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4.10.4: Replicative Cycle of HIV - Biology

4.10.4: Replicative Cycle of HIV - Biology


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LEARNING OBJECTIVES

  • Compare and contrast HIV replication to other viruses

Human immunodeficiency virus (HIV) is a lentivirus (a member of the retrovirus family) that causes acquired immunodeficiency syndrome (AIDS). AIDS is a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive. HIV can infect dendritic cells (DCs). DCs are one of the first cells encountered by the virus during sexual transmission. They are currently thought to play an important role by transmitting HIV to T-cells when the virus is captured in the mucosa by DCs. HIV enters macrophages and T cells by the adsorption of glycoproteins on its surface to receptors on the target cell. This is followed by fusion of the viral envelope with the cell membrane and the release of the HIV capsid into the cell.

Shortly after the viral capsid enters the cell, an enzyme called reverse transcriptase liberates the single-stranded (+)RNA genome from the attached viral proteins and copies it into a complementary DNA (cDNA) molecule. The process of reverse transcription is extremely error-prone, and the resulting mutations may cause drug resistance or allow the virus to evade the body’s immune system. The reverse transcriptase also has ribonuclease activity that degrades the viral RNA during the synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that creates a sense DNA from the antisense cDNA. Together, the cDNA and its complement form a double-stranded viral DNA that is then transported into the cell nucleus.

This integrated viral DNA may then lie dormant, in the latent stage of HIV infection. To actively produce the virus, certain cellular transcription factors need to be present. The most important of these is NF-κB (NF kappa B), which is upregulated when T-cells become activated. This means that those cells most likely to be killed by HIV are those currently fighting infection. During viral replication, the integrated DNA provirus is transcribed into mRNA, which is then spliced into smaller pieces. These small pieces are exported from the nucleus into the cytoplasm, where they are translated into the regulatory proteins Tat (which encourages new virus production) and Rev.

As the newly produced Rev protein accumulates in the nucleus, it binds to viral mRNAs and allows unspliced RNAs to leave the nucleus, where they are otherwise retained until spliced. At this stage, the structural proteins Gag and Env are produced from the full-length mRNA. The full-length RNA is actually the virus genome; it binds to the Gag protein and is packaged into new virus particles. The final step of the viral cycle, assembly of new HIV-1 virions, begins at the plasma membrane of the host cell. The Env polyprotein goes through the endoplasmic reticulum and is transported to the Golgi complex. There, it is cleaved by HIV protease and processed into the two HIV envelope glycoproteins, gp41 and gp120. These are transported to the plasma membrane of the host cell where gp41 anchors gp120 to the membrane of the infected cell. The Gag (p55) and Gag-Pol (p160) polyproteins also associate with the inner surface of the plasma membrane along with the HIV genomic RNA as the forming virion begins to bud from the host cell.

Maturation occurs either in the forming bud or in the immature virion after it buds from the host cell. During maturation, HIV proteases cleave the polyproteins into individual functional HIV proteins. This cleavage step can be inhibited by protease inhibitors. The various structural components then assemble to produce a mature HIV virion. The mature virion is then able to infect another cell.

Key Points

  • First the HIV viron binds to host cell, after binding the virus and cell fuse, which releases the various enzymes HIV needs to reverse transcribe and integrate into the host genome.
  • The reverse transcription of HIV viral RNA to DNA is error prone, causing HIV to have a high mutation rate. This makes it difficult to design treatments against HIV.
  • The HIV provirus can stay dormant in the host genome for years. It may become active when the host T cell is itself activated by fighting an infection that the body is facing.
  • Understanding the HIV life cycle will help in providing effective treatments against HIV.

Comprehensive, up-to-date information on HIV/AIDS treatment and prevention from the University of California San Francisco

Bringing the global HIV epidemic under control will require more effective approaches to prevent the spread of the retrovirus, as well as broader use of existing and future antiretroviral drugs. These interventions must be applicable in the developing world, where HIV has the most severe impact. Understanding the dynamic interplay of HIV with its cellular host provides the biological basis for controlling the epidemic. This chapter reviews current understanding of the HIV life cycle, with particular attention to the interactions between viral proteins and cellular machinery, and highlights promising future points of attack.

The genetic material of HIV, an RNA molecule 9 kilobases in length, contains 9 different genes encoding 15 proteins. Considerable insights have been gained into the function of these different gene products.(Figure 1) To productively infect a target cell, HIV must introduce its genetic material into the cytoplasm of this cell. The process of viral entry involves fusion of the viral envelope with the host cell membrane and requires the specific interaction of the envelope with specific cell surface receptors. The two viral envelope proteins, gp120 and gp41, are conformationally associated to form a trimeric functional unit consisting of three molecules of gp120 exposed on the virion surface and associated with three molecules of gp41 inserted into the viral lipid membrane. Trimeric gp120 on the surface of the virion binds CD4 on the surface of the target cell, inducing a conformational change in the envelope proteins that in turn allows binding of the virion to a specific subset of chemokine receptors on the cell surface.(1)(Figure 2) These receptors normally play a role in chemoattraction, in which hematopoietic cells move along chemokine gradients to specific sites. Although these receptors, which contain seven membrane-spanning domains, normally transduce signals through G proteins,(2) signaling is not required for HIV infection.

