNA/RNA polymerases, strange replication process from RNA template by viral reverse transcriptase
general viral RNA genomes
usually linear, single-stranded, + or - stand (sometimes both), some are double stranded
RNA polymerase and RNA viruses
host has no RNA-dependent RNA polymerase so RNA viruses must encode one in their genome (these produce both RNA genomes and mRNA from RNA templates)
2 segments of dsRNA in genome, infects vertebrates
problem with translation of dsRNA
dsRNA cannot be translated by ribosomes, the minus strand must be used as the template strand to produce + strand mRNA
unique reoviruses lifestyle
dsRNA where viral polymerase is part of the capsid, all replication and mRNA synthesis occurs inside capsid, mRNA is squirted out of capsid through one of 12 turrets into the cytoplasm, translation on host ribosomes, new viral mRNA packaged inside new capsid, viral polymerase copies mRNA to produce dsRNA for genome
(+) strand ssRNA viruses
most plentiful on planet; include picorna, calici, astro, corona, arteri, flavi, retro, toga
translation of (+) strand RNA genomes
translated directly into protein by host ribosomes (must occur before any RNA replication or mRNA synthesis can occur)
(+) ssRNA genome replication steps
(+) strand genome copied into full length (-) strand which is copied back to the original (+) strand RNA, sometimes short "sub-genome" mRNA is formed by only replicating part of the anti-genome back to (+) strand
retroviral genome strategy
RNA is copied into DNA then back to RNA some of which is packaged into virions (converted from (+) ssRNA to dsDNA by viral reverse transcriptase), mRNA genome is never used as a message!!!!!!!!
proviral DNA in retroviruses
dsDNA from intermediate in replication integrates into host chromosome via integrase, serves as template for viral mRNA and genome RNA synthesis, uses CELLULAR RNA polymerase to make mRNA (some is translated and some is packaged as genome)
reverse transcriptase
responsible for the conversion of (+) ssRNA to (-) ssDNA AND to dsDNA
viral families with (-) sense RNA genomes
paramyxo, rhabdo, borna, filo, orthomyxo (mammalian); either single/non-segmented or segmented
(-) ssRNA genome translation
cannot be directly translated, must be copied to make + strand mRNA to be translated (use viral encoded RNA polymerase found inside the capsid)
host cell enzymes and (-) ssRNA viruses
do not have an RNA-dependent RNA polymerase so virus brings it in allready packaged into particle
replication of (-) ssRNA
RNA polymerase must produce + strand RNA that is not mRNA but templates for making the - strand genome
ambisense definition
some viral genome segments are neither plus nor minus strands (used by some (-) ss RNA genomes like arena and bunya), both + and - strand information is on a single RNA strand
ambisense strategy
the 3' end can be minus and 5' end can be plus (not a message without 5' cap and polyA), the 3' end can undergo one round of copying to become mRNA, the 5' end needs two rounds of copying for mRNA
viral genome structure inside a virion capsid
varies between viruses, some highly compacted, some naked/highly condensed, some associated with viral nucleic acid binding proteins (no histones like host)
nucleosomes in virions
polyoma and papilloma are like mini-chromosomes bound in nucleosomes inside virions
computer-predicted RNA genome structures
many stems and loops, genetic and biochemical methods predict importance, may be signals for copying + and - strands and for translation, compared to DNA (which does not do this secondary structure) RNA genomes can store more information based on structure alone
definition of small viral DNA genomes
less than 15 genes, some with only two ORFs, double or single stranded, circular or linear, use host DNA poly (don't encode their own), can only replicate in cells in S phase of cell cycle
double stranded DNA viral replication
DNA is transcribed at different times (early and late), input genome can make mRNA and replicated DNA can make mRNA
single stranded DNA viral replication
genome cannot make mRNA, DNA is replicated into duplex and then either the + or - or both strands is added to the capsid, host RNA poly II transcribes dsDNA
ways small DNA viral genomes attract proper host DNA poly
encode unique replication of origin, encode a unique origin binding protein (binds host replication proteins)
cell cycle and small DNA viral genomes
most host cells are terminally differentiated (no DNA synthesis); either encode viral proteins to move cell into S phase (polyoma, papilloma), wait quietly until cycle starts naturally (circo, parvo)
small DNA genomes clock of gene expression
early genes transcribed by host RNA polymerase from early promoter, makes large and small T antigens, large T antigen stimulates viral DNA replication, then host RNA poly transcribes late genes to make capsid proteins in large quantities
late promoter and viral DNA synthesis
late promoters require DNA replication to occur first, when viral DNA synthesis is blocked then late gene expression is too
4 small animal virus DNA genome families
circo, parvo, papilloma, polyoma; all replicate in host cell nucleus and use host cell replication proteins
small, T=1, naked, ss circular DNA, 3 proteins (VP1,2,3), can't be activated in quiescent cell as they have ssDNA (must be replicated to dsDNA then to mRNA), differential mRNA splicing and overlapping ORFs
smaller, linear ssDNA, T=1, very stable capsid (trypsin resistant!!!), grow only in rapidly dividing cell (S phase)
genome structure of parvoviridae
linear but with hairpin inverted repeats at both 3' and 5' ends
3 genera of parvoviridae
parvovirus (non-defective); dependovirus (defective, even in S phase cells) require unrelated helper virus (adeno-associated virus as adeno is the best helper, gene therapy); densovirus (non-defective) autonomous insect viruses
unusual aspects of parvovirus
linear/non-segmented ssDNA (~5000 nucleotides), compact genome coding with only 2 ORFs (either rep/trans NS1,NS2 proteins or capsid proteins), use several mRNAs with splicing to make multiple proteins from 2 ORFs
ends of parvo genome
looks gapped so induces repair enzymes to viral DNA, usually about 145 nucleotides in hairpin, act as origin of replication and packaging site (either package the +, -, or both strand), terminal repeats can bind host trans factors
strand selective packaging of parvo ITRs
if 3' and 5' sequences aren't identical (autonomous parvoviruses) then only one strand is packaged, if they are identical then both strand are packaged (dependoviruses)
parvo 3' terminal repeat
acts as primer for DNA synthesis (origin of replication)
parvo 5' terminal (autonomous parvoviruses)
