VIRAL LIFE CYCLE

Lentiviruses have a characteristic conical core structure that distinguishes them from type C retroviruses. In addition to Gag, Pol, and Env, three to six additional proteins with diverse regulatory roles contribute to their genetic complexity.

Early Events

The main molecular events of HIV-1 (Figure 1.1) entry are now well understood, although the role of the viral envelope protein in cell death continues to be enigmatic.^59,60 The initial event in virus entry is the binding of the processed viral envelope glycoprotein (gp120) to the CD4 molecule on the cell surface. However, for years, it was known that introduction of human CD4 into nonhuman cells was insufficient to confer entry. The implicated second receptor was determined to be a group of chemokine co-receptors in 1996—CCR5 and CXCR4 predominated.⁶¹ Subsequently, great structural detail has been revealed. After CD4 binding, a conformational shift seems to occur within gp120, which exposes a chemokine co-receptor binding site.⁶² The dominant co-receptors used by HIV-1 are CXCR4 and CCR5. CXCR4-using (or X4) viruses efficiently infect T cells and are also referred to as T cell tropic. CCR5 (or R5) viruses efficiently infect cells of the monocyte/macrophage lineage and are referred to as macrophage tropic.

The limited exposure of the key surfaces within the critical intermediate structures that occur during gp120 binding and fusion is a main reason for the failure to generate neutralizing antibodies

FIGURE 1.1 The HIV-1 life cycle. Entry is illustrated in a temporal sequence beginning at upper left, with assembly and budding at the lower right. The pre-integration complex (PIC) is depicted as a U-shaped intermediate with integrase protein molecules, presumably multimers, at the termini.

capable of controlling the infection.⁶³ Binding to the co-receptor induces further conformational changes within gp120 that dislocate it from gp41 and expose the gp41 fusogenic domain,⁶⁴ an amino-terminal hydrophobic peptide that inserts into the cell membrane. The gp41 then collapses into a trimer-of-hairpins structure that brings the two membranes close and mediates fusion.⁶⁵ Chemokine co-receptor usage seems to be fundamental to lentiviral biology, because FIV also uses CXCR4.⁶⁶'⁶⁷ The recent identification of feline CD134⁶⁸ as the primary receptor rather than feline CD4 (or CD9⁶9) explains the additional tropism of FIV for B cells and CD8 cells. Humans with a homozygous mutation of the CCR5 gene that prevents the receptor from being presented at the cell surface are less likely to be infected by HIV, and if infected, they progress to AIDS less rapidly.^70-72

Cell-cell fusion mediated by gp120 (syncytium formation) and cell surface signal transduction triggered by gp120 can be shown to cause cell death in vitro; their roles in vivo remain unclear.⁷³ It is clear, however, that inhibition of entry can be used therapeutically. T-20 (enfurtivide) is an FDA-approved synthetic peptide that targets the amino-terminal region of gp41, which becomes exposed in the transient extended state.⁷⁴ This drug must be combined with other antiviral agents targeted at different stages of the life cycle or resistance develops rapidly.

Uncoating and Transport to the Nucleus

The uncoating, trafficking, and maturation steps that follow the entry of the viral core into the target cell cytoplasm constitute a poorly understood interval in the HIV-1 life cycle.

Controversy persists about the underlying mechanisms by which lentiviral PICs transit the nucleopore complexes of mitotically inactive cells.⁹² Proposed effectors have included peptide determinants in matrix,^93-100 Vpr,⁹⁶,¹⁰¹,¹⁰² and integrase.⁹⁸,¹⁰³ The central DNA flap^104-106 has also been implicated.¹⁰⁴,¹⁰⁵ Importin-7 has been suggested to mediate PIC translocation in semipermeabilized cell assays.¹⁰⁷ Countervailing views or reassessments exist for some of these proposals.⁹²,^108-111,¹¹² (See references¹¹¹,¹¹² for reviews.)

Integration

The determinants of subsequent intranuclear preintegration PIC trafficking are not understood at all.