Twelve chemokine receptors can function as HIV coreceptors in cultured cells, but only two are known to play a role in vivo.(2) One of these, CCR5, binds macrophage-tropic, non-syncytium-inducing (R5) viruses, which are associated with mucosal and intravenous transmission of HIV infection. The other, CXCR4, binds T-cell-tropic, syncytium-inducing (X4) viruses, which are frequently found during the later stages of disease.(3) 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.(4,5) Individuals homozygous for this mutation (1-2% of the Caucasian population) are almost completely resistant to HIV infection.(4,5) These observations emphasize the pivotal role of CCR5 in the spread of HIV and suggest that small molecules that prevent HIV interaction with CCR5 might form a promising new class of antiretroviral drugs.

Both CD4 and chemokine coreceptors for HIV are found disproportionately in lipid rafts in the cell membrane.(6) These cholesterol- and sphingolipid-enriched microdomains likely provide a better environment for membrane fusion, perhaps by mirroring the optimal lipid bilayer of the virus.(7) Removing cholesterol from virions, producer cells, or target cells greatly decreases the infectivity of HIV.(8) 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 development of effective microbicides represents an important component of future HIV prevention strategies.

The binding of surface gp120, CD4, and the chemokine coreceptors produces an additional radical conformational change in gp41.(9) 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 domains then form hairpin-like structures that draw the virion and cell membranes together to promote fusion, leading to the release of the viral core into the cell interior.(9) 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.(10) 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.(11) 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.

Once inside the cell, the virion undergoes uncoating, likely while still associated with the plasma membrane.(Figure 2) This poorly understood process may involve phosphorylation of viral matrix proteins by a mitogen-activated protein (MAP) kinase(12) and additional actions of cyclophilin A(13) and the viral proteins Nef(14) and Vif.(15) Nef associates with a universal proton pump, V-ATPase,(16) which could promote uncoating by inducing local changes in pH in a manner similar to that of the M2 protein of influenza.(17) After the virion is uncoated, the viral reverse transcription complex is released from the plasma membrane.(18) This complex includes the diploid viral RNA genome, lysine transfer RNA (tRNA Lys ) which acts as a primer for reverse transcription, viral reverse transcriptase, integrase, matrix and nucleocapsid proteins, viral protein R (Vpr), and various host proteins. The reverse transcription complex docks with actin microfilaments.(19) This interaction, mediated by the phosphorylated matrix, is required for efficient viral DNA synthesis. By overcoming destabilizing effects of a recently identified protein termed CEM15/APOBEC3G, Vif stabilizes the reverse transcription complex in most human cells.(15-20)

Reverse transcription yields the HIV preintegration complex (PIC), composed of double-stranded viral cDNA, integrase, matrix, Vpr, reverse transcriptase, and the high mobility group DNA-binding cellular protein HMGI(Y).(21) The PIC may move toward the nucleus by using microtubules as a conduit.(22) 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. How the switch from actin microfilaments to microtubules is orchestrated remains unknown.

Recent studies have revealed a mechanism by which the target cell defends against the HIV intruder.(23,24) 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, a protein present in promyelocytic oncogenic domains, translocate from the nucleus into the cytoplasm.(24)(Figure 2) 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.(24) The binding of integrase to integrase interactor 1 may be a viral adaptation that recruits additional chromatin remodeling factors. Whether these complexes influence the site of viral integration or improve subsequent proviral gene expression is not known.

Unlike most animal retroviruses, HIV can infect nondividing cells, such as terminally differentiated macrophages.(25) This requires an ability to cross the intact nuclear membrane. With a Stokes radius of approximately 28 nm or roughly the size of a ribosome, the PIC is roughly twice as large as the maximal diameter of the central aqueous channel in the nuclear pore.(26) The 3 µm contour length of viral DNA must undergo significant compaction, and the import process must involve considerable molecular gymnastics.

One of the most contentious areas of HIV research involves the identification of key viral proteins that mediate the nuclear import of the PIC. Integrase,(27) matrix,(28) and Vpr(29) have been implicated.(Figure 2) Because plus-strand synthesis is discontinuous in reverse transcription, a triple helical DNA domain or "DNA flap" results that may bind a host protein containing a nuclear targeting signal.(30) Matrix contains a canonical nuclear localization signal that is recognized by the importins alpha and beta, which are components of the classical nuclear import pathway. However, a recent publication calls into question the contributions both of the nuclear import signal in integrase and of the DNA flap to the nuclear uptake of the PIC.(31) The HIV Vpr gene product contains at least three noncanonical nuclear targeting signals.(32) 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,(33) it enhances viral infection in nondividing macrophages.(34) The finding that both matrix(35) and Vpr(32) shuttle between the nucleus and cytoplasm explains their availability for incorporation into new virions.