the NS1 protein binds to the 5' terminal repeat, required for replication acts as an ATPase, a nicking enzyme, a helicase (nick allows for repair proteins)
adeno-associated virus
needs helper virus, spliced and unspliced mRNA with 2 different ORFs (both make different length proteins), one ORF is for replication and the other for capsid, same genomic organization of non-defective parvoviruses, can have replication stimulated by UV light/cycohexamide/some carcinogens
AAV in absence of helper virus
genome is replicated, mRNA, Rep 78/68 protein is made and brought into nucleus to integrate dsDNA into chromosome 19
AAV in presence of helper virus
capsid proteins are made and both minus and plus strands can be packaged and released
dsDNA small viral genome families
polyomaviridae and papillomaviridae (often tumor viruses)
transforming viruses
papilloma and polyoma may allow rare cells to survive infection and become immortalized (tumorous), infection of non-host animals can give rise to rare tumors, can integrate some genome into host genome
SV40 history
1960 contaminant of poliovirus vaccines, SV40 causes transformation of baby hamster cells, polyomaviridae family
SV40 genome
6 genes, 5243 bp, genome condensed by histones then put in icosahedral particles, only uses host DNA poly (only one strand is coding), early and late genes (rep-incoming genomes, cap-replicated genomes), early genes on one side of circular dsDNA and late on other side of origin
SV40 genes
3 late structural genes (capsid VP1, VP2, VP3), 2 early regulation, replication genes and one microRNA (large T and small T antigen are multifunctional proteins produced by alternate splicing, micro RNA controls late expression of T antigen), 1 late maturation protein (LP1)
large T antigen in SV40
binds viral origin as hexamer to unwind and attract host replication proteins, "convinces" cell to go to S phase
SV40 replication cycle (strange step)
strange step where it goes into ER first after entering cell
papillomaviridae spread
commonly spread from shedding squames on skin, must infect basal stem cell and speed up skin cell differentiation process
papilloma characteristics
circular dsDNA, larger than polyoma, use host DNA poly, not easily cultured
bovine papilloma virus
10 genes, 1 coding strand, separates early and late transcription units, 8 early genes (E6, E7 are transforming by interacting with p53 and Rb)
mRNAs from BPV
at least 6 transcription start sites, two strong transcription terminators, multiple splicing, at least 10 proteins
large viral DNA genome families
poxviridae, herpesviridae, adenoviridae
characteristic features of large viral DNA genomes
repeated sequences (herpes), terminal cross-links (pox), covalently attached terminal proteins (adeno, TP protein to 5' end)
temporal cascade regulation of large viral DNA genomes
gene set A turns on set B turns on set C(small genomes use one or two early gene products to turn on DNA replication and late gene expression)
gene classes in large viral DNA genomes
immediate early, early, late
immediate early genes (large DNA genome)
promoters recognized by host RNA polymerase, IE gene encode regulatory proteins and increase transcription of E genes (other viral promoters not recognized by host RNA poly)
early genes (large DNA genome)
promoters require IE gene products to be active, encode genes required to replicate genome, can have genes that modify cell defenses
late genes (large DNA genome)
promoters require DNA replication to be active, encode viral structural proteins (and proteins required for assembly/released of virions)
importance of temporal control
enzymes must be made early as only a small amount is needed and structural proteins are stoichiometric (can't be reused like enzymes and needed in large quantities)
viral DNA replication and structural genes
genome must be replicated first to create many templates and mRNA then is created to make structural proteins in large quantity (DNA that is to be packaged does not play a role)
Splicing in large viral DNA genomes
only occurs in nucleus, herpes/adeno splice pre-mRNA to mRNA but pox does not splice because DNA is replicated/transcribed in cytoplasm
herpesviridae subfamilies
alpha, beta, gamma herpes (also oyster/fish), linear dsDNA, no terminal proteins, repeated DNA segments (long ones at termini, location is unique to different subfamilies)
herpes repeats
subfamilies have large unique sequences flanked by repeats that can be from 30bp to 10,000bp
herpes genes
usually around 70-200 genes encoded, conserved genes between different herpes viruses (structural, membrane, regulatory, DNA metabolism, DNA synthesis (have own machinery), protein processing/modifying
herpes genes not used for replication
proteins that enable virus to counter host defenses and spread in infected host (pathogenesis/virulence genes), genes for a type of latent infection (productive vs. latent infection)
herpes genome replication
all DNA replicated in nucleus after de-envelopment and uncoating at plasma membrane, capsid delivers DNA at nuclear pore, host RNA poly transcribes (IE genes first), viral enzymes for replication (standard fork method), genome package by packaging sequences
latent infection of herpes viruses
no viral particles made, immune defenses are modulated actively/passively, occurs every time a host cell is infected, depends on cell type, if host survives primary infection then it will have a lifelong parasite, can reactivate lytic cycle at times to infect other hosts (stimulates immune system to control infection)
alphaherpes virus latency
infects neurons (HSV, VZV)
betaherpes virus latency
monocyte/macrophage precursors (HCMV)
gammaherpes virus latency
B and T lymphocytes (EBV)
adenovirus facts
common infection of mammals/birds, 80 serological types, permanent resident of populations, not latent (persistent, always replicating)
adenovirus genome strategy
midsize, linear dsDNA (30-38kb), 30-40 genes, terminal sequence is inverted repeat (contain origin of replication), protein attached to 5' end (primer), own DNA poly, packaging site near left side of genome
adenovirus gene expression
not simple early/late groupings, cluster of genes expressed from a limited number of shared promoters, a lot of splicing (multiply spliced mRNA and alternative splicing to make a variety of polypeptides)
vaccinia virus
a poxvirus, used as a live vaccine against smallpox virus, vector system for gene delivery
poxviruses and immune defense
carry many genes whose proteins counter host innate immune response, concerns about bioterrorism
differences of pox virus to other large DNA viruses
very large in size, replicates in cytoplasm and not nucleus (little need for nucleus), make viral mRNA in cytoplasm and replicate viral DNA in cytoplasm (is it possible pox viruses predated the nucleus?)
