HIV MOLECULAR BIOLOGY

Contents

• Figure 1 : An overview of the organization of the (approximately) 9-kilobase genome of the HIV provirus and a summary of the functions of its nine genes encoding 15 proteins

• Figure 2: Schematic description of early events occurring after HIV infection of a susceptible target cell, including interactions between gp120, CD4, and chemokine receptors (CCR5 or CXCR4) leading to gp41-mediated fusion followed by virion uncoating, reverse transcription of the RNA genome, nuclear import of the viral preintegration complex, and integration of the double-stranded viral cDNA into the host chromosome, thus establishing the HIV provirus

• Figure 3: A summary of two different mechanisms potentially underlying post-integration HIV latency contrasted with the central role of Tat in promoting productive infection of target cells.

• Figure 4: A summary of late events in the HIV-infected cell culminating in the assembly of new infectious virions. Highlighted are the roles of various viral proteins in optimizing the intracellular environment for viral replication including downregulation of CD4 and MHC I and inhibition of apoptosis by Nef, and the induction of G2 cell-cycle arrest by Vpr. A key action of the HIV Rev protein in promoting nuclear export of incompletely spliced viral transcripts that encode the structural and enzymatic proteins as well as the viral genome of new virions is also illustrated.

• Figure 5: A summary of late events in the HIV-infected cell culminating in the assembly of new infectious virions. Highlighted are the roles of various viral proteins in optimizing the intracellular environment for viral replication including downregulation of CD4 and MHC I and inhibition of apoptosis by Nef, and the induction of G2 cell-cycle arrest by Vpr. A key action of the HIV Rev protein in promoting nuclear export of incompletely spliced viral transcripts that encode the structural and enzymatic proteins as well as the viral genome of new virions is also illustrated.

• BINDING AND ENTRY 

• CYTOPLASMIC EVENTS

• NUCLEAR PENETRATION

• INTEGRATION

• TRANSCRIPTION CONTROL

• VIRAL TRANSCRIPT

• HIV REPLICATION

• VIRAL ASSEMBLY 

• VIRAL BUDDING

• SUMMARY

• Animation of Molecular Biology of HIV life cycle

Figure 1: An overview of the organization of the (approximately) 9-kilobase genome of the HIV provirus and a summary of the functions of its nine genes encoding 15 proteins 

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Figure 2 : Schematic description of early events occurring after HIV infection of a susceptible target cell, including interactions between gp120, CD4, and chemokine receptors (CCR5 or CXCR4) leading to gp41-mediated fusion followed by virion uncoating, reverse transcription of the RNA genome, nuclear import of the viral preintegration complex, and integration of the double-stranded viral cDNA into the host chromosome, thus establishing the HIV provirus

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BINDING AND ENTRY (chemokine receptors, lipid raft, conformational change, endocytosis of dendritic cell) [Summary]

12 chemokine receptors can function as HIV coreceptors (Fig 2) in cultured cells, but only two are known to play a role in vivo.

• CCR5, binds macrophage-tropic, non-syncytium-inducing (R5) viruses,  associated with mucosal and intravenous transmission of HIV infection.  In up to 13% of individuals of northern European descent, a naturally occurring deletion of 32 base pairs in the CCR5 gene results in a mutant CCR5 receptor that never reaches the cell surface. Individuals homozygous for this mutation (1-2% of the Caucasian population) are  completely resistant to HIV infection. Interrupting  HIV interaction with CCR5 might form a promising new class of antiretroviral drugs.

• CXCR4, binds T-cell-tropic, syncytium-inducing (X4) viruses,  during the later stages of disease.

Both CD4 and chemokine coreceptors for HIV are found disproportionately in lipid rafts (Fig 2) in the cell membrane. These cholesterol- and sphingolipid-enriched microdomains likely provide a better environment for membrane fusion, perhaps by mirroring the optimal lipid bilayer of the virus. Removing cholesterol from virions, producer cells, or target cells greatly decreases the infectivity of HIV. Studies currently under way are exploring whether cholesterol-depleting compounds might be efficacious as topically applied microbicides to inhibit HIV transmission at mucosal surfaces.

