Virus–host interactions (2003)

Introduction The primary goal of a virus is the replication of its genome in an appropriate host cell and the production of progeny virions for the infection of new target cells. This effort is commonly met by antiviral responses by the host organism, which in most cases abolishes or limits virus infections. Viruses have developed different strategies to overcome these restrictions, some causing long-lasting chronic infections, others replicating in fast, lytic cycles. However, all viruses depend to a large degree on specific host factors, from the recognition of specific cell-surface receptors required for virus entry into a target cell to the packaging of cellular factors into virions. HIV penetrates target cells through fusion with the host plasma membrane. This is followed by partial uncoating and reverse transcription of the viral RNA, and subsequent integration of the double-stranded complementary DNA into the host genome. The integrated provirus then serves as a template for the synthesis of viral proteins, which ultimately assemble into progeny virions that are released from the infected host cell. We are far from understanding all of the complex virus–cell interactions that take place during the HIV life cycle, however, our current knowledge suggests that such interactions occur at virtually every step of virus replication. The past few years have brought rapid progress in the identification and characterization of novel host factors supporting HIV replication. In particular, the recent identification of chemokine receptors as HIV co-receptors has significantly advanced our understanding of HIV cell tropism and entry. Less well defined, however, is the extent to which cellular factors are involved in post-entry events required for the establishment of a productive HIV infection. Primate immunodeficiency viruses, including HIV-1, are characterized by the presence of a number of viral accessory genes that encompass vif, vpr, vpx, vpu, and nef. The vif, vpr, and nef genes are expressed in most HIV-1, HIV-2 and SIV isolates. In contrast, the vpu gene is found exclusively in HIV-1 and some SIV isolates. The vpx gene, on the other hand, is not found in HIV-1 isolates but is common to HIV-2 and most SIV isolates. Defects in accessory genes are frequently not correlated with a detectable impairment of virus replication in continuous cell lines, in contrast to primary cell types, which more closely reflect the in-vivo situation. However, it becomes increasingly clear that these proteins exert important functions in their relevant target cells in vivo, and most if not all of the HIV accessory proteins seem to exert multiple independent functions. For most of the HIV accessory and regulatory proteins the precise biochemical mechanisms are still under investigation, however, there is increasing evidence to suggest that none of the HIV accessory or regulatory proteins has catalytic activity on its own. Rather, they appear to function as adapter molecules that connect other viral or cellular factors to various cellular pathways. The goal of the current review is to summarize recent progress in the study of virus–host interactions involving the viral Tat, Rev, and Vif proteins. Vif: overcoming a cellular restriction to virus replication Ever since its identification as a virus infectivity factor in 1987 [1,2], the vif gene, which initially was termed ‘sor’ or ‘A’ [2], was the subject of intense research. However, despite significant progress over the past 16 years, the biochemical function of Vif remains largely unclear. A number of studies have attempted to reveal the biochemical function of Vif, but have produced somewhate conflicting results, and many reported observations regarding Vif remain controversial. It is now generally accepted, however, that Vif-defective viruses are capable of entering target cells but encounter an early block in virus replication before integration of the viral genome into the host genome. It is also now generally accepted that Vif is packaged into viral particles, although its functional significance is still under investigation (Fig. 1, step 2). Nevertheless, a recent analysis of the molecular defect in Vif-defective HIV virions did not detect any apparent abnormalities in Vif-defective virions, except for the lack of Vif [3]. There has been significant discussion regarding the number of Vif molecules packaged into virions, with estimates ranging from less than one molecule per virion to as many as 100 molecules of Vif per virion. In general, studies employing virus from chronically infected cells or from stable cell lines reported lower amounts of virus-associated Vif than studies using virus from productively infected cells [4].Fig. 1.: Regulation of virus infectivity by Vif. Cells restrictive for the replication of Vif-defective HIV express the cytidine deaminase APOBEC3G. In the presence of Vif, intracellular de novo synthesis of APOBEC3G appears to be inhibited through a post-transcriptional mechanism (1). Vif is packaged into virions and associates with the viral nucleoprotein complex. Importantly, Vif inhibits the packaging of APOBEC3G into virions (2) thus increasing virus infectivity. Such viruses can enter a target cell (3), integrate into its host genome and produce infectious progeny virions (4,5). In contrast, the absence of Vif results in the packaging of APOBEC3G into virus particles (6). Such virions are capable of penetrating a target cell and initiate minus-strand cDNA