pox virus inverted repeats
10 kb at each end with the last 3.5 kb with 70bp tandem repeats, covalently attached (denature and get large ss circular DNA), function is in replication and packaging genome, replication initiates in terminal repeats without fork mechanism, own DNA/mRNA synthesis systems
pox particle
over 100 proteins in particle including complete DNA/RNA synthesis proteins, can have several layers of envelopes as it matures
pox gene expression
IE/E/L protein cascade, regulation by viral transcription factors and mRNA stability, many promoters, no splicing
pox gene products (viral, not host)
RNA polymerase, mRNA capping enzyme and polyadenylation enzymes, DNA polymerase, thymidine and thymidylate kinase, DNA ligase, genes to counteract host immune system (block/interfere with cytokine response)
nucelocytoplasmic large DNA viruses (NCLDV virus family)
all replicate in cytoplasm, all carry genes for DNA replication and mRNA synthesis, do not use splicing, collection of genes with little/no homology to others
differnt NCLDV viruses
pox virus, baculovirus, mimivirus, phycodnaviridae, iridoviridae, asfarviridae
two basic genome requirements (RNA or DNA)
genome must be copied end to end without loss of nucleotide sequence, genome must encode viral mRNA that can be translated by host cell machinery
(+) ssRNA families
corona, flavi, picorna, toga (replication in cytoplasm)
picornaviridae viruses
poliovirus, FMDV, HAV
flaviviridae viruses
yellow fever, HCV, west nile
togaviridae viruses
WEE, rubella
coronaviridae viruses
SARS, common cold virus
commonalities between virus family genomes with (+) ssRNA
all are "ribosome ready", purified genomic RNA is infectious when applied to sensitive cells in absence of viral proteins (but low infectivity compared to virion infection), enzymes for replication are made after infection (not already in capsid), RNA-dependent RNA polymerase encoded in (+) strand genome
monopartite tactic for (+) ssRNA genome expression
a single RNA molecule encodes all the proteins (goes against eukaryotic one mRNA, one protein)
tactics for monopartite tactic to work in eukaryotic cell
make huge polyprotein that is cleaved by viral (or cell) proteases (picorna, flavi), make sub-genomic mRNA by copying the genome to the anti-genome and making short mRNAs from this copy (toga, corona)
picorna virus shape
pico=small, rna=RNA, size of ribosome (28-30 nm), have 60 protein subunits (protomers) for T=1 with pseudo-T=3 icosahedral symmetry
common cold virus that belongs to picornaviridae
picornaviridae genomes
covalently attached protein VPg at 5' end, control regions at 5' and 3' UTRs (5' UTR is long and important for translation, RNA replication, and virulence; 3' UTR is short and necessary for anti-genome synthesis), genomes have only one large ORF
picornaviridae polyprotein cleavage
cleaved by 2 viral proteases to make 11 different proteins
VPg in picorna viruses
small basic 23 aa protein, acts as RNA poly primer to initiate minus strand replication, polyuridylylated, complexes with viral RNA poly (complex pairs with polyA tail on 3' end and poly copies template)
picornaviruses and polyA tails
have poly A tails at 3' end but these are encoded in genome not added post-transcriptionally like cellular mRNA
3 major genera of flaviviridae
flavivirus (yellow fever, west nile virus, dengue, from ticks), pestivirus, hepacivirus (HCV)
flavivirus RNA genome parts
5' end has methylated cap, NO polyA tail, one big ORF, cellular/viral proteases cleave into 12-15 individual proteins
toga and flavi twist on polyprotein processing
initially polyprotein is sent to ER like membrane protein (ER, golgi), protease cleaves membrane bound polyprotein into fragments destined for either membrane or cytoplasm, cell signal peptidase cleaves proteins in the lumen
togavirus size and shape
smallest non-defective enveloped virus, membrane acquired from budding through plasma membrane
two togaviridae genera
alphavirus (sinbis virus), rubivirus (rubella)
mosquitoes and ticks and togavirus
replicate in mosquitoes and ticks and then can infect warm-blooded animals, birds are common natural hosts (zoonotic infections); rubiviruses are the exception that are not spread by vectors
togavirus genomes
functional mRNAs, both 5' cap and polyA tail, uses both polyprotein and subgenomic RNA tactics (the subgenomic mRNA is then proteolytically processed too)
togavirus different activities of RNA poly protein
viral replicase (RNA poly) makes antigenome, mRNA, and genome but there can be different cleavages to give four different enzymes from a single sequence polyprotein (different activities appear depending on where cleavage occurs on polyprotein)
nidovirales order
(+) ssRNA with nested set mRNAs with families arteriviridae and coronaviridae
coronaviridae shape
crown of membrane proteins, enveloped with spikes of protein
coronaviridae genera
coronavirus (human common cold, SARS), torovirus
coronoviridae genome
longest of RNA viruses, 5' cap and polyA tail, functions as mRNA, genome is helical nucleocapsid with viral N-protein bound to RNA of genome, more than one ORF
nested set mRNA strategy of corona
make a novel RNA polymerae from a polyprotein encoded in the first ORF, produce MULTIPLE sub-genomic mRNAs with different 5' ends but common 3' ends (nested-set mRNAs
coronavirus individual transcript production
not by splicing as host cell mRNA but by initiating mRNA synthesis on specific parts of antigenome, creates more from monocistronic mRNA (also creates crude timer that makes poly first from full length then capsid proteins from nested)
two types of (-) ssRNA viruses
have one single molecule (monopartite, non-segmented), have several segments of RNA per virion (genome in pieces)
7 (-) ssRNA families
orthomyxo, paramyxo, rhabdo, filo, arena, bunya, borna
6 fundamentals of (-) ssRNA
(-) sense ssRNA genome is NOT mRNA, (-) ssRNA molecules NOT infectious, no 5' cap, no polyA tail, cannot be translated by ribosomes, ALL contain active RNA poly
envelopes and (-) ssRNA
all viruses with (-) ssRNA have envelopes
(-) ssRNA viruses with non-segmented genomes
rhabdo, paramyxo, filo, borna
(-) ssRNA viruses with segmented genomes
orthomyxo, arena, bunya
number of genes in (-) ssRNA viruses
only 5-10, so small but deadly
vesicular stomatitis virus
rhabdoviridae, bovine pathogen, model virus, gene therapy vector
rhabdoviridae, only human pathogen in family
TACTIC 1: polymerase storage in (-) ssRNA viruses
poly is possibly pre-loaded onto RNA during virion packaging and is ready to replicate in permissive cell, begins by adding a 5' capped nucleotide (capping enzyme part of polymerase complex)
TACTIC 2: stuttering polymerase in (-) ssRNA viruses
polymerase makes mRNA from a coding sequence and then stops, adds a bunch of A residues (stuttering) and after about 100 the RNA copy is released to give a single protein, the polymerase remains associated with the genome and re-initiates synthesis of second ORF (occurs for the five rhabdoviridae genes and then falls off to find another template)
