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171 Cards in this Set
- Front
- Back
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What are the receptors HIV uses for entry?
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CD4 - main receptor
CCR5 and CXCR4 - coreceptors |
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Where is genomic RNA reverse transcribed?
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Within the HIV core (which is in the cytoplasm)
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What mediates integration of the provirus into the host genome?
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Integrase
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Which HIV proteins are found in all retroviruseS?
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Gag, pol and env
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What are the HIV regulatory proteins?
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Tat and rev
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What are the HIV accessory proteins?
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Vpr, Nef, Vif, Vpu
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What is TAR RNA?
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Transactivation-responsive RNA
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where is TAR present?
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At the 5' region of all viral RNAs
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Which HIV protein binds Tar?
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Tat
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How is HIV-1 genome organized?
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As nucleosomes - nuc0-4
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What are two types of complexes involved in HIV chromosome remodelling?
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1) ATP-dependent chromatin remodelling
2) Complexes that modify histone acetylation |
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What is an example of an ATP-dependent chromatin remodeling complex?
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SWI/SNF with ATPase subunits
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What are some examples of complexes that modify histone acetylation?
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1) Histone acetyl transferases (HATS) - causes UNFOLDING - transcriptional ACTIVATOR
2) Histone deacetylase - causes reformation of nucleosome - transcriptional REPRESSOR |
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How does Tat affect transcription of HIV genome?
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After some basal transcription, it is necessary to make some Tat which can act as a transactivator- it binds to factors which act on nucleosomes to unfold them
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What factor does Tat interact with?
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pTEFb (transcription elongation factor b)
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What do most transcriptonal activators srecruit?
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TFIID, TFIIB orTFIIH
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What does TFIID do?
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Enahnced binding to nucleosomal templates to increase transcription
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What does TFIIB do?
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Enhances formation of complex formation with TFIIB- allows subsequent loading of RNA PII
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What does TFIIH do?
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Enhanced efficiency of promoter clearance - allows multiple rounds of RNAPII intiation of transcription
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What does Tat interaction with pTEFb do?
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Enhances the efficiency of promoter clearance and elongation, allowing multiple RNAPIIs to intiate transcription and elongation
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What is Tat synthesize from?
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A multiple spliced mRNA - encoded for in part by exon 1, and partially by exon 2
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Which domain is essential for transcription of HIV genome?
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The activation domain - aa 1-58 of Tat
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What is the activation domani composed of?
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Acidic regon, cysteine-rich region, and a core region
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Which regions of the activation domain are absolutely necessary?
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Core and cysteine-rich
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What domain is located next to the AD?
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RNA-binding domain
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What is the structure of Tar like?
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Loops, buldge and stem
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Which sequences of Tar mRNA are required for Tat function?
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In the buldge and loop
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Where does Tat bind?
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Directly to the BULGE of Tar mRNA
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What are the two proteins that make up pTEFb?
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Cyclin T1 and CDK9
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What regulates binding of the Tat/pTEFb complex?
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HATs
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What is the binding like?
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Tat binds to Cyclin T1, and Cyclin T1 binds to CDK9, but Tat does NOT bind CDK9
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What are the steps of Tat action?
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-Binds to a HAT which acetylates Tat
-Increases affinity for pTEFb, which it binds to -Complex has high affinity for Tar, binds to it -HAT acetylates Tat again, resulting in its removal from Tar -Tat relocates to promoter, recruiting factors like TFIID, TFIIB to form an ACTIVE complex -Following TFIIH activity, CDK hyperphosphorylates CTD of RNA P II -CDK9 prevents negative regulatory (phosphatase) form binding |
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What are some differences with the HTLV-1 protein Tax?
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1) Either a DNA OR RNA binding protein
2) CREB binds Tax-responsive elements, CREB binds TxREs, and then Tax binds to CREB |
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What is Rev required for?
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Cytoplasmic expression of unspliced (9kB) and partially spliced (4 kb RNA)
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How does Rev mediate this cytoplasmic expression?
