Lect 45,46 Micro Retroviruses Exam 3

Front 1

Name some human retroviruses

Back 1

• HTLV-1 (human T-cell leukemia virus type 1): causes tropical spastic paraparesis and T-cell leukemia.

• HTLV-2 (human T-cell leukemia virus type 2): causes hairy T-cell leukemia.

• HIV-1 (human immunodeficiency virus type 1): causes AIDS.

• HIV-2 (human immunodeficiency virus type 2): causes AIDS, it is less common and less virulent than HIV-1, it is predominantly found in W. Africa and Cape Verde.

• HFV (human foamy virus): no known disease is associated with this retrovirus.

Front 2

What is HIV-1 organized into?

Back 2

Based on the DNA and amino acid sequences of various viral gene products, HIV-1 has been organized into 8 groups or clades. There appears to be biological differences between viruses from the different clades. For example clade E is transmitted through heterosexual sex better than clade B.

Front 3

Describe the virion structure of retroviruses

Back 3

Retroviruses are enveloped viruses approximately 100nm in diameter. There is both an inner shell (composed of matrix protein) and an inner core (composed of capsid protein). Contained within the core are 2 copies of the retroviral genome which is a positive-stranded RNA. The virion also contains virally encoded proteins that are required for replication. These include reverse transcriptase (RT) and integrase (IN). The fact that there are two copies of RNA per virion allows there to be genetic recombination between different virus strains in mixed infections. This contributes to viral adaptability and pathogenesis.

Front 4

1. Genome of retrovirus? What does it contain?
2. Describe the HIV-1 genome

Back 4

1. The retroviral genome is essentially an mRNA (this is the positive strand). It contains a CAP structure at its 5’ end and a polyA tail at its 3’ end. These structures are features of most eukaryotic mRNAs.
2. The HIV-1 genome is more complex than most retroviruses. In addition to encoding those structural and enzymatic gene products that are found in all replication competent retroviruses, HIV-1 (and all other lentiviruses) encodes additional gene products that regulate virus replication and play a role in pathogenesis.

Front 5

Describe the following:
1. tat
2. nef
3. vpr
4. vif
5. vpu
6. rev

Back 5

tat – transcriptional transactivator, essential for virus replication. Tat can be released from infected cells and alter gene expression in uninfected cells. Tat is thought to induce apoptosis (programmed cell death) in uninfected cells.

nef – membrane associated. Nef downregulates CD4 expression. Nef deficient SIV does not cause disease in adult macaques. So-called long term non-progressors have been identified that harbor HIV-1 variants that are nef deficient (long term non-progressors are individuals who have been infected with HIV-1 for 10-15 years but show no evidence of disease progression). Expressed very early after virus infection and primes T cells for virus replication.

vpr – virion associated protein. Causes T cells to arrest in G2 stage of cell cycle. Causes apoptosis of infected cells.

vif – prevents deamination of viral DNA by the cellular protein, APOBEC3G; facilitates virus spread.

vpu – influences virus release.

rev – post-transcriptional transactivator, essential gene

Front 6

Receptor binding of HIV-1

Back 6

1. The primary receptor for HIV-1 is CD4, which is found on T-helper lymphocytes and macrophages. Attachment of HIV-1 to target cells typically requires the interaction of the viral SU protein, gp120, with CD4. In addition, HIV-1 gp120 must interact with a co-receptor on the surface of the target cell. The two major HIV-1 co-receptors are CCR5 and CXCR4. These molecules normally serve as the receptors for signalling molecules called chemokines. CCR5 is expressed on both CD4+ T cells and macrophages. CXCR4 is preferentially expressed on CD4+ T cells. Different virus strains preferentially use one co-receptor or the other.
2. HIV-1 can also establish a low level of infection in antigen presenting cells such as dendritic cells and Langerhan’s cells. These cells are thought to play an important role is facilitating HIV-1 dissemination from mucosal surfaces to local lymph nodes.
3. Individuals have been identified who have been repeatedly exposed to HIV-1 but have not become infected with the virus. T

Front 7

Step 2: virion entry into the cell

Back 7

The virion enters the cell by the TM (gp41) mediated fusion of the viral envelope with the target cell membrane. This fusion step can be blocked by drugs designed to prevent the TM from adopting a fusion-competent conformation. These “designer drugs” are currently used in the clinic.

Front 8

Step 3: reverse transcription (ssRNA to dsDNA) and uncoating

Back 8

Reverse transcriptase synthesizes a double-stranded DNA using the single-stranded genomic RNA as a template. This process is a major target for anti-retroviral drugs. Nucleoside analogs (such as AZT and ddI) block reverse transcription by substrate competition with the normal nucleoside and by acting as terminators of DNA chain elongation. Nonnucleoside inhibitors bind RT near the active site of the polymerase and block its activity.