regulatory sequences separating genes in stuttering polymerase viral genome
include an AUAC stop code, a poly A (6 U's), a non-transcribed G/CA region, and an mRNA start UUGUC region for the next gene
Le and Tr in (-) ssRNA viruses
leader contains that genomic and trailer contains the anti-genomic polymerase recognition sequences
stuttering polymerase slippage
when it reaches the non-transcribed (intergenic) region it slips and recopies the A residues from the U template until it finally falls off the template
replicating the (-) ssRNA genome in virus with stuttering RNA poly
can't use mRNA's (only single genes), needs to shut off the stuttering activity and make a full length (+) strand (antigenome, not mRNA)
vesicular stomatitus virus and polarity of gene expression
mRNAs toward 3' end of genome are made in much larger quantity than those at the 5' end (due to poly falling off template sometimes before reaching end)
polymerase in viral genome replication with stuttering mRNA production
same polymerase is used for both creation of antigenome and mRNA synthesis but modified with different accessory proteins to prevent stuttering in genome replication
paramyxoviridae viruses
parainfluenza virus, mumps virus
paramyxoviridae groups
morbillivirus group (measles), pneumovirus group (respiratory syncytial virus)
paramyxo gene organization and expression stategy
polymerase in virion, stuttering polymerase
paramyxo gene organization and expression stategy
polymerase in virion, stuttering polymerase
filoviridae virions
pleomorphic in size and shape (strange shape), long and filamentous (800-900 maybe 1400 by 80 nm), sometimes U/6/O shapes, helical nucleocapsid with 50 nm diameter, forms inclusion bodies on cytoplasmic replication, particles bud from the plasma membrane
effects of filoviridae viruses
have the highest fatality rates out of all the hemorrhagic fever viruses
filoviridae mRNA and genome production
use stuttering polymerase with gene boundaries for transcription start and stop, can do mRNA editing (for envelope protein GP)
arena, bunya, orthomyxo number of segments
arena has 2 (many hemorrhagic fever viruses), bunya has 3 (hantaviruses), orthomyxo has either 7 or 8 (influenza virus)
reasons for segmenting a genome
ssRNA is easy to break chemically so segmenting allows for large genome size and decreases physical liability, more efficient decoding with 1 protein per segment, reduction in errors during replication, possible genetic recombination, coordination of segments in mRNA synthesis and replication is not understood
a type of gene exchange only found in segmented minus ssRNA viruses where segments are randomly packaged (2 infecting viruses and 1 cell can create progeny virions with different genomes), rapid information exchange for evolution (pandemic influenza virus)
(-) ssRNA viruses that replicate in host nucleus
orthomyxoviridae and bornaviridae
vesicular stomatitus virus and polarity of gene expression
mRNAs toward 3' end of genome are made in much larger quantity than those at the 5' end (due to poly falling off template sometimes before reaching end)
orthomyxoviridae and influenza
influenza is a virus in this enveloped viral family that bud from the plasma membrane; influenza viruses A and B (8 segments) are major human pathogens while C (7 segments) rarely infects humans
polymerase in viral genome replication with stuttering mRNA production
same polymerase is used for both creation of antigenome and mRNA synthesis but modified with different accessory proteins to prevent stuttering in genome replication
influenza virus nucleocapsid assembly
must occur in nucleus because replication and mRNA must assemble there
paramyxoviridae viruses
parainfluenza virus, mumps virus
cap snatching in (-) ssRNA viruses
viral mRNA is made when subunits of the virion polymerase help to bind host mRNA and cut off 5' ends from random mRNAs (complex endonuclease), the viral polymerase itself cannot synthesize 5' caps which is required for ribosomal translation
splicing tactic of (-) ssRNA viruses
only works for orthomyxo/borna because they replicate in cytoplasm, create pre-mRNA that is spliced by nuclear enzymes
orthomyxoviridae genome rules
each segment gives only 1 protein except #7 which is spliced to two mRNAs, 5' and 3' nucleotide regions are conserved in all 8 segments in influenza A, the terminal sequences is the polymerase recognition for all 8 segments
making polyA tail at the end of viral mRNAs (orthomyxo)
polymerase compelx may be stationary as RNA is thread through and copied, polyU stretch is present 15-22 nt from the 5' end to help add A residues efficiently for a polyA tail
how to make antigenome without adding polyA in (-) ssRNA viruses
no cap snatching for genome replication (viral poly initiates replication de novo), viral poly avoids polyA addition signal in (-) strand (modification of the polymerase and anti-termination by NP)
arenaviridae structure
enveloped, 2 RNA segments, covered by helical nucleocapsids N protein, one large and one small RNA segment (both form covalent circles as ends interact)
arenavirus decoding strategy
polyprotein strategy to make 2 membrane proteins, ambisense coding strategy (get 2 proteins from a single RNA strand)
ambisense in arena
5' end is (+) and 3' is (-), information at the 3' end must copy from - to + and 5' information takes 2 copying rounds of + to - and - to +
enveloped, largest single family of animal viruses, spherical virions with helical nucleocapsids
arenaviruses that can be transmitted by arthropods except hantaviruses that are spread by rodent vectors (humans are often dead-end hosts)
bunyaviridae genome tactics
3 (-) ssRNA segments (small, medium, large); one segment gives one protein, cap stealing (but in the cytoplasm, not the nucleus like orthomyxo), antigenome is used as both mRNA and as replication material to make minus strand, ambisense coding
RNA-dependent RNA polymerase versus RNA depdent mRNA synthesizing enzyme in (-) ssRNA viruses
is the same enzyme but with differential complex formation, protease processing, and/or phosphorylation
stuttering polymerase viruses
all the non-segmented (-) ssRNA viruses
cap snatching viruses
influenza, bunya
nuclear replication and splicing (-) ssRNA viruses
influenza, borna
ambisense genome (-) ssRNA viruses
arena, bunya
retroviral replication strategy
(+) ssRNA to (-) ssDNA to dsDNA to mRNA for proteins or (+) ssRNA (with intact 5' cap and polyA tail
families that use reverse transcription
retroviridae (+ RNA), hepadnaviridae (gapped, ds DNA)
reverse transcription unique to retroviruses
couple reverse transcription with transposition and integration into host DNA (ensure expression of viral proteins and viral genome synthesis)
