Defining the N-linked glycosylation site of Hantaan virus envelope glycoproteins essential for cell fusion

The Hantaan virus (HTNV) is an enveloped virus that is capable of inducing low pH-dependent cell fusion. We molecularly cloned the viral glycoprotein (GP) and nucleocapsid (NP) cDNA of HTNV and expressed them in Vero E6 cells under the control of a CMV promoter. The viral gene expression was assessed using an indirect immunofluorescence assay and immunoprecipitation. The transfected Vero E6 cells expressing GPs, but not those expressing NP, fused and formed a syncytium following exposure to a low pH. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not. We also investigated the N-linked glycosylation of HTNV GPs and its role in cell fusion. The envelope GPs of HTNV are modified by N-linked glycosylation at five sites: four sites on G1 (N134, N235, N347, and N399) and one site on G2 (N928). Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants, which were then expressed in Vero E6 cells. The oligosaccharide chain on residue N928 of G2 was found to be crucial for cell fusion after exposure to a low pH. These results suggest that G2 is likely to be the fusion protein of HTNV.     

Envelope glycoproteins of hantavirus can mediate cell-cell fusion independently

Hantaviruses (HTVs) are enveloped viruses and can induce low PH-dependent cell fusion. In this report we molecularly cloned viral glycoproteins (GPs) cDNA and nucleocapsid (NP) cDNA of two strains of Hantaan virus and one strain of Seoul virus and expressed in Vero E6 cells under control of a CMV promoter. The examinations of viral gene expressions were carried out by IFA and immune-precipitation. After treatment with low PH (PH 5.8) medium the syncytium were observed in the cells transfected with the GPs clones while in the cells transfected with the NP clones we did not find this phenomenon. Furthermore cotransfection of the NP and GPs did not enhance fusion activity. Treatment with anti-GP monoclonal antibodies could inhibit fusion activity whereas the antibodies against NP could not. These results indicated that GPs can mediate cell-cell fusion independently.     

The in vitro and in vivo protective activity of monoclonal antibodies directed against Hantaan virus: potential application for immunotherapy and passive immunization

Hantaan virus (HTNV), a member of the genus Hantavirus, family Bunyaviridae, is an etiologic agent causing a serious human disease, hemorrhagic fever with renal syndrome (HFRS), with a mortality as high as 15% and is also a potential bioterrorism agent. It is urgently needed to develop anti-HTNV-neutralizing monoclonal antibodies (MAbs) for treatment and prevention of HTNV infection. In the present study, 18 murine MAbs directed against HTNV strain Chen were generated and characterized. Among these MAbs, 13 were directed against viral nucleocapsid protein (NP), four recognized the viral envelope glycoprotein G2 and one reacted with both NP and G2. Only those MAbs that recognize the epitopes on G2 were positive in hemagglutination inhibition (HI) test and had in vitro virus-neutralizing activity and in vivo protective activity against HTNV infection of susceptible mice. Since all the mice were protected by administration of the virus-neutralizing MAbs one day before and two days after HTNV challenge, these neutralizing MAbs are potentially useful for pre- and post-exposure prophylaxis and for immunotherapy of HTNV infection. Phase II clinical trials of these neutralizing MAbs for emergent treatment of patients with HTNV infection in early stages of HRFS are carried out in endemic areas in China.     

Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein.

Six monoclonal antibodies directed against respiratory syncytial virus proteins were produced. Each was characterized by immunoprecipitation and indirect immunofluorescence. One was directed against the nucleocapsid protein. NP 44, two were directed against a 37,000-dalton protein, two were directed against the major envelope glycoprotein, GP 90, and one was directed against the 70,000-dalton envelope protein, VP 70. Indirect immunofluorescence stain patterns of infected HEp-2 cells defined GP 90 and VP 70 as viral proteins expressed on the cell surface, whereas NP 44 and the 37,000-dalton protein were detected as intracytoplasmic inclusions. One of the anti-GP 90 antibodies neutralized virus only in the presence of complement but did not inhibit cell-cell fusion. The anti-VP 70 antibody neutralized virus without complement and inhibited cell-cell fusion of previously infected HEp-2 cells, thus identifying VP 70 as the fusion protein.     