synthesis [(−)-cDNA]. However, APOBEC3G causes hypermutation of the viral (−)-cDNA, resulting in the conversion of deoxycytidine to deoxyuridine (7). Deoxyuridine residues in the viral cDNA can be targeted by uracil-DNA glycosylase, which could lead to endonucleolytic cleavage by endonucleases present in the target cell (8). In an alternative model, hypermutated cDNA enters the nucleus (9), and integrates into the host genome but results in the production of defective or aberrant viral proteins (4). This can lead to an impairment of virus assembly or can result in the assembly of non-infectious viruses (5).There is increasing evidence that the packaging of Vif into virus particles is functionally relevant. First, Vif packaging is specific and is mediated through an interaction with viral genomic RNA [5–7]. In addition, virus-associated Vif interacts with Gag or Gag–Pol precursor molecules [8] (K. Strebel, unpublished observation), and is stably associated with the viral nucleoprotein complex [5,9,10]. Finally, virus-associated Vif is proteolytically cleaved by the viral protease at a conserved sequence located near the C-terminus of the protein (residue 150 in HIV-1 Vif) [11]. Intravirion processing of Vif is not restricted to HIV-1 Vif, but was also observed for HIV-2 and some SIV Vif variants (K. Strebel, unpublished observation). Of note is the fact that mutations at or near the processing site that affect Vif processing were also found to affect Vif function, whereas mutations that did not affect Vif processing did not affect Vif function [11]. Interestingly, Vif processing separates the more conserved N-terminus of Vif from its highly variable C-terminal domain, which presumably contains a multimerization domain [12,13] as well as the immunodominant epitope. Removal of the C-terminal region of Vif induces a conformational change in Vif (K. Strebel, unpublished observation) that may expose new functional domains in the protein. Vif is a highly insoluble protein with a strong tendency to aggregate. This characteristic has hampered efforts to purify Vif protein for structural analyses. Therefore, in contrast to most other HIV-encoded proteins, there are currently no structural data available for Vif. Attempts to define functional domains in Vif through biochemical analyses have demonstrated that residues throughout the protein are important for Vif function [14,15]. The only exception appears to be the very C-terminal region of Vif, as evidenced by the fact that a natural variant lacking the C-terminal 19 amino acids was found to be biologically active [16]. These results suggest that Vif contains multiple functional domains that might be important for the interaction with viral or cellular proteins. It is tempting to speculate that Vif, like most other HIV regulatory and accessory proteins, functions as a molecular adapter to connect otherwise unrelated viral or cellular mechanisms. However, the functionally relevant ligands of Vif have not yet been defined. Also, the search for negative trans-dominant Vif variants, which would support such a model, has so far been unsuccessful [17]. Vif functions in a host cell-specific manner. Accordingly, Vif-defective viruses produced in permissive host cells are unrestricted and are thus capable of infecting both permissive and non-permissive target cells. In contrast, Vif-defective viruses grown in non-permissive host cells were unable to infect permissive and non-permissive target cells alike [18–24]. This suggests that host factors play a significant role in restricting virus replication. A number of host factors have been identified as possible targets of Vif. These include vimentin [10], HP68 [25], Hck [26], sp140 [27], and CEM15 [28]. Importantly, the expression of Hck and CEM15 appeared to be associated with the inhibition of viral infectivity in a Vif-dependent manner [26,28]. However, only CEM15 expression was closely linked to non-permissive cellular phenotypes, and, unlike Hck, did not seem to have additional effects on virus production. CEM15 thus represents to date the most promising factor that fits most, if not all, of the characteristics required of a protein associated with Vif-dependent host cell restriction: it appears to be expressed exclusively in non-permissive cells, and furthermore, expression in permissive cells was found to inhibit virus infectivity in the absence but not in the presence of Vif [28,29]. CEM15 is identical to APOBEC3G and is a member of the family of cytidine deaminases [30]. APOBEC3G was found to have DNA cytidine deaminase activity in vitro [31]. The physiological substrates of APOBEC3G are not currently known; however, the tissue-specific expression of APOBEC3G suggests a role in growth or cell cycle control [30]. Interestingly, mutations in the catalytic site of APOBEC3G were associated both with decreased cytidine deaminase activity and a loss of the inhibitory activity of APOBEC3G on HIV replication [32,33]. Most recently, four research groups almost simultaneously reported that APOBEC3G induces the hypermutation of newly synthesized HIV DNA [32–35], thus providing a plausible explanation for the antiviral activity of APOBEC3G. All four reports noted a significant increase in G to A mutations in the viral genome (Fig. 1, step 6). As APOBEC3G-induced C to U mutations will result in a G to A mutation on the complementary strand, the observed G to A changes are most consistent with hypermutation of the minus-strand DNA rather than viral genomic RNA. Consistent with