location of genome replication for reverse transcriptase viral families
the nucleus
three activities of reverse transcriptase
making DNA on an RNA template by RT polymerase, RNase H activity of RT poly to degrade the template RNA after DNA synthesis, makes DNA from DNA template
reverse transcriptase replication mechanism
RNA comes in and polymerase copies it into DNA and after it is copied the RNase activity degrades teh mRNA, at the same time the DNA leaves the enzyme
retroviruses and transformed cells
provirus theory where the viral genome inserts itself into genome to give it a stable inheritance, can make cells immortal by integration into a gene
retroviridae characteristics
enveloped, 80-100 nm, (+) ssRNA, either cone-shaped (lentiviruses) or spherical (non-lenti-retroviruses) nucleocapsids
retroviridae genome
usually packaged as two strands of whole genome, present as mRNA in virion with 5'-cap and polyA tail, contain a cellular tRNA base-paired to each viral RNA genome used as a primer in making DNA from RNA, virions contain about 50-100 molecules of RT per particle, genomes covered with nucleocapsid protein (not capsid)
retroviral genes (simple retrovirus)
three ORFs, often uses polyprotein strategy
(group specific antigen) a polyprotein of capsid, matrix, and nucleocapsid
(polymerase polyprotein) encodes the multifunctional reverse transcriptase, protease, and integrase proteins
(envelope polyprotein) encodes the two membrane proteins
viral mRNA of retroviruses
the RNA genome is NOT a message, mRNA is produced from DNA copy by HOST RNA polymerase II, mRNA is then translated and full length mRNA is packed into virions, NO RNA-dependent RNA polymerase used
long terminal repeats of retrovirus dsDNA
the RT enzyme makes dsDNA with a transmuted copy fusing sequences from the 5' (+) ssRNA to copied sequences from the 3' end and puts these on both ends of the dsDNA (a powerful promoter at both ends to allow for high gene expression in provirus)
process of RNA to dsDNA formation by RT
tRNA acts as primer near 5' end of RNA and small segment of (-) ssDNA is hybridized to the RNA up until 5' end, RNase H activity digests the RNA hybrid on this end, the exposed ssDNA binds to the 3' end of the RNA in a loop and the rest of the minus DNA strand is synthesized and the RNA is digested (the only part that isn't digested acts as plus strand primer), at this RNA primer the plus strand synthesis begins in the opposite direction as the minus strand (but starts before the minus strand is finished), the plus strand eventually reaches the tRNA which is removed by RNase (so is a little piece of RNA primer left over from minus strand synthesis, the strong stop region), the plus strand region where the tRNA was can anneal to the minus strand where the leftover RNA primer was (circular intermediate), the dsDNA can be made circualr by finishing the plus strand and ligating the nick (creates a dead end product), in the productive product the portion of the plus strand first made is used as a template for more minus strand formation and displaces original minus strand, the displaced and unannealed (-) ssDNA is used as a template to make more (+) ssDNA from the partially formed strand and ligating host enzymes repair into linear molecule
RT in uncoating virion
as soon as virion envelope fuses with host plasma membrane, the RT uses the nucleotide substrates in the cytoplasm to begin to copy the RNA into DNA inside the partially uncoated capsid (2 genome strands available for copying)
copy-choice switching of templates
retroviruses with 2 strands, provides explanation why retroviruses are resistant to UV/ionizing radiation (2 copies of genes, use copy-choice to rebuild functional genome); as DNA is synthesized it can switch from one (+) ssRNA to the other to make a normal DNA strand even if both mRNA's are "dead" (recombination), RT switches templates as it copies, both RNA templates are destroyed during reverse transcription (2 mol RNA to 1 mol DNA)
destructive copying
because mRNA genome is destroyed during copying, there is only one chance to get a functional copy of the genome
dsDNA after reverse transcription is complete
dsDNA is transported to nucleus still associated with viral proteins
mitosis and comparing simple vs. complex retroviruses
simple viruses require host cell enter mitosis with dissolution of nuclear membrane before dsDNA can integrate while lentiviruses (complex) don't need mitosis to have DNA enter nucleus
retrovirus DNA integration location
seemingly random location but usually at actively trascribed places, always find short duplication of host DNA at site (integrase integrates at these spots)
three forms of dsDNA following reverse transcription
active linear form with two LTRs surrounding DNA, two circular forms (one with a single LTR and and one with 2 back-to-back LTR's)
integrase dsDNA integration process in retroviruses
integrase holds two LTRs close together around target DNA with the 3'-hydroxyls in proximity to 2 phosphates of the host DNA (processing step), joining step when two host strands join to two different viral strands, repair step is when nucleotides are added and gaps are ligated (never again excised by viral enzymes)
retrovirus genome synthesis after integration
the LTR is a strong DNA promoter that was built during reverse transcription, attracts host RNA poly II to transcribe many viral mRNA copies (either translated into proteins or stuck into capsids); thus only reverse transcription, integration, and transcription (no DNA/RNA replication)
retrovirus pre-mRNA
in nucleus, some spliced and exported for translation and some unspliced and packaged into particle
DNA viruses using reverse transcriptase
hepadnaviruses (hep B) and caulimnoviruses (plant)
hepatitus B genome
minus strand is complete, plus strand is incomplete, 5' end of minus strand is attached to the viral reverse transcriptase, 5' end of the positive strand has an RNA oligonucleotide from the 5' end of pre-genomic RNA, minus strand is the mRNA synthesis template, 3 transcripts can be formed with 4 ORFS
hepadnavirus replication cycle
gapped DNA is taken to the nucleus and repaired to cccDNA (completion of + strand, removal of 5' protein and RNA, ligation), transcribed to long mRNA by host RNA poly II (doesn't stop after one time around so 5' and 3' ends of mRNA are the same), moved into cytoplasm and packed into a new core particle with RT which copies the RNA into (-) ssDNA and acts as its own primer, the plus strand is made next with the 5' end of the preRNA used as a primer
initiatial binding of a host cell by viral particle
initial collision is governed by chance (diffusion), virions can bind to anything including cells, junk, debris..., initial binding is due to electrostatic interactions (pH, ionic strength, specific ions influence binding, binding is generally independent of temperature (though entry does depend on temperature))