Once inside the nucleus, the viral PIC can establish a functional provirus.(Figure 2) Integration of double-stranded viral DNA into the host chromosome is mediated by integrase, which binds the ends of the viral DNA.(21) The host proteins HMGI(Y) and barrier to autointegration (BAF) are required for efficient integration, although their precise functions remain unknown.(36) Integrase removes terminal nucleotides from the viral DNA, producing a two-base recess and thereby correcting the ragged ends generated by the terminal transferase activity of reverse transcriptase.(21) Integrase also catalyzes the subsequent joining reaction that establishes the HIV provirus within the chromosome.

Not all PICs that enter the nucleus result in a functional provirus. The ends of the viral DNA may be joined to form a 2-LTR circle containing long terminal repeat sequences from both ends of the viral genome, or the viral genome may undergo homologous recombination yielding a single-LTR circle. Finally, the viral DNA may auto-integrate into itself, producing a rearranged circular structure. Although some circular forms may direct the synthesis of the transcriptional transactivator Tat or the accessory protein Nef, none produces infectious virus.(37) In a normal cellular response to DNA fragments, the nonhomologous end-joining (NHEJ) system may form 2-LTR circles to protect the cell.(38) This system is responsible for rapid repair of double-strand breaks, thereby preventing an apoptotic response. A single double-strand break within the cell can induce G1 cell-cycle arrest. The ability of the free ends of the viral DNA to mimic such double-strand chromosomal breaks may contribute to the direct cytopathic effects observed with HIV.

Integration can lead to latent or transcriptionally active forms of infection.(39) HIV's transcriptional latency explains the inability of potent antiviral therapies to eradicate the virus from the body. Moreover, despite a vigorous immune response early in infection, these silent proviruses are a reservoir that allows reemergence of HIV when the body's defenses grow weaker. Understanding latency and developing approaches to target latent virus are essential goals if eradication of HIV infection is ever to be achieved.

The chromosomal environment likely shapes the transcriptional activity of the provirus.(40) For example, proviral integration into repressed heterochromatin might result in latency.(Figure 3) Other causes of latency may include cell type differences in the availability of activators that bind to the transcriptional enhancer in the HIV LTR or the lack of Tat. 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 (10 5 -10 6 ) 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.(41) 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 is the transcriptional enhancer, which in HIV-1 binds nuclear factor [kappa]B (NF-[kappa]B), nuclear factor of activated T cells (NFAT), and Ets family members.(42) NF-[kappa]B and NFAT relocalize to the nucleus after cellular activation. NF-[kappa]B is liberated from its cytoplasmic inhibitor, I[kappa]B, by stimulus-coupled phosphorylation, ubiquitination, and proteosomal degradation of the inhibitor.(43) 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.(44) NF-[kappa]B, which is composed of p50 and p65 (RelA) subunits, increases the rates of initiation and elongation of viral transcription.(45) 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

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.(Figure 3) In the process, short nonpolyadenylated transcripts are synthesized, which are stable and persist in cells due to the formation of an RNA stem loop called the transactivation response (TAR) element.(46)

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.(47)(Figure 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.(48) Other targets of P-TEFb include negative transcription elongation factors (N-TEF), such as the DRB-sensitivity inducing (DSIF) and negative elongation (NELF) factors.(48) 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. The arginine-rich motif (ARM) within Tat binds the 5´ bulge region in TAR. A shorter ARM in cyclin T1, which is also called the Tat-TAR recognition motif (TRM), binds the central loop of TAR.(47)

Binding of the Tat cyclin T1 complex to both the bulge and loop regions of TAR strengthens the affinity of this interaction. All of these components are required for Tat transactivation. In the presence of the complex between Tat and P-TEFb, the RNAPII elongates efficiently. Because murine CycT1 contains a cysteine at position 261, the complex between Tat and murine P-TEFb binds TAR weakly.(49) Thus, Tat transactivation is severely compromised in murine cells. Cdk9 also must undergo autophosphorylation of several serine and threonine residues near its C-terminus to allow productive interactions between Tat, P-TEFb, and TAR.(50) Additionally, basal levels of P-TEFb may be low in resting cells or only weakly active due to the interaction between P-TEFb and 7SK RNA.(51) All of these events may contribute to postintegration latency.