The binding of surface gp120, CD4, and the chemokine coreceptors produces radical conformational change in gp41. Assembled as a  trimer  on the virion membrane, this coiled-coil protein springs open, projecting three peptide fusion domains that "harpoon" the lipid bilayer of the target cell. The fusion  leads to the release of the viral core into the cell interior. The fusion inhibitors T-20 and T-1249 act to prevent fusion by blocking the formation of these hairpin structures.

HIV virions can also enter cells by endocytosis. Usually, productive infection does not result, presumably reflecting inactivation of these virions within endosomes. However, a special form of endocytosis has been demonstrated in submucosal dendritic cells. These cells, which normally process and present antigens to immune cells, express a specialized attachment structure termed DC-SIGN. This C-type lectin binds HIV gp120 with high affinity but does not trigger the conformational changes required for fusion. Instead, virions bound to DC-SIGN are internalized into an acidic compartment and subsequently displayed on the cell surface after the dendritic cell has matured and migrated to regional lymph nodes, where it engages T cells. Thus, dendritic cells expressing DC-SIGN appear to act as "Trojan horses" facilitating the spread of HIV from mucosal surfaces to T cells in lymphatic organs

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CYTOPLASMIC EVENTS (phosphorylation, viral protein Nef + Vif, HIV preintegration complex(PIC), microtubule transport, promyleotic oncogene (PML)) [Summary]

Once inside the cell, the virion undergoes uncoating, likely while still associated with the plasma membrane. This poorly understood process may involve phosphorylation of viral matrix proteins by a mitogen-activated protein (MAP) kinase and additional actions of cyclophilin A and the viral proteins Nef and Vif  (Fig 1). 

• Nef associates with a universal proton pump, V-ATPase, which could promote uncoating by inducing local changes in pH in a manner similar to that of the M2 protein of influenza.  

• By overcoming destabilizing effects of a recently identified protein termed CEM15/APOBEC3G, Vif stabilizes the reverse transcription complex in most human cells.

After the virion is uncoated, the viral reverse transcription complex is released from the plasma membrane.

Reverse transcription yields the HIV preintegration complex (PIC) (Fig 2), composed of 

• double-stranded viral cDNA, 

• integrase, 

• matrix, 

• Vpr, 

• reverse transcriptase,  

• high mobility group DNA-binding cellular protein HMGI(Y). 

The PIC may move toward the nucleus by using microtubules as a conduit. Adenovirus and herpes simplex virus 1 also dock with microtubules and use the microtubule-associated dynein molecular motor for cytoplasmic transport. This finding suggests that many viruses use these cytoskeletal structures for directional movement.

Recent studies have revealed a mechanism by which the target cell defends against the HIV intruder. Within 30 minutes of infection, select host proteins including the integrase interactor 1 (also known as INI-1, SNF5, or BAF47), a component of the SWI/SNF chromatin remodeling complex, and PML (Fig 2), a protein present in promyelocytic oncogenic domains, translocate from the nucleus into the cytoplasm. Addition of arsenic trioxide sharply blocks PML movement and enhances the susceptibility of cells to HIV infection raising the possibility that the normal function of PML is to oppose viral infection. 

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NUCLEAR PENETRATION (molecular gymnastics, Matrix vs Vpr shuttle) [Summary]

With a Stokes radius of  28 nm or roughly the size of a ribosome and 3 µm contour length of viral DNA , the PIC is twice as large as the maximal diameter of the central aqueous channel in the nuclear pore and must undergo significant compaction, known as molecular gymnastics.(Fig 2)

The  key viral proteins that mediate the nuclear import of the PIC are Integrase, matrix,and Vpr. 

• Matrix  (Fig 2 inset) contains a canonical nuclear localization signal that is recognized by the importins alpha and beta, which are components of the classical nuclear import pathway.  

• The HIV Vpr (Fig 1) gene product contains at least three noncanonical nuclear targeting signals. Vpr may bypass the importin system altogether, perhaps mediating the direct docking of the PIC with one or more components of the nuclear pore complex. 