RT is not able to correct misincorporation mistakes made during DNA synthesis. This lack of proof-reading activity contributes to the generation of viral sequence variants that accumulate during HIV-1 infection. Some of these variants have a selective growth advantage in the host and a greater pathogenic potential.

Front 9

Step 4: entry of DNA into nucleus

Back 9

Unlike most retroviruses, the lentiviruses, including HIV-1, can productively infect non-dividing cells such as macrophages. For most retroviruses, entry of the DNA into the nucleus requires the breakdown of the nuclear membrane during cell division. In HIV-1, the CA protein facilitates the nuclear transport of DNA in the absence of cell division.

Front 10

Step 5: integration of DNA into host chromosomes

Back 10

Integration of the retroviral DNA into the host chromosome is an obligatory step in the viral life cycle. This process is carried out by the virally encoded IN protein. The integrated retroviral DNA is called the provirus. This step in virus replication can be blocked by drugs such as raltegravir or elvitegravir.

Front 11

Step 6: RNA transcription and processing

Back 11

The provirus is transcribed using the cellular transcription machinery. The HIV-1 tat protein activates virus transcription. Both unspliced and spliced mRNAs are expressed. Unspliced mRNAs serve not only as templates for protein translation but also as the viral genomic RNA.

RNA polymerase II, which is used to transcribe the viral RNA does not have a proof reading activity and therefore contributes to the generation of viral sequence variants.

Front 12

Step 7: Protein translation

Back 12

The different proteins are either translated from alternatively spliced mRNAs or as precursor polyproteins. Many of the viral proteins undergo post-translational modification such as glycosylation or phosphorylation.

Front 13

Step 8: assembly of viral proteins and RNA

Back 13

Unspliced viral mRNA and precursor polyproteins are assembled into virions at the cell surface.

Front 14

Step 9: release of the assembled virion

Back 14

The assembled virions are released from the cell. These virions are immature in that they contain uncleaved polyproteins.

Front 15

Step 10: proteolytic maturation of the virion

Back 15

After release, the virion undergoes a maturation step that involves the proteolytic cleavage of the precursor polyproteins. This is accomplished by the virally encoded PR. PR (protease) is a target for therapeutic intervention. Inhibitors such as Saquinivir and Indinavir bind in the active site of PR and block activity and consequently virion maturation.

Front 16

1. What does HIV-1 infection cause at all stages?

Back 16

During all stages of HIV-1 infection, 108 to 109 virions are generated each day. This high replication rate coupled with the high mutation and recombination rate of HIV-1 results in extensive sequence heterogeneity and ultimately in the outgrowth of viral strains with higher replicative and pathogenic capacities.

Front 17

What's the difference in strains between early infection and later stages of infection of HIV-1?

Back 17

At early stages after infection, most strains of HIV-1 that can be isolated replicate relatively poorly and lack the ability to induce syncytia between cells (NSI variants). The more pathogenic strains that evolve at later stages of infection are typically syncytium inducing (SI strains) and display a T cell tropism. The evolution of multiple strains of HIV-1 at later stages of disease is in part due to the escape of the virus from immune surveillance that results from the destruction of the immune system.

Front 18

What part of the immune system helps control immune responses? How does it do this?

Back 18

CD4+ T lymphocytes help to control immune responses by activating macrophages, inducing cytotoxic or suppressor T cell function and by secreting cytokines that regulate lymphoid cells. During chronic infection, prior to the significant loss of CD4+ T cells seen at later stages of disease, there is impaired T cell function. HIV-1 induced T-cell abnormalities include a loss of response to recall antigens, impaired T cell proliferation in response to stimuli, impaired IL2 receptor expression, altered cytokine expression with a shift from a Th1 to a Th2 profile (IL2 down, IL4, IL10 up).

The primary functions of macrophages include phagocytosis of foreign organisms, antigen presentation and cytokine secretion. During HIV-1 infection, a number of macrophage functions are impaired, including chemotaxis (the movement of a cell toward a signal), phagocytosis, and cytokine production. Impaired macrophage function is in part responsible for AIDS associated opportunistic infections.

Front 19

Name 4 ways in which HIV-1 kill cells?

Back 19

1. direct killing
2. syncytium formation
3. immune attack
4. apoptosis

Front 20

Direct killing

Back 20

During productive HIV-1 infections, there is a very high level of virus gene expression that can result in the death of the infected cell.
This excessive viral expression has at least three consequences:
1) it can interfere with normal cellular gene expression,
2) there is a loss of cell membrane integrity due to excessive virus budding,
3) there is an accumulation of high levels of unintegrated virus DNA in the nucleus which is cytotoxic.

