When interacting with the CD4 receptor, the HIV gp120 envelope glycoprotein undergoes conformational changes that allow binding to the chemokine receptor. Receptor binding is proposed to lead to conformational changes in the gp41 transmembrane envelope glycoprotein involving the creation and͞or exposure of a coiled coil consisting of three heptad repeat (HR) sequences. The subsequent interaction of the HR2 region of gp41 with this coiled coil results in the assembly of a six-helix bundle that promotes the fusion of the viral and target cell membranes. Here we show that CD4 binding to gp120 induces the formation and͞or exposure of the gp41 HR1 coiled coil in a process that does not involve gp120 shedding and that depends on the proteolytic maturation of the gp160 envelope glycoprotein precursor. Importantly, BMS-806 and related HIV-1 entry inhibitors bind gp120 and block the CD4 induction of HR1 exposure without significantly affecting CD4 binding. Moreover, these compounds do not disrupt gp120-chemokine receptor binding or the HR1-HR2 interaction within gp41. These studies thus define a receptor-induced conformational rearrangement of gp120-gp41 that is important for both CD4-dependent and CD4-independent HIV-1 entry and is susceptible to inhibition by low-molecular-weight compounds.

Binding to the CD4 receptor induces conformational changes in the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein. These changes allow gp120 to bind the coreceptor, either CCR5 or CXCR4, and prime the gp41 transmembrane envelope glycoprotein to mediate virus–cell membrane fusion and virus entry. Soluble forms of CD4 (sCD4) and small-molecule CD4 mimics (here exemplified by JRC-II-191) also induce these conformational changes in the HIV-1 envelope glycoproteins, but typically inhibit HIV-1 entry into CD4-expressing cells. To investigate the mechanism of inhibition, we monitored at high temporal resolution inhibitor-induced changes in the conformation and functional competence of the HIV-1 envelope glycoproteins that immediately follow engagement of the soluble CD4 mimics. Both sCD4 and JRC-II-191 efficiently activated the envelope glycoproteins to mediate infection of cells lacking CD4, in a manner dependent on coreceptor affinity and density. This activated state, however, was transient and was followed by spontaneous and apparently irreversible changes of conformation and by loss of functional competence. The longevity of the activated intermediate depended on temperature and the particular HIV-1 strain, but was indistinguishable for sCD4 and JRC-II-191; by contrast, the activated intermediate induced by cell-surface CD4 was relatively long-lived. The inactivating effects of these activation-based inhibitors predominantly affected cell-free virus, whereas virus that was prebound to the target cell surface was mainly activated, infecting the cells even at high concentrations of the CD4 analogue. These results demonstrate the ability of soluble CD4 mimics to inactivate HIV-1 by prematurely triggering active but transient intermediate states of the envelope glycoproteins. This novel strategy for inhibition may be generally applicable to high–potential-energy viral entry machines that are normally activated by receptor binding.

TRIM5␣ is a cytoplasmic protein that mediates a postentry block to infection by some retroviruses. TRIM5␣ contains a tripartite motif (TRIM), which includes RING, B-box 2, and coiled-coil domains, and a C-terminal B30.2 (SPRY) domain. We investigated the contribution of the RING and B-box 2 domains to the antiretroviral activity of rhesus monkey TRIM5␣ (TRIM5␣ rh ), which potently restricts infection by human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus of African green monkeys (SIV agm ). Disruption of the RING domain caused mislocalization of TRIM5␣ rh so that the cytoplasmic level of the protein was decreased compared with that of the wild-type protein. Nonetheless, partial ability to restrict HIV-1 and SIV agm was retained by the RING domain mutants. By contrast, although TRIM5␣ rh mutants with disrupted B-box 2 domains were efficiently expressed and correctly localized to the cytoplasm, antiretroviral activity was absent. The B-box 2 mutants colocalized and associated with wild-type TRIM5␣ rh and exerted dominantnegative effects on the antiretroviral activity of the wild-type protein. Taken together with other data, these results indicate that functionally defective TRIM5␣ rh molecules that retain a coiled coil can act as dominantnegative inhibitors of wild-type TRIM5␣ rh function. The RING domain of TRIM5␣ rh is not absolutely required for retrovirus restriction but can influence cytoplasmic levels of the protein and thus indirectly alter function. The B-box 2 domain, by contrast, appears to be essential for efficient retrovirus restriction.