Transcription of the viral genome results in more than a dozen different HIV-specific transcripts.(52) Some are processed cotranscriptionally and, in the absence of inhibitory RNA sequences (IRS), transported rapidly into the cytoplasm.(53) 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. In addition, the regulator of virion gene expression, Rev, may inhibit splicing by its interaction with alternate splicing factor/splicing factor 2 (ASF/SF2)(54) and its associated p32 protein.(55)

Transport of the incompletely spliced viral transcripts to the cytoplasm depends on an adequate supply of Rev.(53) Rev is a small shuttling protein that binds a complex RNA stem-loop termed the Rev response element (RRE), which is located in the env gene. Rev binds first with high affinity to a small region of the RRE termed the stem-loop IIB.(56)(Figure 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) Of note, the study of Rev was the catalyst for the discovery of such NES in many cellular proteins and led to identification of the complex formed between CRM1/exportin-1 and this sequence.(53)

The nuclear export of this assembly (viral RNA transcript, Rev, and CRM1/exportin 1) depends critically on yet another host factor, RanGTP. Ran is a 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. Conversely, 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.(53)

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.(57)

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.(2)(Figure 4) The absence of Nef in infected monkeys and humans is associated with much slower clinical progression to AIDS.(58,59) This virulence caused by Nef appears to be associated with its ability to affect signaling cascades, including the activation of T-cell antigen receptor,(60) and to decrease the expression of CD4 on the cell surface.(61,62) Nef also promotes the production and release of virions that are more infectious.(63,64) Effects of Nef on the PI3-K signaling cascade--which involves the guanine nucleotide exchange factor Vav, the small GTPases Cdc42 and Rac1, and p21-activated kinase PAK--cause marked changes in the intracellular actin network, promoting lipid raft movement and the formation of larger raft structures that have been implicated in T-cell receptor signaling.(65) Indeed, Nef and viral structural proteins colocalize in lipid rafts.(64,66) Two other HIV proteins assist Nef in downregulating expression of CD4.(67) The envelope protein gp120 binds CD4 in the endoplasmic reticulum, slowing its export to the plasma membrane,(68) and Vpu binds the cytoplasmic tail of CD4, promoting recruitment of TrCP and Skp1p.(Figure 5) These events target CD4 for ubiquitination and proteasomal degradation before it reaches the cell surface.(69)

Nef acts by several mechanisms to impair immunological responses to HIV. In T cells, Nef activates the expression of FasL, which induces apoptosis in bystander cells that express Fas,(70) thereby killing cytotoxic T cells that might otherwise eliminate HIV-1 infected cells. Nef also reduces the expression of MHC I determinants on the surface of the infected cell(71)(Figure 4) and so decreases the recognition and killing of infected cells by CD8 cytotoxic T cells. However, Nef does not decrease the expression of HLA-C,(72) which prevents recognition and killing of these infected cells by natural killer cells.

Nef also inhibits apoptosis. It binds and inhibits the intermediate apoptosis signal regulating kinase-1 (ASK-1)(73) that functions in the Fas and TNFR death signaling pathways and stimulates the phosphorylation of Bad leading to its sequestration by 14-3-3 proteins.(74)(Figure 4) Nef also binds the tumor suppressor protein p53, inhibiting another potiential initator of apoptosis.(75) Via these different mechanisms, Nef prolongs the life of the infected host cell, thereby optimizing viral replication.

Other viral proteins also participate in the modification of the environment in infected cells. Rev-dependent expression of Vpr induces the arrest of proliferating infected cells at the G2/M phase of the cell cycle.(76) Since the viral LTR is more active during G2, this arrest likely enhances viral gene expression.(77) 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.(78)(Figure 4) Intermittently, these herniations rupture, causing the mixing of soluble nuclear and cytoplasmic proteins. Either alterations in the lamina structure or the inappropriate mixing of cell cycle regulators that are normally sequestered in specific cellular compartments could explain the G2 arresting properties of Vpr.

New viral particles are assembled at the plasma membrane.(Figure 5) Each virion consists of roughly 1500 molecules of Gag and 100 Gag-Pol polyproteins,(79) two copies of the viral RNA genome, and Vpr.(80) 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.(81) 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 a natural inhibitor of HIV replication.(20,82) Recently this factor, initially termed CEM15/APOBEC3G, was identified(83) and shown to share homology with APOBEC1, an enzyme involved in RNA editing. Whether the intrinsic antiviral activity of CEM15 involves such an RNA editing function remains unknown. CEM15 is expressed in non-permissive but not in permissive cells and when introduced alone is sufficient to render permissive cells nonpermissive.

The Gag polyproteins are subject to myristylation,(84) and thus associate preferentially with cholesterol- and glycolipid-enriched membrane microdomains.(85) 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.(7)

The budding reaction involves the action of several proteins, including the "late domain"(86) sequence (PTAP) present in the p6 portion of Gag.(87)(Figure 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.(88,89) 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).(90) 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.

As the AIDS pandemic continues, advances in antiretroviral therapies have slowed its advance in the industrialized world, but have had little effect in developing countries. Because of its high rate of mutation, HIV is able to refine and optimize its interactions with various host proteins and pathways, thereby promoting its growth and spread. The virus ensures that the host cell survives until the viral replicative cycle is completed. Possibly even more damaging, HIV establishes stable latent forms that support the chronic nature of infection. Eradication of the virus appears unlikely until effective methods are developed to purge these latent viral reservoirs.

