Lessons from the cat: feline immunodeficiency virus as a tool to develop intervention strategies against human immunodeficiency virus type 1.
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AIDS Vaccines↗
Biomedical subjects
M H Malim
Publications and source records attributed to M H Malim.
Evidence for a newly discovered cellular anti-HIV-1 phenotype.
Animal cells have developed many ways to suppress viral replication, and viruses have evolved diverse strategies to resist these. Here we provide evidence that the virion infectivity factor protein of human immunodeficiency virus type 1 (HIV-1) functions to counteract a newly discovered activity in human cells that otherwise inhibits virus replication. This anti-viral phenotype is shown by human T cells, the principal in vivo targets for HIV-1, and, based on our present understanding of virion infectivity factor action, is presumed to act by interfering with a late step(s) in the virus life cycle. These observations indicate that the inhibition of virion infectivity factor function in vivo may prevent HIV-1 replication by 'unmasking' an innate anti-viral phenotype.
Animals↗
Interaction of the human immunodeficiency virus type 1 Vpr protein with the nuclear pore complex.
The Vpr protein of human immunodeficiency virus type 1 (HIV-1) performs a number of functions that are associated with the nucleus. Vpr enhances the nuclear import of postentry viral nucleoprotein complexes, arrests proliferating cells in the G2 phase of the cell cycle, and acts as a modest transcriptional activator. For this paper, we have investigated the nuclear import of Vpr. Although Vpr does not encode a sequence that is recognizable as a nuclear localization signal (NLS), Vpr functions as a transferable NLS both in somatic cells and in Xenopus laevis oocytes. In certain contexts, Vpr also mediates substantial accumulation at the nuclear envelope and, in particular, at nuclear pore complexes (NPCs). Consistent with this, Vpr is shown to interact specifically with nucleoporin phenylalanine-glycine (FG)-repeat regions. These findings not only demonstrate that Vpr harbors a bona fide NLS but also raise the possibility that one (or more) of Vpr's functions may take place at the NPC.
Biological Transport↗
The HIV-1 Rev protein.
The nuclear export of intron-containing HIV-1 RNA is critically dependent on the activity of Rev, a virally encoded sequence-specific RNA-binding protein. Rev shuttles between the nucleus and the cytoplasm and harbors both a nuclear localization signal and a nuclear export signal. These essential peptide motifs have now been shown to function by accessing cellular signal-mediated pathways for nuclear import and nuclear export. HIV-1 Rev therefore represents an excellent system with which to study aspects of transport across the nuclear envelope.
Alternative Splicing↗
Differential regulation of CXCR4 and CCR5 endocytosis.
The chemokine receptors CCR5 and CXCR4 are major co-receptors/receptors for the CD4-dependent and CD4-independent entry of human and simian immunodeficiency viruses. The chemokines that bind and activate these receptors can inhibit the entry of viruses that use the respective co-receptor molecules. Chemokine-induced co-receptor internalisation is a significant component of the mechanism through which chemokines inhibit virus entry. CXCR4 internalisation is induced by the CXCR4 ligand stromal cell derived factor-1 (SDF-1), phorbol esters and, in T cells, cellular activation. Here we show that CXCR4 endocytosis can be mediated through either one of two distinct internalisation signals. A COOH-terminal serine rich domain is required for ligand- but not phorbol ester- induced CXCR4 internalisation. However, a Ser/IleLeu motif, similar to that required for the endocytosis of CD4 and the T cell receptor/CD3 complex, is required for phorbol ester-induced, but not ligand-induced, CXCR4 endocytosis. By contrast, CCR5 internalisation is induced by the beta-chemokine RANTES but not by phorbol esters. CCR5 lacks the Ser/IleLeu sequence required for phorbol ester-induced uptake of CXCR4. Together these results indicate that distinct mechanisms can regulate CXCR4 and CCR5 endocytosis and trafficking.
Amino Acid Sequence↗
Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kappaB control.