Production and characterization of a recombinant single-chain antibody against Hantaan virus envelop glycoprotein

Hantaan virus (HTNV) is the type of Hantavirus causing hemorrhagic fever with renal syndrome, for which no specific therapeutics are available so far. Cell type-specific internalizing antibodies can be used to deliver therapeutics intracellularly to target cell and thus, have potential application in anti-HTNV infection. To achieve intracellular delivery of therapeutics, it is necessary to obtain antibodies that demonstrate sufficient cell type-specific binding, internalizing, and desired cellular trafficking. Here, we describe the prokaryotic expression, affinity purification, and functional testing of a single-chain Fv antibody fragment (scFv) against HTNV envelop glycoprotein (GP), an HTNV-specific antigen normally located on the membranes of HTNV-infected cells. This HTNV GP-targeting antibody, scFv3G1, was produced in the cytoplasm of Escherichia coli cells as a soluble protein and was purified by immobilized metal affinity chromatography. The purified scFv possessed a high specific antigen-binding activity to HTNV GP and HTNV-infected Vero E6 cells and could be internalized into HTNV-infected cells probably through the clathrin-dependent endocytosis pathways similar to that observed with transferrin. Our results showed that the E. coli-produced scFv had potential applications in targeted and intracellular delivery of therapeutics against HTNV infections.     

汉滩病毒核蛋白的分段表达及抗原表位分析

在大肠杆菌中对汉滩病毒Ｓ基因４种不同长度片段的重组表达质粒进行诱导表达。结果表明表达的４种ＧＳＴ－ＮＰ融合蛋白均以不溶性包含体形式存在于茵体细胞内,表达量分别占菌体蛋白总量的２９－３６％,分子量分别约为７２ｋＤ、６６ｋＤ、５４ｋＤ和４４ｋＤＤ。Ｗｅｓｔｅｒｎ ｂｌｏｔ显示５４ｋＤ和７２ｋＤ融合蛋白用酶标记汉滩病毒ＮＰＭｃＡｂｌＡ８和抗ＧＳＴ ＭｃＡｂ ３Ｃ１１染色呈阳反应。６６ｋＤ和４４ｋＤ融合蛋     

Analysis of immune responses against nucleocapsid protein of the Hantaan virus elicited by virus infection or DNA vaccination

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2K(b) restricted T-cell epitopes of NP. The NP-specific CD8(+) T cell response was analyzed using a (51)Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8(+) T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8(+) T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 approximately 4 weeks after immunization and maximized at 6 approximately 8 weeks. NP-specific CD8(+) T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.     

Fusion proteins encoded by orf 129L of ectromelia and orf A30L of smallpox viruses cross-react with neutralizing monoclonal antibodies

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.     

Cell fusion activity of hepatitis C virus envelope proteins

To examine the cell fusion activity of hepatitis C virus (HCV) envelope proteins (E1 and E2), we have established a sensitive cell fusion assay based on the activation of a reporter gene as described previously (O. Nussbaum, C. C. Broder, and E. A. Berger, J. Virol. 68:5411-5422, 1994). The chimeric HCV E1 and E2 proteins, each consisting of the ectodomain of the E1 and E2 envelope protein and the transmembrane and cytoplasmic domains of the vesicular stomatitis virus G glycoprotein, were expressed on the cell surface. Cells expressing the chimeric envelope proteins and T7 RNA polymerase were cocultured with the various target cell lines transfected with a reporter plasmid encoding the luciferase gene under the control of the T7 promoter. After cocultivation, the cell fusion activity was determined by the expression of luciferase in the cocultured cells. The induction of cell fusion requires both the chimeric E1 and E2 proteins and occurs in a low-pH-dependent manner. Although it has been shown that HCV E2 protein binds human CD81 (P. Pileri, Y. Uematsu, S. Campagnoli, G. Galli, F. Falugi, R. Petracca, A. J. Weiner, M. Houghton, D. Rosa, G. Grandi, and S. Abrignani, Science 282:938-941, 1998), the expression of human CD81 alone is not sufficient to confer susceptibility to cell fusion in the mouse cell line. Treatment of the target cells with pronase, heparinase, or heparitinase reduced the cell fusion activity induced by the chimeric envelope proteins. These results suggest (i) that both HCV E1 and E2 proteins are responsible for fusion with the endosomal membrane after endocytosis and (ii) that certain protein molecules other than human CD81 and some glycosaminoglycans on the cell surface are also involved in the cell fusion induced by HCV.     