The multiple nuclear targeting signals within the PIC may function in a cooperative manner or play larger roles individually in different target cells. For example, while Vpr is not needed for infection of nondividing, resting T cells, it enhances viral infection in nondividing macrophages. The finding that both matrix and Vpr shuttle between the nucleus and cytoplasm explains their availability for incorporation into new virions

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INTEGRATION (integrase, HMGI(Y), BAF,  PIC enter nucleus outcome, NHEJ) [Summary]

Once inside the nucleus, the viral PIC can establish a functional provirus. Integration (Fig 2) of double-stranded viral DNA into the host chromosome is mediated by :

• integrase,
  • binds the ends of the viral DNA.
  •  removes terminal nucleotides from the viral DNA, producing a two-base recess,  correcting the ragged ends generated by the terminal transferase activity of reverse transcriptase.
  • catalyzes the subsequent joining reaction that establishes the HIV provirus within the chromosome.
• host proteins HMGI(Y) 
• barrier to autointegration (BAF)  

Not all PICs that enter the nucleus result in a functional provirus. Other outcomes are:

• the ends of the viral DNA may be joined to form a 2-LTR  (Fig 1) circle containing long terminal repeat sequences from both ends of the viral genome, 
• the viral genome may undergo homologous recombination yielding a single-LTR circle. 
• the viral DNA may auto-integrate into itself, producing a rearranged circular structure. 

In a normal cellular response to DNA fragments, the nonhomologous end-joining (NHEJ) system may form 2-LTR circles to protect the cell.(Fig 2) This system is responsible for rapid repair of double-strand breaks, thereby preventing an apoptotic response. 

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Figure 3 : A summary of two different mechanisms potentially underlying post-integration HIV latency contrasted with the central role of Tat in promoting productive infection of target cells.

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TRANSCRIPTION CONTROL (active / latency transcription, chromosomal environment, transcriptional enhancer, TAT function) [Summary]

Integration can lead to 

• transcriptionally active 
• latent forms of infection. Its transcriptional latency explains the inability of early vigorous immune response and potent antiviral therapies to eradicate the virus from the body. These silent proviruses are a reservoir that allows reemergence of HIV when the body's defenses grow weaker. 

The chromosomal environment likely shapes the transcriptional activity of the provirus. For example, latency might be caused by

• integration proviral into repressed heterochromatin (Fig 3) 
• cell type differences in the availability of activators that bind to the transcriptional enhancer in the HIV LTR 
• the lack of Tat. (Fig 1)

However, of the multiple copies of provirus that are usually integrated in a given infected cell, at least one is likely to be transcriptionally active. This fact may explain why the number of latently infected cells (10⁵-10⁶) in infected patients is small.

In the host genome, the 5´ LTR functions like other eukaryotic transcriptional units. It contains downstream and upstream promoter elements, which include the initiator (Inr), TATA-box (T), and three Sp1 sites. These regions help position the RNA polymerase II (RNAPII) at the site of initiation of transcription and to assemble the preinitiation complex. Slightly upstream of the promoter 

•  the transcriptional enhancer, which in HIV-1 binds (Fig 3)
  • nuclear factor [kappa]B (NF-[kappa]B), 
    • NF-[kappa]B is liberated from its cytoplasmic inhibitor, I[kappa]B, by stimulus-coupled phosphorylation, ubiquitination, and proteosomal degradation of the inhibitor.
    • composed of p50 and p65 (RelA) subunits, 
    • increases the rates of initiation and elongation of viral transcription.
    • Since NF-[kappa]B is activated after several antigen-specific and cytokine-mediated events, it may play a key role in rousing transcriptionally silent proviruses
  • nuclear factor of activated T cells (NFAT), 
    • NFAT is dephosphorylated by calcineurin (a reaction inhibited by cyclosporin A) and, after its nuclear import, assembles with AP1 to form the fully active transcriptional complex
  • Ets family members. .

When these factors engage the LTR, transcription begins, but in the absence of Tat described below the polymerase fails to elongate efficiently along the viral genome.(Fig 3) 

Tat significantly increases the rate of viral gene expression. With cyclin T1 (CycT1), Tat binds to the TAR RNA stem-loop structure and recruits the cellular cyclin-dependent kinase 9 (Cdk9) to the HIV LTR. (Fig 3) Within the positive transcription elongation factor b (P-TEFb) complex, Cdk9 phosphorylates the C-terminal domain of RNAPII, marking the transition from initiation to elongation of eukaryotic transcription. Other targets of P-TEFb include negative transcription elongation factors (N-TEF). The high efficiency with which the HIV LTR attracts these negative transcription factors in vivo may explain why the LTR is a poor promoter in the absence of Tat. 