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Through mediating mRNA export
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Which HIV RNA segment can leave the nucleus (before Rev is produced)?
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2kb
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What does this 2kb HIV mRNA segment encode?
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Tat, Rev and Nef
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How does Rev mediate export?
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By binding to RRE (Rev responsive elements) present on the 9kb and 4kb RNA, mediating their transport out of the nucleus through the CRM-1 pathway
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What regions does Rev have?
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-Arginine-rich region (NLS + RRE binding domain)
-Mutlimerization sites flanking the NLS -Leucine rich region (activation/nuclear export domain) |
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What happens when Rev binds to the HIV-1 mRNA?
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It binds to CRM-1 which binds to RAN-GTP, and then the whole complex leaves the nucleus through the NPC
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What happens once this complex reaches the cytoplasm?
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RNA released, proteins dissociate. RAN-GTP is hydrolyzed to RAN-GDP, Rev is recycled
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How does HTLV-1 Rex compare?
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Same function and uses same mechanism - except additional factor used by HTLV-1 is DDX3
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What do other viruses use for nuclear export?
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The Tap/Nxt sysem
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What are two ways to use the Tap/Nxt system?
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Bind directly to mRNA, or first to another ptoein ICP27
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What effect does TAR RNA have on HIV-1 translation?
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Inhibitory!!!
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How does TAR inhibit HIV-1 translation?
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-Activates PKR which phosphorylates eIF2-alpha, inhibiting translation
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What are some factors that mediate this TAR inhibition through PKR activation?
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PACT (PKR activator)
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How does HIV-1 circumvent thsi HIV-1 mediated inhibition?
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HIV replicates in cells with high amounts of TRBP (TaR RNA bindnig protein) which binds PKR directly and inhibits autophosphorylation. It also inhibits PACT
-ADAR1 also was shown tocomlpex with TRBP to inhibit PKR |
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What is the difference in TRBP and ADAR1?
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HIV doesnt induce higher amounts of TRBP but simply replicates in cells with higher amounts
-Conversely, it induces higher production of ADAR-1 |
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What is another mechanism by which HIV-1 translation can occur?
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Through using an internal ribosome entry site (IRES) - a cap independent process
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What does VPR do?
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Induces G2 cycle arrest and nuclear import of the pre-integration complex
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What does NEF do?
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Down regulates cell-surface CD4 and MHC-I
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What does VIF do?
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Counteracts APOBEC
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What does VPU do?
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Mediates degradation of CD4
Counteracts Tethrin |
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Which cells have receptors for Tat?
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Macrophages, T lymphocytes, monocytes, DCs and endothelial cells
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What are some cell surface receptors for Tat?
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Integrins, CXCR4, Growth factor receptor, CD26, FGF, LRP
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What are some mechanisms that extracellular Tat induces in its target cells?
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Proliferation, apoptosis, angiogenesis, etc..
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What are Kaposi's sarcoma lesions characterized?
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New blood vessel formation, inflammatory infiltrate, proliferation of spindle-shaped cell population -> tumor cell population
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What role has Tat been shown to play in KS lesions?
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-Growth factor for KS cells and endo cells
-Induce migration of KS and endo cells -Induce angiogenesis |
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What role does Tat have in KS (overall)?
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It is a contributing factor - HHV8 is the initial cause
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What receptor does Tat interact with to induce angiogenesis?
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Vascular endothelial growth factor tyrosine kinase receptor 2
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What signalling pathway does Tat interaction with VEGF activate?
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MAPK
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What role does exogeneous Tat have on T cells?
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-Partially activates T cells to increase HIV internalization
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What does Tat bind to on T cells?
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CD26 or CCR2
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What are some target genes that Tat induces upregulation of?
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TNFalpha, TGFbeta, MCP-1, IL-2, IL-6, IL-8 and IL4R
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What are some target genes that Tat induces DOWNregulation of?
