Biomedical subjects

Zhihai Si

Publications and source records attributed to Zhihai Si.

12 recordsLinked to original sources

PubMed2006

Unique features of TRIM5alpha among closely related human TRIM family members.

The tripartite motif (TRIM) protein, TRIM5alpha, restricts some retroviruses, including human immunodeficiency virus (HIV-1), from infecting the cells of particular species. TRIM proteins contain RING, B-box, coiled-coil and, in some cases, B30.2(SPRY) domains. We investigated the properties of human TRIM family members closely related to TRIM5. These TRIM proteins, like TRIM5alpha, assembled into homotrimers and co-localized in the cytoplasm with TRIM5alpha. TRIM5alpha turned over more rapidly than related TRIM proteins. TRIM5alpha, TRIM34 and TRIM6 associated with HIV-1 capsid-nucleocapsid complexes assembled in vitro; the TRIM5alpha and TRIM34 interactions with these complexes were dependent on their B30.2(SPRY) domains. Only TRIM5alpha potently restricted infection by the retroviruses studied; overexpression of TRIM34 resulted in modest inhibition of simian immunodeficiency virus (SIV(mac)) infection. In contrast to the other TRIM genes examined, TRIM5 exhibited evidence of positive selection. The unique features of TRIM5alpha among its TRIM relatives underscore its special status as an antiviral factor.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Requirements for capsid-binding and an effector function in TRIMCyp-mediated restriction of HIV-1.

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.

Animals↗

PubMed2006

Evolution of a cytoplasmic tripartite motif (TRIM) protein in cows that restricts retroviral infection.

Primate tripartite motif 5alpha (TRIM5alpha) proteins mediate innate intracellular resistance to retroviruses. In humans, TRIM5 is located in a paralogous cluster that includes TRIM6, TRIM34, and TRIM22. Although TRIM6 and TRIM34 orthologs are found in other mammals, TRIM5 has to date been identified only in primates. Cow cells exhibit early blocks to infection by several retroviruses. We identify a cytoplasmic TRIM protein encoded by LOC505265 that is responsible for the restriction of infection by several lentiviruses and N-tropic murine leukemia virus in cow cells. Susceptibility of N-tropic murine leukemia virus to 505265-mediated restriction is determined primarily by residue 110 of the viral capsid protein. Phylogenetically, cow LOC505265 segregates with the TRIM5/TRIM6/TRIM34 group, but is not an ortholog of known TRIM genes. The B30.2/SPRY domain of 505265 exhibits long variable regions, a characteristic of the proteins encoded by this paralogous group, and shows evidence of positive selection. Apparently, cows have independently evolved a retroviral restriction factor from the same TRIM family that spawned TRIM5 in primates. Particular features of this subset of cytoplasmic TRIM proteins may be conducive to the convergent evolution of virus-restricting factors.

Animals↗

PubMed2005

The impact of envelope glycoprotein cleavage on the antigenicity, infectivity, and neutralization sensitivity of Env-pseudotyped human immunodeficiency virus type 1 particles.

Endoproteolytic processing of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoproteins is an obligate part of the biosynthetic pathway that generates functional, fusion-competent Env complexes, which are then incorporated into infectious virions. We have examined the influence of cleavage on Env-specific antibody reactivity, Env incorporation into pseudovirions, and the infectivity and neutralization sensitivity of Env-pseudotyped viruses. To do so, we have used both incompletely processed wild-type (Wt) Env and engineered, cleavage-defective Env mutants. We find that there is no simple association between antibody reactivity to cell surface-expressed Env, and the ability of the same antibody to neutralize virus pseudotyped with the same Env proteins. One explanation for the absence of such an association is the diverse array of Env species present on the surface of transiently transfected cells. We also confirm that cleavage-defective mutants are antigenically different from Wt Env. These findings have implications for the use of Env binding assays as predictors of neutralizing activity, and for the development of cleavage-defective Env trimers for use as subunit immunogens.

Antibodies, Monoclonal↗

PubMed2005

The contribution of RING and B-box 2 domains to retroviral restriction mediated by monkey TRIM5alpha.