Members of the NF-kappaB/Rel and inhibitor of apoptosis (IAP) protein families have been implicated in signal transduction programs that prevent cell death elicited by the cytokine tumor necrosis factor alpha (TNF). Although NF-kappaB appears to stimulate the expression of specific protective genes, neither the identities of these genes nor the precise role of IAP proteins in this anti-apoptotic process are known. We demonstrate here that NF-kappaB is required for TNF-mediated induction of the gene encoding human c-IAP2. When overexpressed in mammalian cells, c-IAP2 activates NF-kappaB and suppresses TNF cytotoxicity. Both of these c-IAP2 activities are blocked in vivo by coexpressing a dominant form of IkappaB that is resistant to TNF-induced degradation. In contrast to wild-type c-IAP2, a mutant lacking the C-terminal RING domain inhibits NF-kappaB induction by TNF and enhances TNF killing. These findings suggest that c-IAP2 is critically involved in TNF signaling and exerts positive feedback control on NF-kappaB via an IkappaB targeting mechanism. Functional coupling of NF-kappaB and c-IAP2 during the TNF response may provide a signal amplification loop that promotes cell survival rather than death.
Apoptosis↗
HIV-1 infection of non-dividing cells: evidence that the amino-terminal basic region of the viral matrix protein is important for Gag processing but not for post-entry nuclear import.
Human immunodeficiency virus type-1 (HIV-1) is able to infect non-dividing cells such as tissue macrophages productively because post-entry viral nucleoprotein complexes are specifically imported into the nucleus in the absence of mitosis. Although it has been proposed that an amino-terminal region of the viral matrix (MA, p17Gag) protein harbors a basic-type nuclear localization sequence (NLS) that contributes to this process, utilization of three distinct nuclear import assays failed to provide any direct supporting evidence. Instead, we found that disruption of this region (26KK-->TT) reduces the rate at which the viral Gag polyprotein (p55Gag) is post-translationally processed by the viral protease. Consistent with the fact that appropriate proteolytic processing is essential for efficient viral growth in all cell types, we also show that the 26KK-->TT MA mutation is equivalently deleterious to the replication of a primary macrophage-tropic viral isolate in cultures of non-dividing and dividing cells. Taken together, these observations suggest that proteins other than MA supply the NLS(s) that enable HIV-1 to infect non-dividing cells.
Amino Acid Sequence↗
The Vif and Gag proteins of human immunodeficiency virus type 1 colocalize in infected human T cells.
The Vif protein of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses is required for efficient replication in primary cells and certain immortalized cell lines in vitro and, in all likelihood, for the establishment of pathogenic infections in vivo. Current hypotheses concerning Vif's mechanism of action posit that it operates in virus-expressing cells during virion assembly, budding, or maturation such that released virions are modified in a manner that enables them to undergo productive infection in subsequent viral challenges. To gain further insight into the mechanism of action of lentivirus Vif proteins, we have performed a variety of in situ localization and biochemical fractionation studies using cells in which Vif is essential for efficient replication. Double-label immunofluorescence analyses of cells productively infected with HIV-1 or feline immunodeficiency virus revealed dramatic patterns of colocalization between Vif and the virally encoded Gag proteins. Subcellular fractionations of human T cells expressing HIV-1 Vif performed in the absence of any detergent demonstrated that greater than 90% of Vif is associated with cellular membranes. Additional purification using a continuous density gradient indicated that the majority of the membrane-bound Vif copurifies with the plasma membrane. Taken together, these observations suggest that lentivirus Vif and Gag proteins colocalize at the plasma membrane as virion assembly and budding take place. As a result, Vif is able to exert its modulatory effect(s) on these late steps of the virus life cycle.
Animals↗
Human placental alkaline phosphatase as a marker for gene expression.
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Alkaline Phosphatase↗
Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 Rev.
The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of late HIV-1 mRNAs. This function is dependent on the mutationally defined Rev activation domain, which also forms a potent nuclear export signal. Transcription factor IIIA (TFIIIA) binds to 5S rRNA transcripts and this interaction has been proposed to play a role in the efficient nuclear export of 5S rRNA in amphibian oocytes. Here it is reported that amphibian TFIIIA proteins contain a sequence element with homology to the Rev activation domain that effectively substitutes for this domain in inducing the nuclear export of late HIV-1 mRNAs. It is further demonstrated that this TFIIIA sequence element functions as a protein nuclear export signal in both human cells and frog oocytes. Thus, this shared protein motif may play an analogous role in mediating the nuclear export of both late HIV-1 RNAs and 5S rRNA transcripts.
Amino Acid Sequence↗
Human immunodeficiency virus type 1 Vif does not influence expression or virion incorporation of gag-, pol-, and env-encoded proteins.