汉坦病毒陈株S基因编码区的克隆,序列分析及表达

从汉坦病毒陈株感染的ＶｅｒｏＥ６细胞裂解液中提取病毒ＲＮＡ,经逆转录ＰＣＲ获得病毒Ｓ基因编码区约１．３ｋｂｃＤＮＡ片段,克隆该片段后进行核苷酸序列测定,并与汉坦病毒７６１１８株进行同源性比较,结果二者核苷酸序列同源性为８６％,推导的氨基酸序列同源性为９７％。将该基因片段插入原核表达载体ｐＧＥＸ４Ｔ１,在大肠杆菌中获得高效表达。表达产物为ＧＳＴＮＰ融合蛋白。ＳＤＳＰＡＧＥ检测表达蛋白分子约７２ｋＤ左右。Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ和ＥＬＩＳＡ试验结果表明,表达产物可与多株抗汉坦病毒核蛋白的ＭｃＡｂ发生反应,其抗原表位及ＭｃＡｂ反应谱与７６１１８株相比存在某些差异。     

Characterization of an immunodominant antigenic site on GB virus C glycoprotein E2 that is involved in cell binding

GB virus type C (GBV-C) is a human flavivirus that may cause persistent infection, although most infected individuals clear viremia and develop antibodies to the envelope glycoprotein E2. To study GBV-C E2 antigenicity and cell binding, murine anti-E2 monoclonal antibodies (MAbs) were evaluated to topologically map immunogenic sites on GBV-C E2 and for the ability to detect or block recombinant E2 binding to various cell lines. Five competition groups of MAbs were identified. Groups I and II did not compete with each other. Group III competed with both groups I and II. Group IV did not compete with group I, II, or III. One MAb competed with all of the other MAbs, suggesting that the epitopes bound by these MAbs are intimately related. Individually, none of the MAbs competed extensively with polyclonal human convalescent antibody (PcAb); however, combinations of all five MAb groups completely blocked PcAb binding to E2, suggesting that the epitopes bound by these MAbs form a single, immunodominant antigenic site. Only group I and III MAbs detected purified recombinant E2 bound to cells in binding assays. In contrast, group II MAbs neutralized the binding of E2 to cells. Both PcAb and MAbs were conformation dependent, with the exception of one group II MAb (M6). M6 bound to a five-amino-acid sequence on E2 if the peptide included four C-terminal or eight N-terminal residues, suggesting that the GBV-C E2 protein contains a single immunodominant antigenic site which includes a complex epitope that is involved in specific cellular binding.     

Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein

Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E protein. Moreover, in cells expressing Fc gamma receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E protein variants, we identified 25 E protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.     

Herpes simplex virus type 1 DNA cleavage and encapsidation require the product of the UL28 gene: isolation and characterization of two UL28 deletion mutants.