** Because murine CycT1 contains a cysteine at position 261, the complex between Tat and murine P-TEFb binds TAR weakly. Thus, Tat transactivation is severely compromised in murine cells. 

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Fig 4 : A summary of late events in the HIV-infected cell culminating in the assembly of new infectious virions. Highlighted are the roles of various viral proteins in optimizing the intracellular environment for viral replication including downregulation of CD4 and MHC I and inhibition of apoptosis by Nef, and the induction of G2 cell-cycle arrest by Vpr. A key action of the HIV Rev protein in promoting nuclear export of incompletely spliced viral transcripts that encode the structural and enzymatic proteins as well as the viral genome of new virions is also illustrated.

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VIRAL TRANSCRIPT (splicing, Rev, RanGTP,exportin) [Summary]

Transcription of the viral genome results in more than a dozen different HIV-specific transcripts. 

• Some are processed cotranscriptionally and, in the absence of inhibitory RNA sequences (IRS), transported rapidly into the cytoplasm. These multiply spliced transcripts encode Nef, Tat, and Rev. 
• Other singly spliced or unspliced viral transcripts remain in the nucleus and are relatively stable. 

These viral transcripts encode the structural, enzymatic, and accessory proteins and represent viral genomic RNAs that are needed for the assembly of fully infectious virions.

Incomplete splicing likely results from suboptimal splice donor and acceptor sites in viral transcripts.  Rev, may inhibit splicing by its interaction with alternate splicing factor/splicing factor 2 (ASF/SF2) and its associated p32 protein.

Transport of the incompletely spliced viral transcripts to the cytoplasm depends on an adequate supply of Rev. Rev:

• small shuttling protein that binds a complex RNA stem-loop termed the Rev response element (RRE), which is located in the env gene (Fig 1) 
• Rev binds first with high affinity to a small region of the RRE termed the stem-loop IIB.(Fig 4) 
• This binding leads to the multimerization of Rev on the remainder of the RRE. 
• In addition to a nuclear localization signal, Rev contains a leucine-rich nuclear export sequence (NES).(53) 

The nuclear export of this assembly (viral RNA transcript, Rev, and CRM1/exportin 1) depends critically on  another host factor, RanGTP. Ran 

• small guanine nucleotide-binding protein that switches between GTP- and GDP-bound states. 
• RanGDP is found predominantly in the cytoplasm because the GTPase activating protein specific for Ran (RanGAP) is expressed in this cellular compartment. 
• the Ran nucleotide exchange factor, RCC1, which charges Ran with GTP, is expressed predominantly in the nucleus. 
• The inverse nucleocytoplasmic gradients of RanGTP and RanGDP produced by the subcellular localization of these enzymes likely plays a major role in determining the directional transport of proteins into and out of the nucleus. 

Outbound cargo is only effectively loaded onto CRM1/exportin-1 in the presence of RanGTP. However, when the complex reaches the cytoplasm, GTP is hydrolyzed to GDP, resulting in release of the bound cargo. The opposite relationship regulates the nuclear import by importins alpha and beta, where nuclear RanGTP stimulates cargo release.

For HIV infection to spread, a balance between splicing and transport of viral mRNA species must be achieved. If splicing is too efficient, then only the multiply spliced transcripts appear in the cytoplasm. Although required, the regulatory proteins encoded by multiply spliced transcripts are insufficient to support full viral replication. However, if splicing is impaired, adequate synthesis of Tat, Rev, and Nef will not occur. In many non-primate cells, HIV transcripts may be overly spliced, effectively preventing viral replication in these hosts.

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Fig 5 : A summary of late events in the HIV-infected cell culminating in the assembly of new infectious virions. Highlighted are the roles of various viral proteins in optimizing the intracellular environment for viral replication including downregulation of CD4 and MHC I and inhibition of apoptosis by Nef, and the induction of G2 cell-cycle arrest by Vpr. A key action of the HIV Rev protein in promoting nuclear export of incompletely spliced viral transcripts that encode the structural and enzymatic proteins as well as the viral genome of new virions is also illustrated.