Basic science will clearly play a leading role in future attempts to solve the mysteries of viral latency and replication. A small-animal model that recapitulates the pathogenic mechanisms of HIV is sorely needed to study the mechanisms underlying viral cytopathogenesis. Virally induced cell death is not limited to infected targets but also involves uninfected bystander cells.(91) Murine cells support neither efficient virion assembly nor release of virions from the cell surface.(92) Currently, this defect represents a major impediment to the successful development of a rodent model of AIDS.

Proposed mechanisms for HIV killing of T cells include the formation of giant cell syncytia through the interactions of gp120 with CD4 and chemokine receptors,(93) the accumulation of unintegrated linear forms of viral DNA, the proapoptotic effects of the Tat,(94) Nef,(95) and Vpr(96) proteins, and the adverse effects conferred by the metabolic burden that HIV replication places on the infected cell.(97) Of note, expression of Nef alone as a transgene in mice recapitulates many of the clinical features of AIDS, including immunodeficiency and loss of CD4-positive cells.(98) All of these mechanisms suggest potential points of therapeutic intervention. Finally, future therapies will likely target viral proteins other than the reverse transcriptase, protease, and integrase enzymes. Clinical trials are already underway to study small molecules or short peptides that block the binding of HIV to cell-surface chemokine receptors or interfere with the machinery of viral-host cell fusion. Although not as advanced in development, small molecules have been found that block Tat transactivation(99) and Rev-dependent export of viral transcripts from the nucleus to the cytoplasm.(100) As a proof of principle, dominant-negative mutants of Tat, Rev, and Gag proteins have been shown to block viral replication. By increasing the number of antiviral compounds available to target different steps in the viral replicative cycle, in particular drugs that can be deployed in developing countries, research at the cellular level can serve to extend survival and to improve the quality of life for infected individuals, and to inhibit the spread of AIDS.

Warner C. Greene thanks Gary Howard and Stephen Ordway for editorial support, Robin Givens for administrative support, and the National Institutes of Health (R01 AI45234-02, R01 CA86814-02, P01 HD40543), the UCSF California AIDS Research Center (C99-SF-002), the James B. Pendleton Charitable Trust, and the J. David Gladstone Institutes for funding support.

B. Matija Peterlin thanks the National Institutes of Health (R01-AI38532, R01-AI46967, RO1-AI49104, and R01-AI51165-01) and the Universitywide AIDS Research Program (R00-SF-006) for funding support.


Introduction

The life cycle of retroviruses is arbitrarily divided into two distinct phases: the early phase refers to the steps of infection from cell binding to the integration of the viral cDNA into the cell genome, whereas the late phase begins with the expression of viral genes and continues through to the release and maturation of progeny virions (see Figure 1 for a schematic view of the retroviral life cycle). During the long journey from the cell surface to the nucleus, retroviruses will face multiple obstacles, since in addition to finding a path through the cytoplasm to the nucleus they have to cross two main barriers, the plasma and nuclear membranes, whilst at the same time avoiding or counteracting cellular defences that can interfere with many of these steps. The surge in Human Immunodeficiency Virus (HIV) research in order to identify new therapeutic targets has led to a better understanding of the retroviral life cycle. However, in comparison with the later events of retrovirus infection (for a review, see [1, 2]), early steps are still poorly understood (for reviews, see [3, 4]).

The retroviral life cycle. A schematic view of early and late stages of the retroviral replication cycle is represented. Examples of cellular factors interfering with early steps are indicated: Lv1/Ref1 CEM15, also known as APOBEC3G (apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G) Fv2 Fv1. The question marks indicates the exact step affected by the restriction factors has not precisely been determined. Lv1 and Ref1 block incoming particles before reverse-transcription whereas Fv1 and Fv2 act at a stage between reverse-transcription and integration. See text for detailed discussion. Abbreviations: RTC, reverse transcription complex PIC, pre-integration complex.

In the case of HIV entry, for example, while the mechanisms of receptor binding, conformational changes and fusion appear to be relatively well defined, the involvement of attachment molecules and the importance of lipid rafts in fusion or in recruitment of coreceptors remain uncertain. Similarly, though the molecular process of reverse transcription is well described, very little is known about the concurrent uncoating process. One of the most poorly understood steps is the trafficking of pre-integration complexes (PICs) from the cell surface to the vicinity of the nucleus, despite a growing body of knowledge arising from the study of other viral models such as adenoviruses (Ad) [5] or Herpes simplex viruses (HSV) [6]. Much has been learned regarding nuclear entry, but the cellular proteins involved are still unknown and the exact role of each viral component remains controversial [7]. Finally, the molecular mechanisms of integration, the last event of the early phase of retroviral life cycle, are now well understood, but the choice of target site remains mysterious. Thus, while certain of these steps have been characterized, we are still far from obtaining a complete picture of these processes.