The herpes simplex virus type 1 UL28 gene contains a 785-amino-acid open reading frame that codes for an essential protein. Studies with temperature-sensitive mutants which map to the UL28 gene indicate that the UL28 gene product (ICP18.5) is required for packaging of viral DNA and for expression of viral glycoproteins on the surface of infected cells (C. Addison, F. J. Rixon, and V. G. Preston, J. Gen. Virol. 71:2377-2384, 1990; B. A. Pancake, D. P. Aschman, and P. A. Schaffer, J. Virol. 47:568-585, 1983). In this study, we describe the isolation of two UL28 deletion mutants that were constructed and propagated in Vero cells transformed with the UL28 gene. The mutants, gCB and gC delta 7B, contained deletions of 1,881 and 537 bp, respectively, in the UL28 gene. Although the mutants synthesize viral DNA, they fail to form plaques or produce infectious virus in cells that do not express the UL28 gene. Transmission electron microscopy and Southern blot analysis demonstrated that both mutants are defective in cleavage and encapsidation of viral DNA. Analysis by cell surface immunofluorescence showed that the UL28 gene is not required for expression of viral glycoproteins on the surface of infected cells. A rabbit polyclonal antiserum was made against an Escherichia coli-expressed Cro-UL28 fusion protein. This antibody reacted with an infected-cell protein having an apparent molecular mass of 87 kDa. The 87-kDa protein was first detected at 6 h postinfection and was expressed as late as 24 h postinfection. No detectable UL28 protein was synthesized in gCB- or gC delta 7B-infected Vero cells.     

JNK and PI3k/Akt signaling pathways are required for establishing persistent SARS-CoV infection in Vero E6 cells

Persistence was established after most of the SARS-CoV-infected Vero E6 cells died. RNA of the defective interfering virus was not observed in the persistently infected cells by Northern blot analysis. SARS-CoV diluted to 2 PFU failed to establish persistence, suggesting that some particular viruses in the seed virus did not induce persistent infection. Interestingly, a viral receptor, angiotensin converting enzyme (ACE)-2, was down-regulated in persistently infected cells. G418-selected clones established from parent Vero E6 cells, which were transfected with a plasmid containing the neomycin resistance gene, were infected with SARS-CoV, resulting in a potential cell population capable of persistence in Vero E6 cells. Our previous studies demonstrated that signaling pathways of extracellular signal-related kinase (ERK1/2), c-Jun N-terminal protein kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3'-kinase (PI3K)/Akt were activated in SARS-CoV-infected Vero E6 cells. Previous studies also showed that the activation of p38 MAPK by viral infection-induced apoptosis, and a weak activation of Akt was not sufficient to protect from apoptosis. In the present study, we showed that the inhibitors of JNK and PI3K/Akt inhibited the establishment of persistence, but those of MAPK/ERK kinase (MEK; as an inhibitor for ERK1/2) and p38 MAPK did not. These results indicated that two signaling pathways of JNK and PI3K/Akt were important for the establishment of persistence in Vero E6 cells.     

Processing of the envelope glycoprotein gp160 in immunotoxin-resistant cell lines chronically infected with human immunodeficiency virus type 1.

We describe the isolation and characterization of variant cell lines which are chronically infected with the human immunodeficiency virus (HIV) and resistant to the action of immunotoxins directed against the HIV envelope protein. These variants all produce normal levels of HIV proteins, budding virions, and the envelope protein precursor gp160. Two of the variants, 10E and 11E, contain a mutation within the env gene which results in the production of a truncated precursor and altered processing and transport of the protein to the cell surface. Variants B9 and G4 are defective in gp160 cleavage and do not efficiently transport the envelope protein to the cell surface. There are no mutations in the expressed viruses of B9 and G4. These cell lines express higher levels of CD4 protein and mRNA than H9/NL4-3. Thus, 10E, 11E, B9, and G4 have escaped immunotoxin action by downmodulating the envelope protein from their cell surfaces. None of these variants produce infectious HIV. Two other immunotoxin-resistant variants, E9-3 and 41-17, produce normal levels of gp160, efficiently transport the cleaved and processed subunits to the cell surface, and secrete infectious HIV. These studies identify alterations in gp160 processing that underscore the importance of the relationship between HIV and the cell that it infects.     