Human immunodeficiency virus type 1 (HIV-1)pre-mRNA splicing is regulated in order to maintain pools of unspliced and partially spliced viral RNAs as well as the appropriate levels of multiply spliced mRNAs during virus infection. We have previously described an element in tat exon 2 that negatively regulates splicing at the upstream tat 3 splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, Mol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicing enhancer (ESE) elements, we have termed this element an exon splicing silencer (ESS). We show evidence for another negative cis-acting region within tat-rev exon 3 of HIV-1 RNA that has sequence motifs in common with a 20-nt ESS element in tat exon 2. This sequence is juxtaposed to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream tat-rev 3 splice site. Inhibition of the splicing of substrates containing the ESS element in tat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing mediated by the ESS can be specifically abrogated by the addition of competitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and tat-rev exon 3.Alternative splicing of mRNA precursors plays a critical role in the regulation of gene expression. In metazoan cells, splicing of pre-mRNA is mediated by cis-acting signals which include 5Ј and 3Ј splice sites, branchpoint sequences, and polypyrimidine tracts preceding 3Ј splice sites (for a review, see reference 19). However, the mechanisms by which alternative splice site selection is regulated are not well understood. There are numerous examples of sequences within introns that act to either enhance or inhibit splicing (3,7,10,16,21,25,35,40,43,65). Some of these intron sequences have been shown to bind cellular factors (21,35,40,43). Exon sequences have also been shown to play a role in alternative splicing. Positive-acting exon sequences and purine-rich regions or exon splicing enhancer (ESE) elements have been reported for a number of different cellular and viral genes (4,5,8,23,30,36,50,51,53,56,(58)(59)(60). Some of these positive-acting exon sequences are binding sites for cellular factors (5,23,30,50,58). A family of factors called SR proteins are required for splicing and, in some cases, have been shown to regulate alternative splice site selection in a concentration-dependent manner (14,17,28,33,61). Recent reports have shown that the SR proteins selectively bind to purine-rich splicing elements present in cellular exons (30,49,50). There are also several examples of negative-acting exon splicing elements (2,4,18,45,55). To date, factors interacting with negative-acting exon splicing elements affecting alternative 3Ј splice site usage in metazoan cells have not yet been reported.Human immunodeficie...

The retrovirus restriction factor TRIM5␣ targets the viral capsid soon after entry. Here we show that the TRIM5␣ protein oligomerizes into trimers. The TRIM5␣ coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5␣ mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5␣, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5␣ to interact with threefold pseudosymmetrical structures on retroviral capsids.TRIM5␣ is a constitutively expressed cytoplasmic protein that allows the cells of primates to resist infection by particular retroviruses, including human immunodeficiency virus type 1 (HIV-1) (10,14,25,31,32,37). TRIM5␣ is thought to target the incoming retroviral capsid soon after entry into the cells (9,11,13,15,19,21,22,29,34). The specific mechanism by which TRIM5␣ restricts retroviral infection remains unknown.TRIM5␣ is a member of the tripartite motif (TRIM) family of proteins which contain RING, B-box, and coiled-coil domains (26). Many TRIM proteins self-associate to form homooligomers; less frequently, hetero-oligomerization is observed (26). Structural predictions suggest that the coiled coils of TRIM proteins exhibit a propensity to form both dimers and trimers (6,7,17). There is only limited information available about the oligomeric state of TRIM proteins. Oligomerization has been shown to be important for the function of the nuclear TRIM28 (KAP-1) protein (23,24). In this case, the RING, B-box, and coiled-coil domains were shown to contribute to trimerization. The coiled coil of TRIM7 is essential for oligomerization (39). Here we examine the oligomeric state of TRIM5.The hemagglutinin (HA)-tagged TRIM5 variants in Fig. 1A were expressed transiently in 293T cells or stably in HeLa cells. Cells were washed in phosphate-buffered saline (PBS) and lysed in NP-40 lysis buffer (0.5% Nonidet P40 , 1 ϫ complete EDTA-free protease inhibitor [Roche Diagnostics] in PBS) for 45 min at 4°C. Lysates were centrifuged at 14,000 ϫ g for 15 min at 4°C. The cleared lysates were not stored or frozen but rather were directly cross-linked. Approximately 100 to 200 l of cleared lysates was diluted with PBS plus 1 mM EDTA to a final volume of 400 l. Lysates were cross-linked with various concentrations (up to 10 mM) of glutaraldehyde (GA) for 5 min at room temperature and centrifuged briefly in a table-top centrifuge. The reaction mix was quenched with 0.1 M Tris-HCl, pH 7.5, and briefly centrifuged. The cleared, cross-linked lysates were precipitated with the anti-HA antibody HA.11 (Covance) and protein A-Sepharose beads (Amersham) for 2 h at 4°C; final volumes for the immunoprecipitation were greater than 700 l. The beads were washed four times with NP-40 wash buffer (10 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.5% NP-40) and boiled in LDS sample buffer (106 mM Tris-HCl, 141 mM Tris base, pH 8.5, 0.51 mM EDTA, 10% glycerol, 2% LDS, 0.22 mM ...

In owl monkeys, a retrotransposition event replaced the gene encoding the retroviral restriction factor TRIM5alpha with one encoding TRIMCyp, a fusion between the RING, B-box 2 and coiled-coil domains of TRIM5 and cyclophilin A. TRIMCyp restricts human immunodeficiency virus (HIV-1) infection by a mechanism dependent on the interaction of the cyclophilin A moiety and the HIV-1 capsid protein. Here, we show that infection by retroviruses other than HIV-1 can be restricted by TRIMCyp, providing an explanation for the evolutionary retention of the TRIMCyp gene in owl monkey lineages. The TRIMCyp-mediated block to HIV-1 infection occurs before the earliest step of reverse transcription. TRIMCyp-mediated restriction involves at least two functions: (1) capsid binding, which occurs most efficiently for trimeric TRIMCyp proteins that retain the coiled-coil and cyclophilin A domains, and (2) an effector function that depends upon the B-box 2 domain.

Primate tripartite motif 5␣ (TRIM5␣) proteins mediate innate intracellular resistance to retroviruses. In humans, TRIM5 is located in a paralogous cluster that includes TRIM6, TRIM34, and TRIM22. capsid ͉ convergent evolution ͉ restriction factor ͉ retrovirus ͉ ungulates

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