Fully elucidating the early steps of retrovirus replication is therefore crucial not only for identifying new antiretroviral drugs, but also for improving the design of retroviral vectors for gene therapy. Cellular inhibitors that interfere with these steps can represent useful tools for better characterizing the molecular processes involved and, in this respect, the recent discovery of cellular factors that block the lentiviral cycle at an early stage in primates provides novel directions for AIDS research [8].

In this review, we will summarise our current understanding of the early steps of the retroviral cycle, focussing particularly on the most recent and controversial findings in the field.


Current status of gene therapy strategies to treat HIV/AIDS

Progress in developing effective gene transfer approaches to treat HIV-1 infection has been steady. Many different transgenes have been reported to inhibit HIV-1 in vitro. However, effective translation of such results to clinical practice, or even to animal models of AIDS, has been challenging. Among the reasons for this failure are uncertainty as to the most effective cell population(s) to target, the diffuseness of these target cells in the body, and ineffective or insufficiently durable gene delivery. Better understanding of the HIV-1 replicative cycle, host factors involved in HIV-1 infection, vector biology and application, transgene technology, animal models, and clinical study design have all contributed vastly to planning current and future strategies for application of gene therapeutic approaches to the treatment of AIDS. This review focuses on the newest developments in these areas and provides a strong basis for renewed optimism that gene therapy will have an important role to play in treating people infected with HIV-1.


HIV-1 Replication

In general terms, the replication cycle of lentiviruses, including HIV-1, closely resembles that of other retroviruses. 1 There are, however, a number of unique aspects of HIV replication for example, the HIVs and SIVs target receptors and coreceptors distinct from those used by other retroviruses. Lentiviruses encode a number of regulatory and accessory proteins not encoded by the genomes of the prototypical “simple” retroviruses. Of particular interest from the gene therapy perspective, lentiviruses possess the ability to productively infect some types of non-dividing cells. This chapter, while reiterating certain points discussed in Chapter 1, will attempt to focus on issues unique to HIV-1 replication.

The HIV-1 genome encodes the major structural and non-structural proteins common to all replication-competent retroviruses (Fig. 1, and Chapter 1). From the 5′- to 3′-ends of the genome are found the gag (for group-specific antigen), pol (for polymerase), and env (for envelope glycoprotein) genes. The gag gene encodes a polyprotein precursor whose name, Pr55 Gag , is based on its molecular weight. Pr55 Gag is cleaved by the viral protease (PR) to the mature Gag proteins matrix (also known as MA or p17), capsid (CA or p24), nucleocapsid (NC or p7), and p6. Two spacer peptides, p2 and p1, are also generated upon Pr55 Gag processing. The pol-encoded enzymes are initially synthesized as part of a large polyprotein precursor, Pr160 GagPol , whose synthesis results from a rare frameshifting event during Pr55 Gag translation. The individual pol-encoded enzymes, PR, reverse transcriptase (RT), and integrase (IN), are cleaved from Pr160 GagPol by the viral PR.

The envelope (Env) glycoproteins are also synthesized as a polyprotein precursor (Fig. 1). Unlike the Gag and Pol precursors, which are cleaved by the viral PR, the Env precursor, known as gp160, is processed by a cellular protease during Env trafficking to the cell surface. gp160 processing results in the generation of the surface (SU) Env glycoprotein gp120 and the transmembrane (TM) glycoprotein gp41. gp120 contains the determinants that interact with receptor and coreceptor, while gp41 not only anchors the gp120/gp41 complex in the membrane (Fig. 2), but also contains domains that are critical for catalyzing the membrane fusion reaction between viral and host lipid bilayers during virus entry. Comparison of env sequences from a large number of virus isolates revealed that gp120 is organized into five conserved regions (C1-C5) and five highly variable domains (V1-V5). The variable regions tend to be located in disulfide-linked loops. gp41 is composed of three major domains: the ectodomain (which contains determinants essential for membrane fusion), the transmembrane anchor sequence, and the cytoplasmic tail.

In addition to the gag, pol, and env genes, HIV-1 also encodes a number of regulatory and accessory proteins. Tat is critical for transcription from the HIV-1 LTR and Rev plays a major role in the transport of viral RNAs from the nucleus to the cytoplasm. Vpu, Vif, Vpr and Nef have been termed “accessory” or “auxiliary” proteins to reflect the fact that they are not uniformly required for virus replication. The functions of these very interesting proteins will be discussed in more detail at the end of this chapter.

HIV replication proceeds in a series of events that can be divided into two overall phases: “early” and “late” (Fig. 3). 1 Although some events occur in a concerted or simultaneous fashion, the replication cycle can be viewed most simply as proceeding in an ordered, step-wise manner. In this chapter, each step in virus replication will be considered additional information can be obtained from the more detailed reviews and primary references that are cited.