TRIM5alpha is a cytoplasmic protein that mediates a post-entry block to infection by some retroviruses. TRIM5alpha 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 TRIM5alpha (TRIM5alpharh), which potently restricts infection by human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus of African green monkeys (SIVagm). Disruption of the RING domain caused mislocalization of TRIM5alpharh 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 SIVagm was retained by the RING domain mutants. By contrast, although TRIM5alpharh 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 TRIM5alpharh and exerted dominant-negative effects on the antiretroviral activity of the wild-type protein. Taken together with other data, these results indicate that functionally defective TRIM5alpharh molecules that retain a coiled coil can act as dominant-negative inhibitors of wild-type TRIM5alpharh function. The RING domain of TRIM5alpharh 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.

Animals↗

PubMed2005

Functional mimicry of a human immunodeficiency virus type 1 coreceptor by a neutralizing monoclonal antibody.

Interaction of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein with the primary receptor, CD4, promotes binding to a chemokine receptor, either CCR5 or CXCR4. The chemokine receptor-binding site on gp120 elicits CD4-induced (CD4i) antibodies in some HIV-1-infected individuals. Like CCR5 itself, the CD4i antibody 412d exhibits a preference for CCR5-using HIV-1 strains and utilizes sulfated tyrosines to achieve binding to gp120. Here, we show that 412d binding requires the gp120 beta19 strand and the base of the V3 loop, elements that are important for the binding of the CCR5 N terminus. Two gp120 residues in the V3 loop base determined 412d preference for CCR5-using HIV-1 strains. A chimeric molecule in which the 412d heavy-chain third complementarity-determining loop sequence replaces the CCR5 N terminus functioned as an efficient second receptor, selectively supporting the entry of CCR5-using HIV-1 strains. Sulfation of N-terminal tyrosines contributed to the function of this chimeric receptor. These results emphasize the close mimicry of the CCR5 N terminus by the gp120-interactive region of a naturally elicited CD4i antibody.

Amino Acid Sequence↗

PubMed2005

Retroviral restriction factor TRIM5alpha is a trimer.

The retrovirus restriction factor TRIM5alpha targets the viral capsid soon after entry. Here we show that the TRIM5alpha protein oligomerizes into trimers. The TRIM5alpha coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5alpha mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5alpha, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5alpha to interact with threefold pseudosymmetrical structures on retroviral capsids.

Animals↗

PubMed2004

Small-molecule inhibitors of HIV-1 entry block receptor-induced conformational changes in the viral envelope glycoproteins.

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 and/or 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 and/or 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.

Cell Line↗

PubMed2004

Mode of action for linear peptide inhibitors of HIV-1 gp120 interactions.

The linear peptide 12p1 (RINNIPWSEAMM) was previously isolated from a phage display library and was found to inhibit interaction of HIV-1 gp120 with both CD4 and a CCR5 surrogate, mAb 17b [Ferrer, M., and Harrison, S. (1999) J. Virol. 73, 5795-5802]. In this work, we investigated the mechanism that leads to this dual inhibition of gp120 binding. We found that there is a direct interaction of 12p1 with gp120, which occurs with a binding stoichiometry of 1:1. The peptide inhibits binding of monomeric YU2 gp120 to both sCD4 and 17b at IC(50) values of 1.1 and 1.6 microM, respectively. The 12p1 peptide also inhibited the binding of these ligands to trimeric envelope glycoproteins, blocked the binding of gp120 to the native coreceptor CCR5, and specifically inhibited HIV-1 infection of target cells in vitro. Analyses of sCD4 saturation of monomeric gp120 in the presence or absence of a fixed concentration of peptide suggest that 12p1 suppression of CD4 binding to gp120 is due to allosteric inhibitory effects rather than competitive inhibition of CD4 binding. Using a panel of gp120 mutants that exhibit weakened inhibition by 12p1, the putative binding site of the peptide was mapped to a region immediately adjacent to, but distinguishable from, the CD4 binding footprint. In the case of the peptide, the effects of single-12p1 residue substitutions and various peptide truncations indicate that the side chain of Trp7 and other structural elements of 12p1 are critical for gp120 binding or efficient inhibition of binding of a ligand to gp120. Finally, 12p1 was unable to inhibit binding of sCD4 to a gp120 mutant that is believed to resemble the CD4-induced conformation of gp120. These results suggest that 12p1 preferentially binds gp120 prior to engagement of CD4; binding of the peptide to gp120 limits the interaction with ligands (CD4 and CCR5) that are generally crucial for viral entry. More importantly, these results indicate that 12p1 binds to a unique site that may prove to be a prototypic target for novel CD4-gp120 inhibitors.