Front 21

Syncytium formation

Back 21

Syncytia are large, multinucleated cells that are generated when uninfected CD4 expressing cells bind to and fuse with HIV-1 infected cells expressing gp120 on their surface. Cells that are fused to form syncytia ultimately die. The appearance of SI strains of HIV-1 coincides with disease progression.

Front 22

Immune attack

Back 22

Viral gp120 shed from HIV-1 infected cells can bind to uninfected CD4 expressing cells. These gp120-bound, uninfected cells can then be killed by antibody dependent cell-mediated cytotoxicity (ADCC).
Infected cells can also be killed during the normal immune response to viral infection, especially by a CTL response during the early stages of infection.

Front 23

Apoptosis

Back 23

HIV-1 infected cells can release the viral tat protein. Tat can enter uninfected cells and affect cellular gene expression. One consequence can be the induction of genes involved in the initiation of apoptosis (programmed cell death).

Front 24

Epidemiology of HIV

Back 24

A. Worldwide epidemic has epicenters in Sub-Saharan Africa, India, China, and Russia
B. Important differences exist between incidence and prevalence
C. Prevention, education, and treatment interventions are difficult in resource-poor settings, but improvements are occurring
D. HAART (highly-active antiretroviral therapy) has dramatically altered the epidemiology in the US by reducing the number of deaths and the occurrence of opportunistic infections
E. HIV infection in the US disproportionately affects minority populations

Front 25

Transmission of HIV

Back 25

A. The modes of HIV transmission have been well known since the early days of the epidemic. Just as importantly ways that HIV is not transmitted are well understood.
1. Sexual contact
2. Perinatal (and breast milk)
3. Blood and blood products (needle sharing)
4. Organ tissue transplantation

B. The efficiency of transmission is dependent on the amount of virus in any particular body fluid or tissue, the infectiousness of a particular virus strain, the integrity of the host mucosal surfaces, and the host immune response.

Front 26

Symptoms of acute HIV infection

Back 26

fever, rash, adenopathy, pharyngitis, hepatosplenomegaly, meningitis)

Front 27

Describe rapid HIV testing

Back 27

A twenty-minute assay has recently been introduced that detects the presence of anti-HIV antibody; this may often replace the screening ELISA prior to confirmatory Western blot testing.

Front 28

Describe Western blot diagnosis

Back 28

As a confirmatory assay for a patient whose serum has tested positive by ELISA, a confirmatory Western blot assay is performed using a viral lysate that is separated by electrophoresis. Patient serum is allowed to bind. The presence of bands corresponding to each of the three major coding regions is considered a positive test result.

Front 29

HIV RNA (viral load) testing

Back 29

The amount of virus in plasma, or other body compartments, is measured by quantifying the amount of HIV RNA using branched-chain DNA (bDNA) assay, RT PCR, or nucleic acid sequence-based assay (NASBA). These assays utilize mixtures of primers to various coding regions in the HIV genome. These mixtures determine how well these assays measure the different clades of HIV.

Front 30

CD4 cell count determination

Back 30

The absolute number and percentage of CD4 cells in whole blood is used as a measure of immune function during HIV infection. The absolute number of CD4 cells is determined as the product of the CD4 cell percentage, the total white blood cell count, and the percentage of lymphocytes obtained using a complete blood count (CBC) with differential.

Front 31

Genotypic and phenotypic resistance testing

Back 31

HIV develops resistance to antiretroviral (ARV) drugs very rapidly with ongoing HIV replication in the presence of drug therapy. Two types of assays exist to measure ARV drug resistance. In genotypic resistance testing, the sequence of nucleotides in the reverse transcriptase and protease coding regions is determined and correlations with known mutations that confer resistance to specific drugs, using large databases, are reported. In phenotypic resistance testing, molecular constructs of the quasi-species that exist in a particular patient are made, and these constructs are grown in the presence of ARV drugs. The extent of replication is a measure of ARV drug resistance.

Front 32

Antiretroviral drug therapy..
1. What events are targeted by drugs?

Back 32

a) Main life-cycle events targeted by currently FDA-approved drugs include virus cell membrane fusion, reverse-transcription, protease enzyme cleavage, and integrase enzyme function.

b) Life-cycle events targeted by investigational drugs currently in clinical trials include co-receptor binding, reverse-transcription, and protease enzyme cleavage.

c) Life-cycle events targeted by drugs in pre-clinical or early clinical development include CD4-gp120 blockers, integrase inhibitors, and agents that affect accessory protein function.

Front 33

What are some strategies for anti-HIV therapy?

Back 33

1. When to start, stop, switch treatment; treatment during acute, primary HIV infection and at later times (clinical breakpoints of CD4 cell counts (500, 350, 200, 100, 75, 50)
2. When to interrupt treatment
3. How to initiate treatment
4. How to deal with adverse drug reactions
5. Strategic Therapy Interruptions (STIs)
6. Which drugs to choose and why
a) treatment-naïve patients
b) treatment-experienced patients

Front 34

What are the benefits of early therapy in asymptomatic HIV+ patients?