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HIV REPLICATION ( Nef, low CD4 expression, Vpr ) [Summary]

In contrast to Tat and Rev, which act directly on viral RNA structures, Nef modifies the environment of the infected cell to optimize viral replication. (Fig 4) The absence of Nef in infected monkeys and humans is associated with much slower clinical progression to AIDS. This virulence caused by Nef appears to be 

• affect  activation of T-cell antigen receptor,
• decrease the expression of CD4 on the cell surface. It is assisted by two other HIV proteins : 
  •  The envelope protein gp120 binds CD4 in the endoplasmic reticulum, slowing its export to the plasma membrane,
  •  Vpu binds the cytoplasmic tail of CD4, promoting recruitment of TrCP and Skp1p.(Fig 5) These events target CD4 for ubiquitination and proteasomal degradation before it reaches the cell surface.
•  promotes the production and release of more infectious virions. 
•  promoting lipid raft movement  

Nef  impairs immunological responses to HIV. 

• activates the expression of FasL, which induces apoptosis in bystander cells that express Fas, thereby killing cytotoxic T cells that might otherwise eliminate HIV-1 infected cells.
•  reduces the expression of MHC I determinants on the surface of the infected cell (Fig 4) and  decreases the recognition and killing of infected cells by CD8 cytotoxic T cells. 

** However, Nef does not decrease the expression of HLA-C,which prevents recognition and killing of these infected cells by natural killer cells.

Nef also inhibits cell apoptosis by binding  to 

• signal regulating kinase-1 (ASK-1)t
• tumor suppressor protein p53

Hence, Nef prolongs the life of the infected host cell, thereby optimizing viral replication.

Rev-dependent expression of Vpr (Fig 1) induces the arrest of proliferating infected cells at the G2/M phase of the cell cycle. Since the viral LTR is more active during G2, this arrest likely enhances viral gene expression. These cell-cycle arresting properties involve localized defects in the structure of the nuclear lamina that lead to dynamic, DNA-filled herniations that project from the nuclear envelope into the cytoplasm. (Fig  4) Intermittently, these herniations rupture, causing the mixing of soluble nuclear and cytoplasmic proteins. 

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VIRAL ASSEMBLY (plasma membrane, ViF, molecular chaperone, CEM15/APOBEC3G) [Summary]

New viral particles are assembled at the  plasma membrane . (Fig 5) Each virion consists of 

• 1500 molecules of Gag 
• 100 Gag-Pol polyproteins
• two copies of the viral RNA genome
•  Vpr

Several proteins participate in the assembly process, including Gag polyproteins and Gag-Pol, as well as Nef and Env. A human ATP-binding protein, HP68 (previously identified as an RNase L inhibitor), likely acts as a molecular chaperone, facilitating conformational changes in Gag needed for the assembly of viral capsids.

In primary CD4 T lymphocytes, Vif plays a key but poorly understood role in the assembly of infectious virions. In the absence of Vif, normal levels of virus are produced, but these virions are noninfectious, displaying arrest at the level of reverse transcription in the subsequent target cell. Heterokaryon analyses of cells formed by the fusion of nonpermissive (requiring Vif for viral growth) and permissive (supporting growth of Vif-deficient viruses) cells have revealed that Vif overcomes the effects of  CEM15/APOBEC3G :

• natural inhibitor of HIV replication
• shown to share homology with APOBEC1, an enzyme involved in RNA editing. 

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VIRAL BUDDING (Gag protein, TSG 101, ubiquitination, multivesicular body) [Summary]

The Gag polyproteins (Fig 1) are subject to myristylation, and thus associate preferentially with cholesterol- and glycolipid-enriched membrane microdomains. Virion budding occurs through these specialized regions in the lipid bilayer, yielding virions with cholesterol-rich membranes. This lipid composition likely favors release, stability, and fusion of virions with the subsequent target cell.