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MHC-I, MnSoD, IL-12
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What is MnSoD?
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Important anti-oxidant enzyme - inhibiton leads to oxidative stress
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What is AIDS dementia associated with?
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Monocyte/macrophage infiltration in the brain
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How does Tat play a role in AIDS dementia?
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It acts as a cytokine and a chemoattractant for monocytes
It induces excitation of neurons and neurotoxicity |
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Which cells can HIV infect in the brain?
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Brain astrocytes, macrop0hages/microglia, NOT neurons
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What happens as a result of Tat induced cytokine release in the brain?
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Microglia activation, astrocyte activation, neuron damage and apoptosis
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What size is Nef?
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27 kDa
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Where is Nef localized to following production?
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Plasma membrane and cytoskeleton
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What is required for Nef to be anchored to the membrane?
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To be MYRISTOYLATED
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What are the components of Nef?
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SH3 domain, 2 kinases (PACS or Pak), beta-COP, and 2 adaptor proteins
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Which molecules does Nef induce downregulation of?
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CD4, MHC-I, IL-2R, CD28
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What molecule does Nef upregulate?
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FasL
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How does Nef mediate internalization of CD4 and MHC-I?
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Can interact with cytoplasmic tails of MHC-I, CD4, and can ALSO interact with adaptor protein AP2 in clathrin-coated pits - uiltimately results in degradation in degradative lysosomes
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Which Src family kinase members does Nef bind to and what is the result?
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Nef binds to Lck and Fyn in T lymphocytes and inhibits their activity - results in failure to mediate signals from CD4, CD8, IL-2R and TCR
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What does Nef binding to Src family kinase in MDM do?
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Nef binds to Lyn and Hck in MDM and upregulates their activity - results in increased production of IL-6, TNF-alpha and IL-1beta
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How does nef play a role in apoptosis?
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Nef binds to ASK1 and inhibits it
Nef binds to PAK and activates it Results in DECREASED apoptosis |
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How many copies of Nef are incorporated into viral particles?
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10-100/virion
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Where is Nef myristoylated?
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N-terminal
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When does Nef act?
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Only in the next infection cycle- since nef is activated witin the viron by pro-mediated cleavage
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How does the Env protein result in downregulation of the primary receptor CD4?
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Retention in ER- Env binds CD4 in the ER and prevents its migration to the surface
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How does Vpu mediate downregulation of CD4?
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Degradation in the proteasome - recruits ubiquitin-proteasome pathway through interactions with beta-TrCP and Akp1p
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Why is decreased CD4 important?
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For full viral infectivity - need it for a functional envelope, to prevent cell surface viral aggregation, or to prevent envelope sequestration
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What are RNA decoys?
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RNA molecular decoys are RNA molecules that mimic natural viral RNA structures
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How do RNA decoys block viral replication?
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They bind to cellular and viral factors, outcompeting the viral RNA and these factors become unavailable for viral expression
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What are three HIV RNA structures that can be used as RNA decoys?
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TAR
Psi RRE |
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How can one improve the efficacy of the TAR RNA?
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Combining it with the U16 snoRNA
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What are antisense oligonucleotides?
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DNA or RNA molecules that hybridize with an mRNA (target mRNA) and inhibit its function
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What are two mechanisms by which antisense oligonucleotides inhibit?
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1) RNA degradation by RNAse H (antisense DNA) or RNAse 1 (antisense RNA)
2) Translation inhibition |
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How can antisense oligonucleotide inhibit translation?
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Combine it with another molecule to create a steric antisense oligo nucleotide - these will prevent translational machinery from binding
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What is a ribozyme?
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An enzyme that is an RNA molecule - carries out the catalytic activity. Thes can cleave themselves (self-cleavage) or cleave a target (trans-cleavage)
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What are some naturally existing catalytic RNAs?
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Hammerhead, hairpin, HDV RNA, group I and group II introns, and RNA subunit of RNAse P
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How can we increase the specificity of ribozymes for their viral target?