Allosteric Regulation↗

PubMed2004

Envelope glycoprotein determinants of increased entry in a pathogenic simian-human immunodeficiency virus (SHIV-HXBc2P 3.2) passaged in monkeys.

Passage of a nonpathogenic simian-human immunodeficiency virus (SHIV-HXBc2) in monkeys resulted in changes in the viral envelope glycoproteins that are responsible for a dramatic increase in replication and pathogenicity in vivo. Here, we show that the envelope glycoproteins of the pathogenic SHIV-HXBc2P 3.2 mediate virus entry into rhesus monkey peripheral blood mononuclear cells (PBMC) more efficiently than the parental SHIV-HXBc2 envelope glycoproteins, and study the basis for this increase. Both parental and pathogenic SHIVs exclusively use CXCR4 as a coreceptor. The determinants of the increased entry associated with the SHIV-HXBc2P 3.2 envelope glycoproteins are located in both the gp120 and gp41 subunits. Changes in the gp120 V3 variable loop specify a decreased sensitivity to SDF-1, consistent with an increase in the affinity of the HXBc2P 3.2 gp120 glycoprotein for CXCR4. Thus, multiple changes in the gp120 variable loops and the gp41 ectodomain of a pathogenic SHIV cooperate to allow enhanced replicative capacity, which in part results from increased chemokine receptor binding.

Animals↗

PubMed2004

CD4-induced T-20 binding to human immunodeficiency virus type 1 gp120 blocks interaction with the CXCR4 coreceptor.

The synthetic peptide T-20, which corresponds to a sequence within the C-terminal heptad repeat region (HR2) of the human immunodeficiency virus type 1 (HIV-1) gp41 envelope glycoprotein, potently inhibits viral membrane fusion and entry. Although T-20 is thought to bind the N-terminal heptad repeat region (HR1) of gp41 and interfere with gp41 conformational changes required for membrane fusion, coreceptor specificity determined by the V3 loop of gp120 strongly influences the sensitivity of HIV-1 variants to T-20. Here, we show that T-20 binds to the gp120 glycoproteins of HIV-1 isolates that utilize CXCR4 as a coreceptor in a manner determined by the sequences of the gp120 V3 loop. T-20 binding to gp120 was enhanced in the presence of soluble CD4. Analysis of T-20 binding to gp120 mutants with variable loop deletions and the reciprocal competition of T-20 and particular anti-gp120 antibodies suggested that T-20 interacts with a gp120 region near the base of the V3 loop. Consistent with the involvement of this region in coreceptor binding, T-20 was able to block the interaction of gp120-CD4 complexes with the CXCR4 coreceptor. These results help to explain the increased sensitivity of CXCR4-specific HIV-1 isolates to the T-20 peptide. Interactions between the gp41 HR2 region and coreceptor-binding regions of gp120 may also play a role in the function of the HIV-1 envelope glycoproteins.

Amino Acid Sequence↗

PubMed2003

Effects of HIV type 1 envelope glycoprotein proteolytic processing on antigenicity.

Passaged simian-human immunodeficiency virus (SHIV)-HXBc2P 3.2 exhibits resistance to neutralization by most antibodies and soluble CD4 compared with the parental SHIV-HXBc2; these SHIVs are neutralized equivalently by 2G12 antibody. 2G12 antibody bound proteolytically processed, cell surface envelope glycoproteins from these viruses equivalently; by contrast, other antibodies bound less efficiently to HXBc2P 3.2 envelope glycoproteins than to HXBc2 envelope glycoproteins. We have examined the influence of proteolytic processing of the envelope glycoprotein precursor on antigenicity, comparing antibody binding to cleaved and uncleaved cell surface envelope glycoproteins and to uncleaved soluble trimeric envelope glycoproteins. All envelope glycoproteins bound neutralizing antibodies better than nonneutralizing antibodies, suggesting that their general topology is similar. Differences between cleaved HXBc2 and HXBc2P 3.2 envelope glycoproteins in binding a given antibody, which correlated with susceptibility to neutralization, were not evident in uncleaved envelope glycoproteins. These results indicate that proteolytic processing allows subtle but biologically important adjustments in the conformation of HIV-1 envelope glycoproteins.

Antibodies, Monoclonal↗