The budding reaction involves the action of several proteins, including the "late domain" sequence (PTAP) present in the p6 portion of Gag.(Fig 5) The p6 protein also appears to be modified by ubiquitination. The product of the tumor suppressor gene 101 (TSG101) binds the PTAP motif of p6 Gag and also recognizes ubiquitin through its ubiquitin enzyme 2 (UEV) domain. 

**The TSG101 protein normally associates with other cellular proteins in the vacuolar protein sorting pathway to form the ESCRT-1 complex that selects cargo for incorporation into the multivesicular body (MVB). The MVB is produced when surface patches on late endosomes bud away from the cytoplasm and fuse with lysosomes, releasing their contents for degradation within this organelle. 

In the case of HIV, TSG101 appears to be "hijacked" to participate in the budding of virions into the extracellular space away from the cytoplasm

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SUMMARY 

BINDING AND ENTRY 

 HIV entry into cell mediated through 

• chemokine recpetors (CCR5,CXCR4). 1-2% Caucasian with deletion of CCR5 is immune to HIV.
•  lipid raft. Depleting lipid compound could prevent entry. 
• gp41 conformational change into hairpin-like sructure 
• endocytosis

CYTOPLASMIC EVENTS

After entry, viral uncoating release viral reverse transcription complex which produce HIV preintegration complex (PIC). Two viral protein

• Nef – promote uncoating by inducing conducive microenvironment
• Vif – stabilize PIC

PIC ‘docked’ on microtubules (cytoskeletal structure), traveling towards nucleus.

Host promyelolytic oncogen (from nucleus to cytoplasm) might prevent PIC movement

 NUCLEAR PENETRATION

 Molecular gymnastics occur as PIC is 2x larger than nuclear pore. Viral proteins involved

• Integrase

• Matrix – classical nuclear import pathway with host importins a and b
• Vpr – direct docking

INTEGRATION

 Integration (viral DNA into hos chromosome) to form provirus helped by

•  Integrase

• host protein HMGI(Y)
• BAF (barrier autoregulation)

Not all become  functional provirus

NHEJ (non homologous endjoining) system involved in DNA fragment repair

TRANSCRIPTION CONTROL

Integration leads to active / latency infection.

Chromosomal environment induce latency:

•  heterochromatin

•   low transcriptional enhancer

  • nuclear factor [kappa]B (NF-[kappa]B) - ­ rate elongation / initiation replication

  • nuclear factor of activated T cells (NFAT) – form active complex

  •  Ets family members

•    lack of viral protein, Tat

  • binds cyclin T1 (Cyc T1) forming cellular cyclin-dependent kinase 9 (Cdk9) needed for effective elongation

  • Murine cell impair Tat function

VIRAL TRANSCRIPT

 Transcription produces spliced transcripts Nef, Tat, Rev

Transporting these from nucleus o cytoplasm required:

• Viral protein Rev

  •  Binds RRE (Rev Response Element, located in Env gene)

  • Has leucine-riched nuclear export system

• Host factor RanGTP

  • Gradient RanGTP (nucleus) vs RanGDP (cytoplasm) determine direction movement

•  Exportin 1 – ‘cargo'

HIV REPLICATION

 Tat, Rev acts on viral RNA

 Nef acts on environment of infected cells

 Nef virulence: 

•  CD4 expression on cell surface. Helped by

  •  Gp120 – stop CD4 movement to cell surface by binding to them

  • Vpu – enhance CD4 proteosomal degradation

• activate Fasl, which kill cytotoxic T cell

•   inhibit cell apaptosis, prolonging life of infected cell, hence optimizing viral replication

Fortunately, Nef is no expressed in infected human cell which slow progression to AIDS.

Vpr arrest replication of infected cell by inducing DNA-filled herniation to rupture, producing mixing nucleus and cytoplasm protein

VIRAL ASSEMBLY 

 Assembled in plasma membrane   

Vif overcomes host CEM15/APOBEC3G for effective assembly 

VIRAL BUDDING

 Budding needs enriched cholesterol and lipid environment.

 Gag proteins involved by ‘hijacking’ TSG101 through ubiquitination to form MVB

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ANIMATED MOLECULAR BIOLOGY of  HIV CYCLE

• JOHN HOPKINS
• ROCHE
• HIVinsite

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