HIV-1 Replication and Pathogenesis Flashcards
How does Australia’s pattern of HIV transmission/distribution differ from the worldwide trends?
Australia: mostly MSM
Worldwide: mostly heterosexual women
List 6 behavioural and social factors promoting a heterosexually transmitted HIV epidemic
Little or no condom use Multiple partners Overlapping sexual partners Large sexual networks Age mixing (old men and young girls - due to pervasive myths about treatment of HIV) Women dependent on marriage/prostitution
List 3 biological factors promoting a heterosexually transmitted HIV epidemic
High STI rates (especially HSV-2, which accelerates HIV transmission)
High viral loads (due to lack of access to HAART)
Low rate of male circumcision
What type of virus is HIV?
Lentivirus (from the family retrovirus)
Describe the genome of HIV
Diploid, linear, 9.2kb
+sense ssRNA
What is the origin of HIV?
Zoonosis from chimpanzees
Describe the capsid of HIV
Icosahedral
Does HIV have an envelope?
Yes
What are the 2 glycoproteins located in the envelope of HIV-1 and what are their roles?
SU-surface (gp-120): for cell attachment
TM- transmembrane (gp-41): fusion domain and anchors SU into the membrane
What are the 3 proteins located in the virion interior (Gag proteins)?
MA-matrix: connects to and encloses the cone-shaped capsid
CA-capsid: encloses the nucleocapsid and important enzymes involved in viral replication
NC-nucleocapsid: encloses RNA genome
What are the 3 viral enzymes of HIV and what are their roles?
RT-reverse transcriptase
IN-integrase
PR-protease
How is the envelope of HIV-1 produced?
Lipid bilayer derived from host cell plasma membrane during budding (also contains viral GPs)
What are Gag proteins?
Structural proteins of the capsid, matrix, core and nucleocapsid
What are Pol proteins? How are they expressed?
Viral enzymes including protease, reverse transcriptase, RNAse H, integrase
Expressed as a Gag-Pol polyprotein before autocleavage
How are envelope glycoproteins expressed?
From a spliced mRNA
Describe the HIV-1 replication cycle
Viral particle binds to CD4 receptors on T-lymphocytes and macrophages
Fusion event causes release of the matrix, capsid and core into the host cell
Reverse transcription of the viral +sense ssRNA into cDNA occurs within the capsid
The provirus cDNA is incorporated into the host cell DNA
Provirus is transcribed, spliced and translated into protein to produce virions and genomic RNA
Virion is assembled and buds out from the host cell, taking part of the plasma membrane as its envelope
Virion matures (aided by activity of viral protease)
What is 1 important mechanism by which HIV is able to evade immunity and how does it achieve this?
High degree of variability for Gag and Env proteins means it is difficult for the host to produce a specific immune response to the virus
Mutations are introduced during reverse transcription of the virus +sense ssRNA into cDNA
Describe the process of membrane fusion which occurs during entry of HIV-1
Virion attaches to host cell via non-specific cell receptors including C-type lectin receptors (CLRs)
CD4 binding induces conformational changes in gp120
Conformational change exposes chemokine coreceptor sites (including CCR5, CXCR4) which bind to promote gp41 fusion and peptide insertion
Structural rearrangement of gp41 trimers drives membrane fusion to release the core of the virus into the host cell
Give 2 examples of CLRs
DC-SIGN or mannose R on astrocytes
When in the course of an infection is CCR5 used?
Early (CXCR4 late)
Are HIV virions which use CCR5 M-tropic or T-tropic? What does this mean?
M-tropic
Mainly infect macrophages and do not form syncytium
Are HIV virions which use CXCR4 M-tropic or T-tropic? What does this mean?
T-tropic
Mainly infect T cells and can form syncytium
What is syncytium?
A multinucleated cell resulting from the fusion of multiple uninuclear cells
What is the difference in pathogenicity between CCR5 and CXCR4 HIV virions?
CCR5 have moderate virulence
CXCR4 have high virulence
What ligands block HIV with CCR5?
RANTES
MIP-1a/B
What ligands block HIV with CXCR4?
SDF-1
What is the most common functional mutation in CCR5?
CCR5 D32
What is the most functional mutation in CXCR4?
SDF-1 (RNA)
Describe the process of HIV spread throughout the body
Musocal exposure to HIV-1
Selective infection by R5 strains
Virions bind DCs via DC-SIGN
DCs transport virus to regional LNs
Infection spreads to activated CD4+ T lymphocytes
Widespread dissemination occurs, with spread to organs including the brain, spleen, lymph nodes and GALT
What is the 1 of the major barriers to clearing HIV in infected patients?
Resting CD4+ T cells are infected and can become activated at any time
What is the error rate in reverse transcription of the HIV genome? Why is this important?
1:10,000nt
Results in an ~1nt change per infectious cycle, which is the maximum tolerated by nature but which enables the virus to evade the immune system
What processes occur in reverse transcription of the viral genome?
