Michael J. Endres

CD4-independent infection by HIV-2 is mediated by Fusin/CXCR4

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit HIV-1 entry and infection. However, the mechanism by which these molecules serve as CD4-associated cofactors is unclear. In the present report, we show that one member of this family, termed Fusin/ CXCR4, is able to function as an alternative receptor for some isolates of HIV-2 in the absence of CD4. This conclusion is supported by the finding that (1) CD4-independent infection by these viruses is inhibited by an anti-Fusin monoclonal antibody, (2) Fusin expression renders human and nonhuman CD4-negative cell lines sensitive to HIV-2-induced syncytium induction and/or infection, and (3) Fusin is selectively down-regulated from the cell surface following HIV-2 infection. The finding that one chemokine receptor can function as a primary viral receptor strongly suggests that the HIV envelope glycoprotein contains a binding site for these proteins and that differences in the affinity and/or the availability of this site can extend the host range of these viruses to include a number of CD4-negative cell types.

SCH-C (SCH 351125), an orally bioavailable, small molecule antagonist of the chemokine receptor CCR5, is a potent inhibitor of HIV-1 infection in vitro and in vivo

We describe here the identification and properties of SCH-C (SCH 351125), a small molecule inhibitor of HIV-1 entry via the CCR5 coreceptor. SCH-C, an oxime–piperidine compound, is a specific CCR5 antagonist as determined in multiple receptor binding and signal transduction assays. This compound specifically inhibits HIV-1 infection mediated by CCR5 in U-87 astroglioma cells but has no effect on infection of CXCR4-expressing cells. SCH-C has broad and potent antiviral activity in vitro against primary HIV-1 isolates that use CCR5 as their entry coreceptor, with mean 50% inhibitory concentrations ranging between 0.4 and 9 nM. Moreover, SCH-C strongly inhibits the replication of an R5-using HIV-1 isolate in SCID-hu Thy/Liv mice. SCH-C has a favorable pharmacokinetic profile in rodents and primates with an oral bioavailability of 50–60% and a serum half-life of 5–6 h. On the basis of its novel mechanism of action, potent antiviral activity, and in vivo pharmacokinetic profile, SCH-C is a promising new candidate for therapeutic intervention of HIV infection.

In Vivo Attenuation of Simian Immunodeficiency Virus by Disruption of a Tyrosine-Dependent Sorting Signal in the Envelope Glycoprotein Cytoplasmic Tail

ABSTRACT Attenuated simian immunodeficiency viruses (SIVs) have been described that produce low levels of plasma virion RNA and exhibit a reduced capacity to cause disease. These viruses are particularly useful in identifying viral determinants of pathogenesis. In the present study, we show that mutation of a highly conserved tyrosine (Tyr)-containing motif (Yxxφ) in the envelope glycoprotein (Env) cytoplasmic tail (amino acids YRPV at positions 721 to 724) can profoundly reduce the in vivo pathogenicity of SIVmac239. This domain constitutes both a potent endocytosis signal that reduces Env expression on infected cells and a sorting signal that directs Env expression to the basolateral surface of polarized cells. Rhesus macaques were inoculated with SIVmac239 control or SIVmac239 containing either a Tyr-721-to-Ile mutation (SIVmac239Y/I) or a deletion of Tyr-721 and the preceding glycine (ΔGY). To assess the in vivo replication competence, all viruses contained a stop codon innef that has been shown to revert during in vivo but not in vitro replication. All three control animals developed high viral loads and disease. One of two animals that received SIVmac239Y/I and two of three animals that received SIVmac239ΔGY remained healthy for up to 140 weeks with low to undetectable plasma viral RNA levels and normal CD4+ T-cell percentages. These animals exhibited ongoing viral replication as determined by detection of viral sequences and culturing of mutant viruses from peripheral blood mononuclear cells and persistent anti-SIV antibody titers. In one animal that received SIVmac239Y/I, the Ile reverted to a Tyr and was associated with a high plasma RNA level and disease, while one animal that received SIVmac239ΔGY also developed a high viral load that was associated with novel and possibly compensatory mutations in the TM cytoplasmic domain. In all control and experimental animals, the nefstop codon reverted to an open reading frame within the first 2 months of inoculation, indicating that the mutant viruses had replicated well enough to repair this mutation. These findings indicate that the Yxxφ signal plays an important role in SIV pathogenesis. Moreover, because mutations in this motif may attenuate SIV through mechanisms that are distinct from those caused by mutations in nef, this Tyr-based sorting signal represents a novel target for future models of SIV and human immunodeficiency virus attenuation that could be useful in new vaccine strategies.