Back 34

a) Control of viral replication and mutation
b) Reduction of viral burden
c) Prevention of progressive immunodeficiency
d) Reconstitution of “normal” immune system
e) Delayed progression to AIDS and prolongation of life
f) Decreased selection of resistant viruses
g) Decreased drug toxicity
h) Possible decrease of viral transmission

Front 35

What are the risks of early therapy in asymptomatic HIV+ patients?

Back 35

a) Reduction in quality of life from adverse drug effects
b) Inconvenience of prolonged drug therapy
c) Earlier development of drug resistance
d) Transmission of drug-resistant virus
e) Limitation in future choices of HAART due to development of resistance
f) Unknown long-term toxicity of drugs
g) Unknown duration of effectiveness of current drugs

Front 36

Name some nucleoside reverse transcriptase inhibitors

Back 36

a) zidovudine (ZDV or AZT, Retrovir)
b) lamivudine (3TC, Epivir)
c) stavudine (d4T, Zerit)
d) didanosine (ddI, Videx)
e) zalcitabine (ddC, Hivid)
f) abacavir (ABC, Ziagen)
g) emtricitabine (FTC, Emtriva)

Front 37

What is a nucleotide reverse transcriptase inhibitors?

Back 37

a) tenofovir (TDF, Viread)

Front 38

What are some combination formulation drugs ?

Back 38

a) zidovudine/lamivudine (Combivir)
b) abacavir/lamivudine (Epzicom)
c) tenofovir/emtricitabine (Truvada)
d) tenofovir/emtricitabine/efavirenz (Atripla)

Front 39

Non-nucleoside reverse transcriptase inhibitors; non competitive inhibitors of RT

Back 39

a) efavirenz (EFV, Sustiva)
b) nevirapine (NVP, Viramune)
c) delavirdine (DLV, Rescriptor)
d) etravirine (ETR, Intelence)

Front 40

Protease inhibitors of viral protease enzyme..

Back 40

a) saquinavir (SQV, Invirase)
b) indinavir (IDV, Crixivan)
c) nelfinavir (NFV, Viracept)
d) amprenavir (APV, Agenerase)
e) lopinavir/ritonavir (LPV/r, Kaletra)
f) ritonavir (RTV, Norvir)
g) atazanavir (ATV, Reyataz)
h) fos-amprenavir (FPV, Lexiva)
i) tipranavir (TPV, Aptivus)
j) darunavir (DRV, Prezista)

Front 41

What is a fusion inhibitor?

Back 41

enfuvirtide (T-20, Fuzeon)

Front 42

What are some CCR5 inhibitors?

Back 42

a) maraviroc (MVC, Selzentry)
b) vicroviroc

Front 43

What is an integrase inhibitor?

Back 43

Raltegravir (RGV, Isentress)

Front 44

Antiretroviral resistance

Back 44

1. Selective drug pressure results in mutations which change the quasi-species of HIV that are present in a particular body compartment
2. Genotypic and phenotypic resistance assays are used to predict which HAART regimens may be effective
3. Therapeutic drug monitoring (TDM) may be useful in conjunction with resistance assays

Front 45

Preventative vaccines

Back 45

Much effort has been appropriately expended to develop a preventative HIV vaccine. This is a difficult task because of the presence of different clades worldwide, the great variability of quasi-species within clades, dangers associated with the use of live attenuated vaccines, and an incomplete understanding of the immune correlates of protection (cytotoxic T-lymphocytes and neutralizing antibodies). Various strategies under active development include subunit protein vaccines, DNA vaccines, different vector delivery systems (e.g., canarypox or adenovirus vectors), and the use of various adjuvants.

Front 46

Name the prophylactic therapies for opportunistic infections of HIV

Back 46

a) Pneumocystis carinii
1) TMP-SMX
2) dapsone
3) atovaquone

b) Cytomegalovirus
1) ganciclovir
2) valganciclovir

c) Mycobacterium avium intracellulare
1) Azithromycin

Front 47

What are some special therapeutic situations

Back 47

1. Occupational exposures and post-exposure prophylaxis
2. Prevention of vertical transmission

Front 48

Pharmacokinetic drug interactions

Back 48

1. Factors affecting drug absorption
2. Factors affecting drug dosing
a) P450 system interactions
3. Important drug interactions between anti-HIV drugs and between anti-HIV drugs and other agents

Front 49

Adherence issues

Back 49

1. Prevention of drug resistance is directly related to the consistency with which patients take their medications
2. Strategies to improve adherence are an important part of clinical care

Front 50

What are some adverse effects of antiretroviral and HIV therapies

Back 50

1. Drug specific side effects
2. Fat redistribution
3. Hypercholesterolemia