+sense ssRNA converted to proviral cDNA
Sequences at the end of the virual RNA are duplicated to form a DNA structure called the “long terminal repeat” (LTR)
How is the viral cDNA integrated into the host cell DNA?
HIV integrase catalyses the random integration of HIV cDNA into cell DNA by binding the LTR (occurs in resting and terminally differentiated cells)
What is the role of the 5’LTR?
Acts as the HIV gene promoter
Give examples of contexts in which 5’LTR increases or decreases HIV expression
HIV expression silenced soon after initial replication
Increased expression in response to HIV Tat protein
Responds to cellular proteins made during T-cell immune activation to dramatically increase HIV expression
Give an example of a cellular protein made during T-cell activation which dramatically increases HIV expression
NF-kB
How is HIV basal transcription regulated?
By cis- (non-coding DNA, at the integration site) and trans- (coding DNA, expressed during T-cell activation) acting factors
Give an example of cis-acting factors
Chromatin and associated factors (e.g. HDACs, histone methyltransferases)
Give an example of trans-acting factors
Transcription factors (e.g. NFkB, NFAT)
What is TAR?
Trans-activation response RNA element
What is Tat?
Transactivator of HIV transcription through TAR RNA
What is Rev?
Regulator of structural gene expression via binding of the Rev-responsive RNA element (RRE)
How does Tat promote transcriptional elongation of HIV-1?
Bind TAR RNA and recruits Tat-associated kinases (including cyclin T and CDK9) to increase the activity of RNA polymerase, and therefore the number of copies of full length RNA produced
How does Rev work? Contrast initial and late cycles of viral replication
During late replication, Rev has been accumulated and can bind unspliced (9kb) and partially spliced (4kb) RNA transcripts in a complex with RRE, and export them to the cytoplasm, where they can be translated into structural (Gag-Pol) elements
This is in contrast to initial replication where there is low Rev, and RNA transcript is quickly degraded in the nucleus so that only 1.8kb RNA transcripts can be exported to the cytoplasm and transcribed (these smaller transcripts produce proteins including Tat, Nef, Vpr and more Rev)
List 4 HIV-1 regulatory and accessory proteins not essential for replication, and their roles
Vif: promotes infectivity of cell free virus by degrading APOBEC3G
Vpr: role in nuclear import of cDNA, cell growth arrest, and weakly in transcription transactivation
Vpu: regulator of particle release and Env processing, promotes MHC I and CD4 degradation, antagonises tetherin, and inhibits surface expression of CD1d
Nef: multifunctional protein important for in vivo pathogenesis, down-modulates cell MHC I and CD4
How does Nef down-regulate MHC I expression?
By inducing lysosomal degradation of MHC I
How does Vpu down-regulate MHC I expression?
By inducing lysosomal degradation of MHC I and directing MHC I to the proteosome
How does Tat down-regulate MHC I expression?
Inhibits MHC I gene transcription
What is the role of TRIM5a in the immune response to HIV?
Destabilises the viral capsid, causing untimely uncoating leading to proteosomal degradation of the virion
What is the role of APOBEC3G in the immune response to HIV?
A cytidine deaminase that induces lethal G>A hyper-mutations of the viral genome while it is still ssDNA
What is the role of tetherin in the immune response to HIV?
Inhibits virus release
What events occur during maturation of HIV particles?
Icosahedral core matures to form a complex rod shape after budding
Protease activity of Gag-Pol precursor protein causes cleavage of polyproteins into individual proteins
What is the half-life of a cell infected with HIV? Of virus particles? Of resting infected T cells?
1 day
6 hrs
Months-yrs
What cells make up the main latent HIV reservoir?
Central and transitional memory T-cells
What is the effect of HAART on the latent HIV reservoir?
Patients successfully treated with HAART for >10 years have no appreciable decrease in the size of the reservoir; suggests the virus will proliferate immediately following removal of treatment
Identify 7 factors which prevent HIV production in latent host cells
Cellular factors: limited transcriptional activators
Viral factors: integration in active genes, low acetylation, high methylation, transcriptional interference from host DNA
Impaired RNA export from nucleus
HIV-specific host microRNA
How does Tat activate RNA expression?
Recruits histone acetyl-transferase (HATs), which displace HDACs leading to acetylation of histones in chromatin
This opens up the structure of the DNA to allow the promoter to be accessed by RNA polymerase and various TFs
What determines whether HIV provirus is being actively transcribed, is attenuated or is being completely silenced?
Level of Tat activity (critical threshold takes provirus from silenced to attenuated state)
What can be measured in a productive infection vs. a latent infection?
Productive: can measure RNA in a single copy assay (SCA), viral DNA (integrated or unintegrated)
Latent: can measure integrated DNA
What are the units used when measuring integrated HIV DNA?
IUPM: infectious units of provirus per million cells