The Vif protein of human immunodeficiency virus type 1 is required for productive replication in peripheral blood lymphocytes and a limited number of immortalized T-lymphoid lines (nonpermissive cells). In contrast, Vif is fully dispensable for virus replication in other T-cell lines (permissive cells). Because the infection phenotype of released virions is determined by producer cells and by the presence of Vif in those cells, we have analyzed the protein contents of purified viral particles in an attempt to define compositional differences that could explain the infection phenotype. Surprisingly, we were unable to discern any Vif- or cell-type-dependent quantitative or qualitative difference in the Gag, Pol, and Env proteins of virions or virus-producing cells that correlates with virus infectivity. We were, however, able to demonstrate that Vif itself is present in virions and, using semiquantitative Western blotting (immunoblotting), that there is an average of 30 to 80 molecules of Vif incorporated into each virion. Importantly, parallel analyses of total lysates of the producer cells revealed that the cell-associated expression levels of Vif are close to those of the Gag proteins. Given the dramatically higher abundance of Vif in cells than in virions, we speculate that Vif exerts its principal activity during the processes of virus assembly and budding and that this function could be of a structural-conformational nature.
Gene Deletion↗
Nuclear transport of human immunodeficiency virus type 1, visna virus, and equine infectious anemia virus Rev proteins: identification of a family of transferable nuclear export signals.
The human immunodeficiency virus type 1 Rev trans activator binds directly to unspliced viral mRNA in the nucleus and activates its transport to the cytoplasm. In additon to the sequences that confer RNA binding and nuclear localization, Rev has a carboxy-terminal region, the activation domain, whose integrity is essential for biological activity. Because it has been established that Rev constitutively exits and reenters the nucleus and that the activation domain is required for nuclear exit, it has been proposed that Rev's activation domain is a nuclear export signal (NES). Here, we used microinjection-based assays to demonstrate that the activation domain of human immunodeficiency virus type 1 Rev imparts rapid nuclear export after its transfer to heterologous substrates. NES- mediated export is specific, as it is sensitive both to inactivation by missense mutation and to selective inhibition by an excess of the wild-type, but not mutant, activation domain peptide. Examination of the Rev trans activators of two nonprimate lentiviruses, visna virus and equine infectious anemia virus, revealed that their activation domains are also potent NESs. Taken together, these data demonstrate that nuclear export can be determined by positively acting peptide motifs, namely, NESs, and suggest that Rev proteins activate viral RNA transport by providing export ribonucleoproteins with specific information that targets them to the cytoplasm.
Amino Acid Sequence↗
The human immunodeficiency virus type 1 Vif protein modulates the postpenetration stability of viral nucleoprotein complexes.
The vif gene of human immunodeficiency virus type 1 is absolutely required for productive infection of primary cells derived from human blood and certain immortalized T lymphoid cells, for example, H9. Cells with this restrictive phenotype are termed nonpermissive, whereas cell lines in which vif-deficient virus can replicate efficiently are known as permissive. In this paper, we describe experiments in which virus stocks derived from single-cycle infections of strictly nonpermissive H9 cells were used to determine the fate of vif-deficient infections. By PCR-based approaches, it was found that Vif has no significant impact on the biosynthetic capability of the virion reverse transcriptase in infected C8166 T cells. Specifically, the initial appearance of all DNA species up to and including initiated second (plus) strands as well as the early accumulation of these replicative intermediates is equivalent for wild-type and vif-deficient infections. However, whereas these viral DNAs are stably maintained in wild-type infections and can proceed to establish proviruses, they are largely degraded by the later time points of vif-deficient infections and, as a result, are prevented from forming proviruses. Subcellular fractionation analyses indicated that the majority of viral DNA is localized to the nucleus within 2 h of infection and that the turnover of reverse transcripts that occurs in these vif-deficient infections presumably takes place in the nucleus. Given that the ultimate infection phenotype of the virions is determined during virus production, we propose that Vif is required for an aspect of virus assembly and/or maturation that endows penetrating viral nucleoprotein cores with the ability to mature into functional preintegration complexes that can proceed to provirus establishment. In contrast, viruses that are produced in the absence of Vif give rise to nucleoprotein complexes that disassemble prematurely in challenged cells and fail to protect their RNA/DNA contents from nucleolytic destruction.
Cell Nucleus↗
The human T-cell leukemia virus type 1 posttranscriptional trans-activator Rex contains a nuclear export signal.