CD4-independent utilization of the CXCR4 chemokine receptor by HIV-1 and HIV-2

HIV entry is mediated by an interaction between CD4 and members of the chemokine receptor family of proteins. It is likely that CD4 induces conformational changes in the viral envelope glycoproteins that facilitate a subsequent interaction with the chemokine receptor. To understand these events, variants of HIV-2 and HIV-1 have been derived that are able to interact directly with CXCR4 in the absence of CD4. One HIV-2 variant. termed HIV-2/vcp, has an expanded host range that includes CXCR4+/CD4- lymphoid and nonlymphoid cell lines. In contrast to T-tropic isolates of HIV-1, HIV-2/vcp was shown to induce > 95% downregulation of CXCR4 on chronically infected cells and was able to superinfect HIV-1-infected cells. A variant of HIV-1/IIIB termed HIV-1/IIIBx was also derived that is both replication competent and fusogenic for a CD4-negative subclone of SupT1 cells, termed BC7. Infection of BC7 cells by HIV-1/IIIBx was resistant to anti-CD4 monoclonal antibodies but inhibited by the anti-CXCR4 mAb, 12G5. HIV-1/IIIBx was highly fusogenic on 3T3 cells expressing CXCR4 in the absence of CD4. In contrast to HIV-2/vcp, the host range of HIV-1/IIIBx was highly restricted and replication in several CD4+/CXCR4+ lymphoid cell lines was reduced compared to HIV-1/IIIB. In addition, HIV-1/IIIBx failed to downregulate CXCR4 on chronically infected cells. These studies indicate that HIV-1 and HIV-2 variants can be derived in vitro that utilize CXCR4 in the absence of CD4. Although the mechanism(s) for these changes remain unclear, possibilities include an increased avidity of the viral envelope glycoprotein for CXCR4 and/or the increased exposure of the chemokine receptor binding site. Further biochemical and molecular analysis of the envelope glycoproteins from these viruses should be helpful in addressing these and other possibilities.

The Large Viral RNA Segment of California Serogroup Bunyaviruses Encodes the Large Viral Protein

Reassortant bunyaviruses derived from two members of the California serogroup (La Crosse/original and Tahyna/181-57) viruses were used to demonstrate that the large Mr viral protein (L) is encoded by the L RNA segment. Radiolabelled viral proteins were analysed by discontinuous SDS-PAGE. The L protein of La Crosse virus was observed to migrate ahead of its Tahyna virus counterpart when electrophoresed through a 5% acrylamide resolving gel. Among the reassortant viruses, the L protein phenotype segregated with the viral L RNA segment. After confirming the genotype of the viruses used in this study, it was concluded that the L RNA species of California serogroup viruses codes for the L protein, the presumed viral polymerase.

The Differential Sensitivity of Human and Rhesus Macaque CCR5 to Small-Molecule Inhibitors of Human Immunodeficiency Virus Type 1 Entry Is Explained by a Single Amino Acid Difference and Suggests a Mechanism of Action for These Inhibitors

ABSTRACT AD101 and SCH-C are two chemically related small molecules that inhibit the entry of human immunodeficiency virus type 1 (HIV-1) via human CCR5. AD101 also inhibits HIV-1 entry via rhesus macaque CCR5, but SCH-C does not. Among the eight residues that differ between the human and macaque versions of the coreceptor, only one, methionine-198, accounts for the insensitivity of macaque CCR5 to inhibition by SCH-C. Thus, the macaque coreceptor engineered to contain the natural human CCR5 residue (isoleucine) at position 198 is sensitive to HIV-1 entry inhibition by SCH-C, whereas a human CCR5 mutant containing the corresponding macaque residue (methionine) is resistant. Position 198 is in CCR5 transmembrane (TM) helix 5 and is not located within the previously defined binding site for AD101 and SCH-C, which involves residues in TM helices 1, 2, 3, and 7. SCH-C binds to human CCR5 whether residue 198 is isoleucine or methionine, and it also binds to macaque CCR5. However, the binding of a conformation-dependent monoclonal antibody to human CCR5 is inhibited by SCH-C only when residue 198 is isoleucine. These observations, taken together, suggest that the antiviral effects of SCH-C and AD101 involve stabilization, or induction, of a CCR5 conformation that is not compatible with HIV-1 infection. However, SCH-C is unable to exert this effect on CCR5 conformation when residue 198 is methionine. The region of CCR5 near residue 198 has, therefore, an important influence on the conformational state of this receptor.

Reassortment of La Crosse and Tahyna bunyaviruses in Aedes triseriatus mosquitoes.

Experiments were conducted to determine if La Crosse (LAC) and Tahyna (TAH) viruses reassort in Aedes triseriatus mosquitoes and to determine the genotypic frequencies of viruses selected by in vivo vector interactions. A molecular hybridization technique was used to analyze progeny viruses. Probes specific for the La Crosse L, M and S segments (pLAC4.16: LAC L RNA; pLAC4.27: LAC M RNA; pLAC4C-26: LAC S RNA) were used to determine the parental origin of the progeny RNA segments. Following infection with a mixture of LAC and TAH viruses, mosquitoes were held for 23 days extrinsic incubation, then assayed for reassortment. Individual progeny viruses were isolated by plaque assay and propagated in BHK-21 cells. Cytoplasmic RNA was extracted from the cells, blotted in triplicate to Nytran, and each blot was hybridized with 32P-labelled pLAC4.16, pLAC4.27 or pLAC4C-26 to determine the parental origin of each RNA segment. High frequency reassortment occurred in these mosquitoes. All of the expected genotypes resulting from a cross of LAC and TAH were obtained from these mosquitoes. Genotypic frequencies of 708 virus isolates from 39 mosquitoes were: LLL, 150 (21%); LLT, 71 (10%); LTL, 39 (5.5%); LTT, 109 (15%); TTT, 259 (36%); TTL, 16 (2.2%); TLT, 55 (7.8%); TLL, 9 (1.2%).