The linear viral cDNA that results from reverse transcription has four principal fates: bona fide integration; formation of 1-LTR circles, probably by homologous recombination¹¹⁶; conversion to 2-LTR circles by the host cell nonhomologous DNA end-joining (NHEJ) pathway¹¹⁷; and autointegration to form defective DNAs.¹¹⁸ Only the linear form is a precursor to integration. Details of integrase catalysis have been reviewed elsewhere.¹¹⁹

There is some evidence that integration intermediates might trigger cell death under some circumstances (e.g., when DNA repair mechanisms are impaired). Early work by Temin and colleagues suggested that transient cytopathic effects correlated temporally with the transient accumulation of large numbers of unintegrated spleen necrosis virus DNA molecules.^120,121 More recently, the nonhomologous end joining (NHEJ) pathway has been implicated in completing the final stages of integration, and these studies have raised the possibility that integration intermediates can be toxic.¹²² The NHEJ pathway is the principal repair system for double-stranded DNA breaks in eukaryotic cells. Such breaks occur during lymphocyte V(D)J recombination. They are also generated by ionizing radiation and are proapoptotic: just a few double-strand DNA breaks can induce p53-dependent G1 arrest.¹²³ The principal components of this pathway are DNA-dependent protein kinase (DNA-PK), Ku, XRCC4 ligase, and DNA ligase IV.^124,125 DNA-PK, which is activated by dsDNA ends, participates in this repair along with the Ku proteins, although the exact role played by DNA-PK has not been established.

Retroviral reverse transcription also generates a double-stranded DNA with blunt ends. Before integration, two nucleotides are removed by integrase from each 3' strand of the linear molecule. The recessed 3' ends are then joined (the strand transfer reaction) to 5' DNA ends in cellular DNA via a direct attack by the 3' hydroxyl group.¹¹⁹

Daniel et al. found that retroviral vector integration was reduced in SCID cells and that high- titer infection induced apoptosis within approximately 12 h of infection.¹²² In contrast, integration­defective vectors did not trigger cell death. They concluded that DNA-PK is required to complete integration, presumably by repairing the DNA gaps. Subsequent reports have led to refinement and some contradiction of this scenario.^117,127,128 In one study, increased cell death was also observed in SCID cells, but only after transduction at quite high multiplicity of infection (MOI) and subse­quent cell division.¹²⁷ At an MOI of less than 1.0, transduction efficiency was actually higher in SCID cells than in control cells, and it proceeded normally in vivo in SCID mice brain without cell death, leading to the conclusion that DNA-PK and another DNA repair enzyme, PARP, are not essential for HIV-1 integration.¹²⁷ Similarly, Li et al. found that lower-titer HIV-1 vector infection proceeded normally in cells mutant for components of the NHEJ pathway.¹¹⁷ Similar to other studies, high-titer infections were found to be toxic in NHEJ-mutant cells, apparently by an apoptotic mechanism.

The roles of DNA-PK and other cellular repair mechanisms in integration and cell death remain uncertain. Its relevance to the actual disease pathology of lentiviruses is not clear, because normal human CD4⁺ T cells are fully competent for the NHEJ pathway.

Latency

The factors that influence PIC trafficking to particular integration sites after nuclear import are unknown, but they are of clinical importance since recent data established that HIV-1 integration sites are not randomly distributed in the genome. This, in turn, may influence the ability of latent proviruses to serve as sources of recrudescence from resting memory T cells. Postintegration proviral latency is an area of intense interest at present. Viral rebound invariably occurs after the discontinuation of HAART, generally within 2 weeks, and virus can be isolated from T cells in vitro during effective HAART'.¹²⁹¹³¹ The capacity to remain transcriptionally silent for extended durations is one factor that permits the virus to evade immune surveillance.¹³² The half-life of the latent reservoir in resting memory T cells is estimated to be approximately 4 years, indicating that eradication by decay on HAART is highly unlikely to be feasible.^133,134 Therefore, understanding the molecular mechanisms controlling integration site preference, silencing, and reactivation of the integrated HIV-1 provirus has important implications for understanding HIV disease pathogenesis and for therapy.^134,135 Chromatin possesses marked structural and functional heterogeneity, and extensive evidence demonstrates that retroviral transcription is strongly affected by the context of integration.^136-139 Epigenetic mechanisms such as histone deacetylation and DNA methylation play major roles in the silencing of viral promoters that occur progressively after integration.^140,141 Methylation-associated silencing is dependent on chromosomal position.¹⁴⁰