this, a direct analysis of RNA from Vif-defective virions produced in non-permissive H9 cells without endogenous reverse transcription did not reveal G to A hypermutation, whereas an analysis of cDNA derived by endogenous reverse transcription of the same viruses revealed G to A hypermutation [32,35]. This suggests that all the factors involved in hypermutation of HIV cDNA are present in virions from non-permissive cells. Consistent with these results, APOBEC3G was found to be packaged into HIV-1 virions [28,29,36]. Details of how APOBEC3G interferes with the replication of Vif-defective HIV have yet to be investigated. However, the observation that APOBEC3G induced defects in Vif-defective virions that became more and more severe with each step of virus replication has led to the proposal that the APOBEC3G-induced inhibition of HIV replication is the cumulative result of multiple defects [33]. It is possible that hypermutation of proviral DNA induces aberrant stop codons or mutates viral proteins (Fig. 1, step 9). More likely, however, seems to be the possibility that deaminated minus strand DNA is targeted by uracil-DNA glycosylase, which could result in the degradation of viral DNA via a uracil-based excision pathway [32,34], and could thus lead to abortive infection typical of Vif-defective viruses (Fig. 1, step 8). The mechanism by which Vif prevents hypermutation by APOBEC3G is still under investigation. Data from several groups, including our own, suggested that Vif inhibits the packaging of APOBEC3G into HIV-1 particles in a dose-dependent manner [29,36] (Fig. 1, step 2). Moreover, the inhibition of APOBEC3G packaging requires biologically active Vif protein, whereas a series of biologically inactive Vif variants, including point mutants and in-frame deletions of larger portions of Vif, did not affect APOBEC3G packaging (Fig. 1, step 6). The inhibition of APOBEC3G packaging by wild-type Vif protein is paralleled by a reduction in the cell-associated expression levels of APOBEC3G protein [29]. Interestingly, Vif did not affect the expression level of APOBEC3G messenger RNA [28,29], suggesting that Vif affects APOBEC3G protein expression via a post-transcriptional mechanism (Fig. 1, step 1). A major focus of future research will undoubtedly be how Vif blocks APOBEC3G-induced deamination of viral cDNA. This should lead to a detailed understanding of Vif function, and may reveal novel targets for antiviral therapy. Tat: making the connections Because of its crucial role in activating viral gene transcription, the HIV Tat protein has been a focus of HIV research for many It is now well accepted that Tat functions as a molecular of the cellular transcription to the viral RNA to the of transcription by the RNA complex. Tat is a protein of amino acids that is expressed from a RNA early during HIV replication. Tat contains several functional residues encompass the domain or domain, whereas the domain located residues and is required for RNA as well as the of Tat In addition, the C-terminal domain of Tat has been in the of HIV-1 through a direct interaction with the In the absence of Tat, transcription from the HIV-1 RNA that include the region In contrast, the expression of Tat results in the production of RNA and in gene expression The of in the absence of Tat is most by the of RNA transcription to the viral and as was initially by an activity of (Fig. step 1). There is a complex the transcription factor and negative transcription factors factor and the negative factor complex A factor in the of RNA is the of its domain of the with of the RNA whereas of the the of the complex of the is by a activity (Fig. step The which was identified as the of the complex was identified as the can with different including and However, Tat was found to into the complex in the of from the HIV (Fig. step of region RNA. residues are to in the by A factor in the of transcription from the by RNA is the of its domain of the with whereas the of the complex. 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Tat was found to the production of such as growth factor or in the and in cells and at one the other hand, Tat was found to the gene in infected primary suggesting that in cell types, Tat expression may to cell of these effects may be by an interaction of Tat with specific cell-surface receptors that the of cellular pathways. However, some of these effects may also be by Tat into cells. Tat was found to to and in the of cells, and activating a pathway but not the of Tat was found to reverse activity during the of viral replication and to increase viral presumably by the synthesis of viral DNA Finally, Tat was found to RNA activity and to the of RNA viral RNA, although the significance of observation for the in-vivo function of Tat remains to be viral RNA in HIV place from a located the The resulting primary functions both as genomic RNA and as for the expression of the and of genes of however, requires of the primary resulting in a complex of or RNA. the Tat, Rev, and which are to the the of or requires the activity of The protein contains an RNA that to a as the located in the gene (Fig. step 1). The same in also as a which is required for the of from the to the The the direct of the protein to the factor which targets the protein complex to the nucleus (Fig. step 2). 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It also remains to be if and how the interaction of Vif with viral RNA, as well as viral and cellular proteins, is to the of Vif to viral infectivity.