three modes of viral binding/attachment to cells
physical contact (usually reversible), rolling on cell surface (electrostatic interactions broken and reformed along surface), irreversible permanent docking involving energy and changes in protein conformation (protein-protein interaction)
specificity in viral cell-binding
adhering to surface is non-specific (universal electrostatic interactions), must attach to specific receptor(s), transfer genome to the inside of the cell (specific receptor/viral ligand interactions); both cell and receptor binding are random events but genome release is not (always follows receptor binding)
triggers to uncoat and initiate viral replication
receptor binding causes structural changes in virion to initiate entry, stimulation of host signal transduction and endocytosis
viral genome interaction with host cytoplasm
may be released completely, may remain internalized inside capsid (mRNA synthesis can occur inside capsid or in cytoplasm and the capsid and/or genome must move to sites of replication)
general entry program for nuclear replicating virus
binding of cell, lateral diffusion to receptor, signaling to cause internalization, vesicular transport to an organelle where the initial PM budding membrane is lost, intracellular transport along cytoskeleton, nuclear import, genome uncoating
diversity/similarity of viral receptors
wide array of surface molecules, picornaviruses can use nine different molecules (no specific rules), different viruses can use the same receptor (adenovirus&picornavirus, herpesvirus&poliovirus), different strains of the same virus may use different receptors (rhinoviruses-3, retroviruses-8)
hepadna genome formation from pregenomic mRNA
terminally redundant mRNA, RT towards 5' end adds - DNA to RNA template on 3' end and RNase activity removes the RNA, minus strand forms up until end where an RNA primer survive degradation on 3' end of - DNA, seldom there is + strand priming from this RNA, usually the primer is translocated towards the minus strand 5' end, priming goes towards minus 5' end and continues along 3' end when a terminal redundant sequence from the - strand pairs with the 5' - DNA strand and the + strand is synthesized to a variable length along the 3' end
retroviral mRNA and splicing
unspliced mRNA encodes capsids/enzyme or is packaged into virions, spliced mRNA encodes envelope proteins
ways to identify cellular genes encoding viral receptors
add genomic or cloned DNA from cells that express the receptor to receptor-negative but permissive cells (some cells will then express receptor); to identify add recombinant virus with a drug resistance gene (infected cells will survive in presence of drug), add recombinant virus with indicator gene like GFP (cells will express indicator if have receptor), or add receptor antibody and then add fluorophore and receptor cells will develop color
sialic acid
receptor for influenza viruses A and B, A and B do not bind the receptor for C viruses, sialic acid is linked to a galactose in a certain way that matters for infection (birds and humans different), viral envelope protein hemagglutinin recognizes sialic acid and viral neuraminidase cleaves off the galactose
molecular structure of influenza hemagglutinin protein
has a globular head and a thinner stem, the sialic acid interacts with the head
viral co-receptors
viruses may bind a receptor for attachment and then another for cellular entry (quality control)
herpes simplex virus receptors
primary receptor is a heparin sulfate proteoglycan in the cell's ECM, multiple secondary receptors (nectin 1 or 2, TNF receptor on T cells)
HIV co-receptors
CD4 and chemokine receptors, infection requires engagement of both (or not susceptible)
HIV order of binding receptors
virion envelope protein gp120 binds to CD4, changes conformation of gp120 to reveal second receptor binding pocket (chemokine, CCR5 binds to the pocket), viral fusion machinery is activated (cell fusion and core proteins are released into cell)
HIV-1 tropism
there is a T-cell strain and a macrophage strain, the T-cell has an alpha-chemokine and the macrophage has a beta-chemokine
attachment of naked virions to receptors
receptor binding sites involve surface features like canyons, loops, and centers of symmetry, antibodies can effectively block viral entry if they cover up an important structural region
human rhinvoviral cellular interaction
interacts with ICAM-1 (cellular adhesion molecule), many molecules binds to ICAM-1 at different regions
viral movement in cytoplasm
it is very crowded so movement does not occur by diffusion, requires cellular transport machinery (diffusion would take a REALLY long time in the cytoplasm compared to in water), actin cables/myosin motors, microtubule tracks with dynein&kinesin motors
normal cellular pathway to bring in outside material (pinocytosis) or to recycle surface membrane proteins to the inside (receptor-mediated endocytosis with a ligand and a receptor); phagocytosis is not endocytosis (particle is surrounded by plasma membrane)
receptor-mediated endocytosis
bring molecules into cell when a complex/ligand/virion binds to a receptor, clathrin-coated pit is formed as endocytosis occurs, uncoating occurs in early endosome, receptor is recycled by vesicle to plasma membrane, the cargo/virus is taken to late endosome then lysosome
different endocytic pathways used by viruses
macropinocytosis, clathrin-independent, clathrin-mediated, caveolar, cholesterol-dependent, dynamin-2-dependent
virion uncoating
exposing the viral genome to the cytoplasm
(+) ssRNA genome uncoating
for + strand RNA genomes, the genome must be translated immediately and the ribsome may actually pull the genome out of the capsid as it translates
poliovirus-mediated pore formation
native virion (160S) binds to receptor and is endocytosed, capsid has conformational change, hydrophobic N-termini of poliovirus VP1 protein are extruded into host plasma membrane to form pore in its carrying vesicle, VpG protein and attached RNA moves through pore
genome uncoating of (-) ssRNA or dsRNA viruses
genome is copied by viral RNA poly already inside particle (actual uncoating may not be required only loosening of capsid to allow influx of nucleotides), mRNA must be released to ribosomes
uncoating of orthomyxo and borna viridae
they replicate in nucleus so must sned nucleocapsids to nucleus but still use the viral RNA polymerase that remains associated with the nucleocapsid
DNA virus uncoating
more complicated, varies for a number of different viruses in hlw they get into the nucleus (except pox, which replicates in cytoplasm)
general pathway for entry/uncoating of nuclear-replicating viruses
attachment, lateral movement on PM, signaling, endocytosis, membrane penetration, cytosolic transport, nuclear import, uncoating
constant flux of the cell surface