The Rex protein of human T-cell leukemia virus type 1 is required for the nuclear export of unspliced viral mRNA and, therefore, for virus replication. In this manuscript, we demonstrate that Rex shuttles between the nucleus and the cytoplasm and that its activation domain constitutes a nuclear export signal that specifies efficient transport to the cytoplasm. These findings are consistent with a model for Rex-mediated trans-activation in which Rex-viral mRNA complexes are targeted for nuclear export by the direct action of the activation domain.
Amino Acid Sequence↗
Complementation of vif-defective human immunodeficiency virus type 1 by primate, but not nonprimate, lentivirus vif genes.
The productive infection of many susceptible human cells, including lymphocytes and macrophages derived from peripheral blood, by the pathogenic lentivirus human immunodeficiency virus type 1 requires expression of the virally encoded vif (for virion infectivity factor) gene. Interestingly, this gene appears to have been conserved among all of the lentiviruses of primates and almost all of the lentiviruses of nonprimates. Using T cells constitutively expressing vif genes derived from diverse sources and virus replication assays, we show that the vif gene of a second primate lentivirus, simian immunodeficiency virus from macaques, complements vif-defective human immunodeficiency virus type 1 but that those of three distinct nonprimate lentiviruses do not. Although the molecular basis for Vif function has yet to be defined, the potential implications of this noted restriction of vif complementarity are discussed.
Animals↗
The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm.
The HIV-1 Rev protein is a nuclear trans-activator essential for the transport of unspliced viral transcripts to the cytoplasm. In this paper we demonstrate that Rev, rather than being confined to the nucleus, is constantly shuttling between the nucleus and the cytoplasm. We also show that inactivation of Rev's leucine-rich activation domain generates mutant proteins that not only fail to induce the nuclear export of viral transcripts but are also unable to enter the cytoplasm. On the basis of this correlation, we propose that Rev activates viral mRNA transport by directly binding to these RNAs and translocating, with them, to the cytoplasm. In addition, these results also identify, for the first time, a peptide sequence that is important for nuclear export.
Amino Acid Sequence↗
Sequence requirements for Rev multimerization in vivo.
Multimerization of the human immunodeficiency virus type 1 (HIV-1) Rev protein is believed to be critical to its biological activity. However, the precise protein sequence requirements for Rev multimerization in vivo, and whether multimerization is facilitated by specific RNA binding or vice versa, has remained controversial. In this report, we describe a sensitive in vivo assay for the multimerization of HIV-1 Rev on its cognate RRE primary RNA binding site. Using this assay, we demonstrate that an intact Rev arginine-rich domain, while critical to specific RNA binding, is dispensable for multimerization on the RRE. Mutations introduced into Rev sequences that flank this basic domain produce a partial multimerization phenotype in vivo even though these mutations are known to block Rev multimerization in vitro. Similarly, mutations introduced into the leucine-rich activation domain of Rev, which appear to have no effect on in vitro multimerization, also markedly inhibit multimerization of Rev on the RRE in vivo. Overall, these data appear consistent with the hypothesis that in vivo formation of the multimeric Rev:RRE ribonucleoprotein complex is facilitated by both the RRE RNA substrate and, as first proposed by Bogerd and Greene U. Virol. 67, 2496-2502, 1993), by bridging by a cellular cofactor for Rev that likely interacts with multiple Rev activation domains.
Amino Acid Sequence↗
Scanning mutagenesis of the arginine-rich region of the human immunodeficiency virus type 1 Rev trans activator.
The structural proteins of human immunodeficiency virus type 1, for example, Gag and Env, are encoded by unspliced and incompletely spliced viral transcripts. The expression of these mRNAs in the cytoplasm, along with their commensurate translation, is absolutely dependent on the virally encoded Rev trans activator. Previous studies have demonstrated that Rev binds directly to its substrate mRNAs via an arginine-rich element that also serves as its nuclear localization sequence. In an attempt to define the specific amino acid residues that are important for in vivo activity, we have constructed a series of missense mutations that scan across this region. Our data demonstrate that all eight arginine residues within this element can, individually, be substituted for either leucine or lysine with no apparent loss of function. Importantly, these findings suggest that no single amino acid within the arginine-rich domain of Rev is, by itself, essential for activity and that considerable functional redundancy is therefore likely to exist within this region. Interestingly, one mutant in which a tryptophan had been substituted for a serine failed to accumulate exclusively in the nucleus but still bound RNA in a manner that was indistinguishable from that of the wild-type protein. This observation indicates that features of the arginine-rich region that are additional to those required for RNA binding are important for Rev's correct accumulation in the nucleus.
Amino Acid Sequence↗