CONCLUSIONS

It is now clear that HIV and SIV prefer to infect activated memory CD4 + T cells that express CCR5 and that most of the T cells of this phenotype reside in the intestine and other mucosal sites. The recognition that progressive HIV and SIV infection is linked to immune activation, which in turn is linked to a leaky gut, has only recently focused intense interest on the effects of HIV and SIV infection on the intestinal epithelial barrier. The details of how infection and loss of intestinal CD4 + T cells leads to a “leaky gut” are unclear, but multiple avenues of investigation have begun to be explored. If it were possible to prevent or decrease the breakdown of the mucosal barrier through therapeutic means, it is possible that this could greatly slow AIDS disease progression, as appears to be the case in natural nonhuman primate hosts of SIV that are persistently infected, suffer acute loss of intestinal CD4 + T cells, but apparently do not have a leaky gut nor chronic immune activation and rarely progress to AIDS.


4.10.4: Replicative Cycle of HIV - Biology

Although the replicative life cycle of HIV within CD4 T cells is understood in molecular detail, less is known about how this human retrovirus promotes the loss of CD4 T lymphocytes. It is this cell death process that drives clinical progression to acquired immune deficiency syndrome (AIDS). Recent studies have highlighted how abortive infection of resting and thus nonpermissive CD4 T cells in lymphoid tissues triggers a lethal innate immune response against the incomplete DNA products generated by inefficient viral reverse transcription in these cells. Sensing of these DNA fragments results in pyroptosis, a highly inflammatory form of programmed cell death, that potentially further perpetuates chronic inflammation and immune activation. As discussed here, these studies cast CD4 T cell death during HIV infection in a different light. Further, they identify drug targets that may be exploited to both block CD4 T cell demise and the chronic inflammatory response generated during pyroptosis.


Molecular biology of the human immunodeficiency virus type 1

The immunodeficiency virus type 1 is a complex retrovirus. In addition to genes that specify the proteins of the virus particle and the replicative enzymes common to all retroviruses, HIV-1 specifies at least six additional proteins that regulate the virus life cycle. Two of these regulatory genes, tat and rev, specify proteins essential for replication. These proteins bind to specific sequences of newly synthesized virus RNA and profoundly affect virus protein expression. Tat and rev appear to be prototypes of novel eukaryotic regulatory proteins. These two genes may play a central role in regulating the rate of virus replication. Three other viral genes, vif, vpu, and vpr, affect the assembly and replication capacity of newly made virus particles. These genes may play a critical role in spread of the virus from tissue to tissue and from person to person. Our understanding of the contribution of each of the virus structural proteins and regulatory genes to the complex life cycle of the virus in natural infections is incomplete. However, enough insight has been gained into the structure and function of each of these components to provide a firm basis for rational antiviral drug development.—Haseltine, W. A. Molecular biology of the human immunodeficiency virus type 1. FASEB J. 5: 2349–2360 1991.


Vaccines and Anti-Viral Drugs for Treatment

Vaccines and anti-viral drugs can be used to inhibit the virus and reduce symptoms in individuals suffering from viral infections.

Learning Objectives

Give examples of treatments with anti-viral drugs

Key Takeaways

Key Points

  • Vaccines can boost an individual’s immune response and control viruses, such as Ebola and rabies, before they become deadly.
  • Anti-viral drugs inhibit the virus by blocking the actions of its proteins they are used to control and reduce symptoms for viral diseases.
  • Tamiflu can reduce flu symptoms by inhibiting the enzyme neuraminidase, which blocks the virus from spreading to uninfected cells.
  • Anti-HIV drugs inhibit and control viral replication at many different phases of the HIV replication cycle, so patients taking these drugs have a higher survival rate.
  • Viruses can develop resistance to individual anti-viral drugs.
  • The treatment of HIV involves a mixture of different drugs (fusion inhibitors, reverse transcriptase inhibitors, integrase inhibitors, and protease inhibitors) in a cocktail viruses have greater difficulty gaining resistance to multiple drugs.

Key Terms

  • virion: a single individual particle of a virus (the viral equivalent of a cell)
  • anti-viral drug: a class of medication, such as antibiotics, that inhibits the virus by blocking the actions of one or more of its proteins
  • Ebola virus: an extremely contagious virus of African origin that causes Ebola fever, spread through contact with bodily fluids or secretions of infected persons and by airborne particles

Vaccines and Anti-viral Drugs for Treatment

In some cases, vaccines can be used to treat an active viral infection. The concept behind this is that by giving the vaccine, immunity is boosted without adding more disease-causing virus. In the case of rabies, a fatal neurological disease transmitted via the saliva of rabies virus-infected animals, the progression of the disease from the time of the animal bite to the time it enters the central nervous system may be two weeks or longer. This is enough time to vaccinate an individual who suspects that they have been bitten by a rabid animaL their boosted immune response is sufficient to prevent the virus from entering nervous tissue. Thus, the potentially-fatal neurological consequences of the disease are averted the individual only has to recover from the infected bite. This approach is also being used for the treatment of Ebola virus, one of the fastest and most deadly viruses on earth. Transmitted by bats and great apes, this disease can cause death in 70–90 percent of infected humans within two weeks. Using newly-developed vaccines that boost the immune response in this way, there is hope that affected individuals will be better able to control the virus, potentially saving a greater percentage of infected persons from a rapid and very painful death.