sampling the outside world, regurgitating intracellular things, moving membrane and membrane proteins in and out, recycling, transport, complex-structure formation, virions that have no receptor may still land on a particularly mobile part of the cell surface and still get it
non-specific uptake
pinocytosis (take up material non-specifically bound at cell surface and electrostatically bound viruses), phagocytosis (active engulfment of viruses/virus-infected cells by macrophages)
Fc receptors
found on macrophages and other phagocytic cells, bind IgG antibodies by constant tail region and whatever the antibody is bound to is internalized by receptor-mediated endocytosis (cleans up viral infection)
danger of antibodies
non-specific infection of macrophages can occur, when virion/antibody complex is taken up by Fc receptors at the cell surface, the cells can be infected even with a good antibody response
backdoor entry event probability
low frequency, not high efficiency mechanisms, do represent novel routes (HIV, Dengue Fever Virus, Measles Virus)
macrophages and Dengue Fever Virus
4 serotypes of the virus, if 1 infects the person usually mounts an Ab response to stop it, but if a second serotype infects the same person the Ab made against the first binds to virions of both serotypes, these enter macrophages by Fc receptors, although not normally infected if macrophages are infected this can be lethal (Dengue Hemorrhagic Shock Syndrome)
MOI and entry/infection
MOI is the number of INFECTIOUS particles added per cell (not total virus); total virus is measured by the electron microscope and infectious is assayed for plaques
particle to PFU ratio
the number of particles it takes to make a plaque forming unit
EOP and entry/infection
efficiency of plating
absolute EOP
the number of physical particles required to infect a cell (related to particle to PFU ratio), number of particles divided by plaque forming uunits
relative EOP
a ratio of the number of infectious units on one cell line compared to another (useful when absolute EOP is unknown or irrelevant), PFU in condition A divided by PFU in condition B, equivalent to plating efficiency (cells have different plating efficiencies for a given virus due to number/types fo receptors and different efficiency of replication)
absolute EOP of 1
almost every particle has a 100% chance of starting an infection that results in a plaque, most viruses have higher EOPs (more than one particle needed to form a plaque)
high particle to PFU ratio on plaque assay
infectious unit
the smallest volume of virus that can elicit a positive response in your assay (plaque, focus-forming, hemagglutinating unit...), electron microscope measures physical particles
high absolute EOPs and related phenomena
efficiency of reversible and irreversible binding, mulitple steps leading up to scoring of a cell as infected (most steps are not 100%), technical issues by lab person (damaged particles), defective particles (biology issues)
7 major viral steps
entry, uncoating, transcription, translation, replication, assembly, egress
after viral mRNA translation, the proteins perform 3 functions
recognize/replicate the genome, encapsidate the new genomes in particles, keep cells alive or tolerant by disabling host defenses
six viral tactics to make or process mRNA
multiple subgenomic mRNAs, nested sets of mRNA, ambisense coding, splicing, mRNA transport out of nucleus, RNA editing
six viral tactics to make or process proteins
IRES, polyproteins, protein processing/conformational change/new activities, leaky scanning, suppression of termination, frame-shifting
two fundamental tenets about viral mRNA
no matter how mRNA is made it must be translated by host ribosomes in the cytoplasm, because host mRNA must be modified before being ribosome-ready so must the viral mRNA
host mRNA processing process
DNA, capping at the beginning of transcription, finishing transcription (pre-mRNA), endonucleolytic cleavage and polyA, splicing away introns, export (sometimes intron mRNAs are exported too), translation/degradation
capping viral messages (done by host)
retroviridae, adenoviridae, herpesviridae (any DNA virus that replicates in nucleus)
capping viral messages (done by viral enzyme)
poxviridae, reoviridae, togaviridae
viruses that steal mRNA caps
orthomyxoviridae, bunyaviridae
strategies for adding polyA tail
host enzymes in nucleus (retro, nuclear DNA viruses), viral enzymes (picorna have polyU stretch in their genome, reiterative copying of short U stretches in template by ortho- and paramyxoviridae, poxviridae mRNA completely done by viral enzymes in cytoplasm), bypass the polyA requirement (some have no polyA, picorna has it in its genome, some flaviviridae)
more than one protein from a single mRNA, processing of one preRNA to make several mRNAs
never go to cytoplasm, never see ribosome, ready to be poly-adenylated and spliced
adenovirus pre-mRNA
transcribed by cellular RNA polymerase II, 5 early transcription units, 2 delayed early units, 1 late, very large unit made only after viral DNA replication has occured
post-transcriptional regulation by differential splicing
each pre-mRNA gives rise to several mRNAs that are differentiated by alternative splicing and use of different polyA sites, introns are removed either constitutively (keep same exon order) or alternatively
exon skipping splicing
certain exons are treated as parts of introns when their splice site is ignored and that exon is not included in some proportion of the final mRNA transcripts
alternative 5' or 3' splice sites
two 5' splice sites present in an exon where if one cuts then part of the exon is included and if the other cuts then that part is removed with the intron (same with 3' but different direction)
alternative polyadenylation and splicing of major late adenoviral transcripts
major late promoter is transcribed into mRNA, alternative polyA tail addition to five different sites before splicing, then alternative splicing of the five different RNA form different mRNAs for translation (all have a tripartite leader sequence)
regulation of mRNA nuclear export
any virus that replicates in the nucleus has export mechanisms for moving mRNA into cytoplasm to be translated (may encode mechanisms to reduce/block host mRNA export)
exporting unspliced HIV RNA
pre-mRNA is made by RNA poly II from integrated provirus, early on the pre-mRNA is spliced but for capsid proteins and genome the mRNA is unspliced (and it gets out of the nucleus)
spliceosome retention hypothesis
mRNAs containing splice sites (the introns) are retained in the nucleus because they are bound to spliceosomes (held back until splicing occurs or until pre-mRNA is degraded)
HIV Rev protein
shuttle protein involved in nuclear export of mRNA with introns (encoded by one of the early, spliced mRNAs), binds and exports unspliced mRNA, specifically interacts on its N-terminal side with an HIV intron sequence at the Rec-responsive element stem-loop, and Rev has a C-terminal nuclear export signal