Recent studies established definitively that several retroviruses do not integrate randomly with respect to genomic loci. MLV and HIV-1 both integrate preferentially into transcriptionally active regions.^{113,114,142, 143} However, the placements of their proviruses seem to differ with respect to functional elements within the cellular transcription units. HIV-1 integration favors regions down­stream of promoters.^113,114,142 In contrast, MoMLV integrations show a predilection for promoter regions, favoring the 2.5 kb surrounding the transcriptional start site.¹¹⁴ In latently infected cells, a bias toward integration in heterochromatic regions, such as alphoid repeats, is also observed.¹⁴⁴ Integration site distributions will receive concentrated study with the newer methods available^113,114,142 because of the implications for HIV-1 latency^{134,136,144-146} and to address the problem of insertional mutagenesis in human gene therapy.¹⁴⁷

Cellular cofactors responsible for retroviral PIC trafficking or integration site specificity have not been identified.¹⁴⁸ Lentiviral integrase proteins localize to cell nuclei and associate with chro­matin when expressed in the absence of other viral components.^98,149-153 These observations have led to efforts to identify a nuclear localization signal (NLS) and to implicate this protein in preintegration complex nuclear translocation. However, nuclear/chromatin targeting of lentiviral integrases now seems to be due not to any nuclear localization signal but rather to interaction with the transcriptional coactivator LEDGF∕p75.^153,154 This interaction, which is lentivirus specific, is clearly not needed for nuclear import of the PIC,¹⁵³ but there is interest in its potential involvement in integration site choice. Interactions of integrase or PIC DNA with other cellular factors have been demonstrated as well.^148,155,156 Examples are BAF,^146,157,158 HMGa1 protein,^159-162 INI1,^155,163 Ku70 and Ku80,¹¹⁷ the aforementioned DNA-PK,¹²² and hRad18.¹⁶⁴ Some are known to enhance in vitro integration reactions,^{155,157,165-169} as does the viral nucleocapsid (NC) protein,^159,169 although the significance in each case to the reaction in the natural life cycle of HIV-1 is not clear. INI1, an essential constituent of the human SWI/SNF chromatin remodeling machine, is incorporated into HIV-1 but not HIV-2, SIV, or other retroviral particles¹⁷⁰ and it seems to be required for efficient HIV-1 budding.¹⁶³ INH also stimulates the DNA-joining activity of HIV-1 integrase in vitro, and a role in targeting the viral DNA to active genes has been hypothesized.¹⁵⁵ Moreover, incoming HIV-1 and murine leukemia virus (MLV) PICs were found to trigger export of INI1 and the nuclear body constituent PML (promyelocytye leukemia protein) to the cytoplasm, which may be part of a cellular antiviral response.¹⁶⁸

Late Events

Two small RNA-binding regulatory proteins, Tat and Rev, govern viral mRNA production. HIV-1 transcription is minimal in the absence of Tat, because transcriptional elongation does not occur efficiently. Tat binds to an RNA stem-loop (TAR) in the 5 end of the nascent viral transcript. The N-terminal domain of Tat interacts with CyclinT1, which, in turn, recruits the cyclin-dependent kinase 9 (CDK-9), leading to hyperphosphorylation of the large subunit of the RNA polymerase II.¹⁷¹,¹⁷² The result is increased elongation efficiency of the Pol II complex.¹⁷²,¹⁷³ Tat’s role in modulating transcription seems to extend to the cellular Iranscriptome^174-176; roles in regulating apoptosis,¹⁷⁷ and class I major histocompatibility complex (MHC) molecule expression have also been reported.¹⁷⁸ Rev binds to a secondary RNA structure in the env region, the Rev response element, and enables the virus to solve a central problem: export of its unspliced genomic RNA against the normal cellular checkpoint that prevents export of intron-containing messages.^179,180 The Nef and Vpr proteins have been reported to play a wide variety of roles in the viral life cycle and to be implicated in cell death; these proteins will be reviewed in Chapters 15 and 14, respectively.

Maturation of the virion via protease cleavage is synchronous with budding. Recently, viral budding has been shown to employ a complex cellular machinery involved in protein sorting and membrane budding at the multivesicular body¹⁸¹; this system is involved in the intracellular budding of HIV-1 virions that is observed in macrophages.^182,183 The HIV-1 protease cleaves the Gag/Pol precursor into virion proteins and was also implicated in cleaving cellular proteins such as caspase- 8,¹⁸⁴ a cell death mechanism that will be reviewed in Chapter 17.