Another way of treating viral infections is the use of antiviral drugs. These drugs often have limited success in curing viral disease, but in many cases, they have been used to control and reduce symptoms for a wide variety of viral diseases. For most viruses, these drugs can inhibit the virus by blocking the actions of one or more of its proteins. It is important that the targeted proteins be encoded by viral genes and that these molecules are not present in a healthy host cell. In this way, viral growth is inhibited without damaging the host. There are large numbers of antiviral drugs available to treat infections, some specific for a particular virus and others that can affect multiple viruses.

Antivirals have been developed to treat genital herpes (herpes simplex II) and influenza. For genital herpes, drugs such as acyclovir can reduce the number and duration of episodes of active viral disease during which patients develop viral lesions in their skin cells. As the virus remains latent in nervous tissue of the body for life, this drug is not curative, but can make the symptoms of the disease more manageable. For influenza, drugs like Tamiflu (oseltamivir) can reduce the duration of “flu” symptoms by one or two days, but the drug does not prevent symptoms entirely. Tamiflu works by inhibiting an enzyme (viral neuraminidase) that allows new virions to leave their infected cells. Thus, Tamiflu inhibits the spread of virus from infected to uninfected cells. Other antiviral drugs, such as Ribavirin, have been used to treat a variety of viral infections, although its mechanism of action against certain viruses remains unclear.

Tamiflu: (a) Tamiflu inhibits a viral enzyme called neuraminidase (NA) found in the influenza viral envelope. (b) Neuraminidase cleaves the connection between viral hemagglutinin (HA), also found in the viral envelope, and glycoproteins on the host cell surface. Inhibition of neuraminidase prevents the virus from detaching from the host cell, thereby blocking further infection.

Anti-HIV Drugs

By far, the most successful use of antivirals has been in the treatment of the retrovirus HIV, which causes a disease that, if untreated, is usually fatal within 10–12 years after infection. Anti-HIV drugs have been able to control viral replication to the point that individuals receiving these drugs survive for a significantly longer time than the untreated.

Anti-HIV drugs inhibit viral replication at many different phases of the HIV replicative cycle. Drugs have been developed that inhibit the fusion of the HIV viral envelope with the plasma membrane of the host cell (fusion inhibitors), the conversion of its RNA genome into double-stranded DNA (reverse transcriptase inhibitors), the integration of the viral DNA into the host genome (integrase inhibitors), and the processing of viral proteins (protease inhibitors).

HIV: HIV, an enveloped, icosahedral virus, attaches to the CD4 receptor of an immune cell and fuses with the cell membrane. Viral contents are released into the cell where viral enzymes convert the single-stranded RNA genome into DNA and incorporate it into the host genome.

When any of these drugs are used individually, the high mutation rate of the virus allows it to easily and rapidly develop resistance to the drug, limiting the drug’s effectiveness. The breakthrough in the treatment of HIV was the development of HAART, highly-active anti-retroviral therapy, which involves a mixture of different drugs, sometimes called a drug “cocktail.” By attacking the virus at different stages of its replicative cycle, it is much more difficult for the virus to develop resistance to multiple drugs at the same time. Still, even with the use of combination HAART therapy, there is concern that, over time, the virus will develop resistance to this therapy. Thus, new anti-HIV drugs are constantly being developed with the hope of continuing the battle against this highly fatal virus.


Question : Part A The Human Immunodeficiency Virus (HIV): The Human Immunodeficiency Virus (HIV): can have its replicative cycle slowed by the introduction of the chemical AZT into its environment. is a retrovirus. uses the enzyme reverse transcriptase to reproduce. uses RNA as its genetic material, not DNA. All of the above. Part B Which of the

Which of the following statements about proteins is FALSE?

Which of the following statements about proteins is FALSE?

Plants & animals store most of their excess energy in the form of protein molecules.
The information needed to make proteins is encoded in DNA genes.
Proteins are needed to synthesize all of the other biomolecules in your body.
Some signaling molecules (hormones & neurotransmitters) are proteins.
Many proteins act as enzymes, catalyzing chemical reactions inside living organisms.

shared by all life on Earth.
not redundant.
stored in the protein molecules in our cells.
unique to each species of a living organism.
different between plants and animals.

Assuming correct nucleotide base-pairing, a double-stranded DNA helix that is composed of 22% Guanine nucleotides will also have:

Assuming correct nucleotide base-pairing, a double-stranded DNA helix that is composed of 22% Guanine nucleotides will also have:


Watch the video: HIV Life Cycle. HHMI BioInteractive Video (June 2022).