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Incorporate the catalytic centers into antisense oligonucleotides
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What is the hammerhead ribozyme cleavage site?
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U followed by C/U/A
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What is the hairpin ribozyme cleaveage site?
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Before GUC
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What is RNAse P?
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Endonuclease that generates the mature ends of tRNAse
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What does the protein component of RNase P do?
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Facilates Rz-substrate binding
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Which part of RNAse P carries out the catalytic activity?
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The RNA subunit
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What is unique about the HDV Rz?
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Sole example of a ribozyme derived from an RNA species found in human cells - and therfore has increased stability because it naturally functions in the presence of human proteins
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What happens once the target RNA is cleaved by HDV Rz?
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It is degraded by host nucleases
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What is the SOFA Rz?
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It is an HDV-Rz with an on-off adaptor
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What needs to be designed in this SOFA-Rz?
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P1 stem (ribozyme)
Biosensor (homologues to target RNA) Blocker stem -blocks ribozyme if target RNA is not present |
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What is required for HDV cleavage?
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Only 7bp formed with the target
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What is RNA interference?
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A natural mechanism used by eukaryotes for gene silencing
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What does RNA interference use?
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The mehcnaaism uses dsRNA identical to a target gene that will degrade its RNA
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What happens when cells are injected with exogeneous LONG dsRNA?
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This activates PKR and IFN -> shuts down cellular and viral translation
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How can we avoid this PKR activation?
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Use small dsRNA (siRNA) which can inactivate target gene expression and do not activate PKR
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Which promoter are siRNA typically expressed from?
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Pol III (U6 or H1)
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How do siRNA differ from ribozymes?
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By themselves, th ey have no intrinsive activity
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Which three proteins are required for siRNA-mediated gene silencing?
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Dicer
Argonaute family member dsRNAbindnig protein (TRBP -TAR RNA binding protein) |
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What compelx do these three proteins form?
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RISC- RNA-induced silencing complex
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Which family does Dicer belong to?
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Rnase III
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What does mammalian Dicer do?
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Cleaves dsRNA to generate siRNA
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What is the mammalian Argonaute member?
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Ago2
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Which family does Dicer belong to?
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Rnase III
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What does Ago2 do?
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Cleaves the RNA hybridized with one of the siRNA strands
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What does mammalian Dicer do?
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Cleaves dsRNA to generate siRNA
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What are the two pathways of RNA interference?
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Endogenous and exogenous
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What is the mammalian Argonaute member?
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Ago2
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What does Ago2 do?
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Cleaves the RNA hybridized with one of the siRNA strands
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What are the two pathways of RNA interference?
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Endogenous and exogenous
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What is the exogenous pathway? (5 steps)
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1) siRNA introduced to the cell, bound by Dicer + TRBP
2) Long dsRNA or shRNA is cleaved by dicer to form short siRNA 3) TRBP, Dicer and Argo2 will bind to siRNA -> strand separation 4) one strand bhydbridaizes to homologous mRNA targout - Argo 2 mediates cleavage 5) RNA degradation |
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What is required for mRNA degradation?
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siRNA is perfectly homologous to the target
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What probably encodes miRNA?
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The "junk NDA"
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What are is the first step of endogenous RNA interference?
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1)miRNA forms primary miRNA in the nucleus
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What happens after primary miRNA is formed in the nucleus?
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It is bound by Drosha and DGCR8
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What does Drosha do?
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Mediates cleavage of primary miRNA int he nucleus, generator precursor miRNAs
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What happens to the precursor miRNAS?
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They exit the nucleus, and in the cytoplasm they are bound by Dicer and TRBP -> then follows same steps are similar to exogenous pathway
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What is an important difference between the endogenous and exogenous pathway?
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miRNA has mistmatches with target mRNA- results in mainly translational repression
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What happens when homology is greater with the target strand?