nuclear export sequences
if covalently attached to any protein then that protein will be exported independent of whether it is or isn't normally, enables Rev to shuttle back and forth
RRE sequence
enables Rev to escort specific RNAs out of nucleus
RNA editing
tactic to process mRNA to obtain new proteins from one encoded ORF, insert new nucleotides into an mRNA not encoded by the viral genome
ways to add new bases to mRNA
viral RNA polymerasse stuttering, modification of a base after mRNA is made (enzymes like adenine deaminase)
paramyxovirus and RNA editing
one ORF makes 3 proteins by polymerase suttering (P, C, V proteins), the a second ORF that encodes protein C is within the P gene and a third ORF encoding V lies within the P gene (a fusion protein is created where the amino terminal is the same but the C-terminus is different)
ebola virus and RNA editing
ebola makes two proteins from one ORF (a secreted glycoprotein from unedited RNA and a membrane glycoprotein after editing and C-terminal fusion)
IRES and 5' cap
IRES are RNA structures that bypass the requirement for a 5' cap on mRNA (poliovirus is not capped, but does have an IRES ribosomal landing pad)
poliovirus and 2a protease
destroys the host cap-binding system so that only RNA with IRES elements can be translated in a polio virus infected cell
cell IRES detection
IRES elements are rare in eukaryotic cells, the cell uses the Rigl protein as a host cell sensor of IRES RNA and subsequent signal cascade induces host antiviral defenses
kozak consensus sequence
there are certain sequences around a start codon that are most correlated with efficieny translation and many viral genomes change this around in ways to regulate the amounts of protein made from a single mRNA
sendai virus (leaky scanning and mRNA editing)
there are a number of start sites that produce different C proteins from the same mRNA due to leaky scanning (the first and second start sites have poor initiation contexts so a number of C-terminal nested set proteins are formed), mRNA editing by stuttering polymerase can shorten the P protein and add either a V or W terminus
ribosomal frameshifting
the ribosome backs up one base and then reads an alternative reading frame after slippage on a certain sequence (small proportion of the time), fuse the first ORF with the second to make a new polyprotein
pseudoknot and ribosomal slippage
downstream of slippery sequence is an inverted repeat that can form a pseudoknot, this structure can cause ribosomes to pause at the slippery sequence to increase probability of frameshifting
termination suppression
when translation reads through a stop codon or a mistake is made by tRNA amino acid addition, makes a fused polyprotein of 2 adjacent ORFs
supression of translation in togaviruses
normally a nonstructural ORF protein is produced, but it can be readthrough to add on the sequence of the RNA-dependent RNA polymerase, which is processed proteolytically to be active
supression of translation in retroviruses
stop codon between gag and pol genes is followed by a pseudoknot that can cause supression of translation to form a fused gag-pol protien
nucleic acid molecules in virions
may not be ready to make mRNA (use cellular or pre-packaged viral enzymes to), the molecule in the virion is made form replication and packaging, mechanisms of priming/initiation of replication are diverse
generalities about copying nucleic acids
add 5' P to 3' OH, usually requires primer (always DNA poly and RT, not all viral RNA poly need primer, host RNA poly DO NOT need primers)
three types of primers that provide the required 3'-OH
RNA primers (as in Okasaki fragments), hairpin primers (encoded in nucleic acid itself), protein primer (covalently attached to 5' end of genome)
RNA primers
requires a primase enzyme to synthesize, must be removed later in newly copied DNA by RNase, DNA at extreme 5' end of linear DNA is lost if RNA primers are used
hairpin primers
strand-displacement synthesis (parvo, pox), encoded in nucleic acid itself
protein primer
attached to 5' end of genome (adeno, hepadna, picorna)
small DNA viral genomes and replication
only encode proteins that direct host replication machinery to origin (large T antigen in polyoma, NS1 protein in parvo)
large DNA viral genomes and replication
have complete DNA replication system
RNA viruses and replication
all have own replication machinery except retroviridae and hep D satellite which use host RNA poly II (the RT does not officially make the packaged genome)
2 modes of DNA synthesis
fork: papilloma, polyoma, herpes

displacement: adeno (protein), parvo (hairpin), pox (hairpin), essentially all RNA viruses
adenoviral replication
pTP 5' protein assembles with DNA poly and uses a serine to bind to a cytosine nucleotide which pairs with a guanine and initiates strand displacement of the 5' end strand (which is held by ss binding proteins), then the other strand is replicated alone
5 common problems in replication of DNA viruses
origin recognition/unwinding, priming, elongation, termination, resolution of intermediates
common problems for either + or - strand RNA genomes
both + or - strands must be made as genome and antigenome, templates for genome production must be exact copies without bases lost or gained (needs precise initiation and can't cap, add polyA, or stutter), both must avoid producing hybrid dsRNA (activates host defenses), all must make many copies of new mRNAs
switching from mRNA synthesis to genome replication in (-) ssRNA viruses
incoming genomes copied by viral polymerase to make either mRNA or antigenome (full length copy used to form full length genome)
switching from mRNA synthesis to genome replication in (+) ssRNA viruses
incoming genome RNA molecules must first be translated by host ribosomes to obtain viral polymerase, viral poly must make antigenome as a tempalte for more genomes (ribosomes reading from 5' end of mRNA genome can collide with RNA poly reading from 3' end of genome)
nucleocapsids and RNA genomes
(-) ssRNA are usually coated with a nucleocapsid (already preloaded with viral RNA poly) while (+) ssRNA are usually uncoated (directly inside icosahedral capsid), retroviridae are coated with nucleocapsid protein as an exception
(+) strand discrimination of mRNA or replication from the antigenome
in some cases depends on the polymerase used like for togaviruses with 3 polymerases with different specificities (because of different cleavages)
(-) strand discrimination of mRNA or replication template
mRNA uses a primer (influenza does cap snatching) but replication may use no primer (de novo initiation)
dsRNA replication
each strand is made in two separate steps in space and time, the + strand is used for translation (passage through viral turret) and packaging and the - strand is replicated from the + strand