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Have more mRNA degradation, less translational repression
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What are P bodies?
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miRNA bound to rpoteins
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What are some advantages to siRNA?
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Easy to design
very effective |
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What are some limitations to using siRNA?
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-Exact match required
-Since short sequence - can observe VIRAL ESCAPE -May induce IFN and PKR -Delivery is difficult |
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What was the original aim of gene therapy?
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To correct a genetic defect by transferring afunctional normal copy of the gene into cells
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What is transduction?
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Delivery of genes with a viral vector
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What is T cell based gene therapy?
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Isolate CD4+ T cells, transduce with gene, reinfuste T cells into patient
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What is stem-cell based gene therapy?
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Transduce stem cells -the vector will be expressed in all cells the stem cell differentiates into
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What are viral vectors?
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Use viral particles to carry nucleic acids (genes) into cells
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What is the rationale behind using viral vectors for gene therapy?
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-Viruses are obligatory intracellular parasites
-Efficinent at transferring their DNA into host cells -Target specific cells -Gene replacement |
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What type of viruses need to be used for gene therapy?
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Replication defective
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What does it mean that a virus is replication defective?
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Cannot replicate beyond the first cycle of infection
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What are the elements needed to generate a replication defective viral vector?
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-Transfer vector
-Packaging vector -Helper vector |
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What are some factors to consider before choosing a viral vector?
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Viral titer
Insert size Manipulation of tropism Immunogenicity |
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What is not so great about adenovirus viral vectors?
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They have high immunogenecity
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What isnt so good about retrovirus vectors?
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They cant infect non-dividing cells
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What is lakcking in AAV and Herpesvirus vectors?
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It is hard to manipulate their tropism
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What is required in the transfer plasmid for retroviral vectors?
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Gene of interest
LTRs Primer binding site RNA packaging signal Polypurine tract |
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What do LTRs contain?
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promoter, poly A, integration, replcication and reverse transcription
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What must the packaging vector contain?
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Gag, Gag-Pol and Env
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What are some problems seen with using retroviral gene vectors?
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-Regeneration of replication competent retrovirus
-Insertional mutagenesis -Limited envelope tropism -Heterologous gene expression wanes over time -Do not ifnect non dividing cells |
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How can we avoid regeneration of competent retroviruses?
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Modify the LITER and insert a non-retroviral promoter (CMV) that prevents this recombination
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How can we overcome the limited retrovirus vector tropism?
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Pseudotype the vector particles with a pantropic Env -> like VSV-G protein
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What is a key difference between retroviral and lentiviral vectors?
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Lentiviral vectors are more complex and require Tat, Rev on the packaging plasmids in addtion to the Gag, pol and env
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What is the most common form of adenoviral vector?
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Ad5
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Which genes are deleted in the Ad5 vector?
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Essential E1 genes
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What is inserted instead of the E1 gene?
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cDNA plus promoter/enhancer sequence
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What is recombinant Ad5 Dna transfected into to generate recombinant viral particles?
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E1-producing 293 cells
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Why is there only transient expression of Ad vectors?
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It does NOT integrate into the host
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What are some advantages of Ad vectors?
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-High titers
-Infects dividing + non dividing cells -wide tropism |
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What are disdvantages of Ad vectors?
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Highly immunogenic
High titers of virus can be toxic |
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What are adenovirus associated vectors?
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Parvovirus ssDNA
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What type of virus is the parvovirus?
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Small ss DNA (4.5 kb)
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What must AAV be cotransfected with?
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Plasmids expressing Rep and Cap
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What do we normally instert into AAV vectors?
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CMV promoter + polaA
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What are some advantages of using AAV vectors?
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Integration + persistent expression
No insertional mutagenesis Infect dividing and nondividing cells Safe |
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What are some disadvantages of AAV?
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Size limitation
low titer |
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What are advantages of using Herpesvirus vectors?
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Insert a large maount of DNA (50 kb of foreign DNA!)
Neutrotropic |