Synthesis and processing of human immunodeficiency virus type 1 envelope proteins encoded by a recombinant human adenovirus.

A recombinant adenovirus was constructed by inserting the human immunodeficiency virus type 1 (HIV-1) envelope gene downstream from the early region 3 (E3) promoter of adenovirus type 5 (Ad5), replacing the coding sequences of E3. The recombinant virus replicated as efficiently as the parent virus in all cell lines tested. Human cells infected with the recombinant virus synthesized the HIV-1 envelope precursor gp160, which was efficiently processed to the envelope glycoproteins gp120 and gp41. A human T-lymphoblast line (Molt-4) infected with the recombinant virus expressed HIV-1 envelope glycoproteins on the cell surface, leading to syncytium formation. The envelope gene was expressed from the E3 promoter at early times after infection and at late times from the major late promoter. When cotton rats were infected with the recombinant virus, antibodies against the HIV-1 envelope glycoproteins could be expressed in an immunoreactive form by the recombinant adenovirus, further illustrating the usefulness of adenoviruses as expression vectors.     

The herpes simplex virus type 1 UL20 protein modulates membrane fusion events during cytoplasmic virion morphogenesis and virus-induced cell fusion

The herpes simplex virus type 1 (HSV-1) UL20 protein is an important determinant for virion morphogenesis and virus-induced cell fusion. A precise deletion of the UL20 gene in the HSV-1 KOS strain was constructed without affecting the adjacent UL20.5 gene. The resultant KOS/UL20-null virus produced small plaques of 8 to 15 cells in Vero cells while it produced wild-type plaques on the complementing cell line G5. Electron microscopic examination of infected cells revealed that the KOS/UL20-null virions predominantly accumulated capsids in the cytoplasm while a small percentage of virions were found as enveloped virions within cytoplasmic vacuoles. Recently, it was shown that UL20 expression was necessary and sufficient for cell surface expression of gK (T. P. Foster, X. Alvarez, and K. G. Kousoulas, J. Virol. 77:499-510, 2003). Therefore, we investigated the effect of UL20 on virus-induced cell fusion caused by syncytial mutations in gB and gK by constructing recombinant viruses containing the gBsyn3 or gKsyn1 mutations in a UL20-null genetic background. Both recombinant viruses failed to cause virus-induced cell fusion in Vero cells while they readily caused fusion of UL20-null complementing G5 cells. Ultrastructural examination of UL20-null viruses carrying the gBsyn3 or gKsyn1 mutation revealed a similar distribution of virions as the KOS/UL20-null virus. However, cytoplasmic vacuoles contained aberrant virions having multiple capsids within a single envelope. These multicapsid virions may have been formed either by fusion of viral envelopes or by the concurrent reenvelopment of multiple capsids. These results suggest that the UL20 protein regulates membrane fusion phenomena involved in virion morphogenesis and virus-induced cell fusion.     

Expression cloning of functional receptor used by SARS coronavirus

We have expressed a series of truncated spike (S) glycoproteins of SARS-CoV and found that the N-terminus 14-502 residuals were sufficient to bind to SARS-CoV susceptible Vero E6 cells. With this soluble S protein fragment as an affinity ligand, we screened HeLa cells transduced with retroviral cDNA library from Vero E6 cells and obtained a HeLa cell clone which could bind with the S protein. This cell clone was susceptible to HIV/SARS pseudovirus infection and the presence of a functional receptor for S protein in this cell clone was confirmed by the cell-cell fusion assay. Further studies showed the susceptibility of this cell was due to the expression of endogenous angiotensin-converting enzyme 2 (ACE2) which was activated by inserted LTR from retroviral vector used for expression cloning. When human ACE2 cDNA was transduced into NIH3T3 cells, the ACE2 expressing NIH3T3 cells could be infected with HIV/SARS pseudovirus. These data clearly demonstrated that ACE2 was the functional receptor for SARS-CoV.     

Characterization and epitope mapping of neutralizing monoclonal antibodies produced by immunization with oligomeric simian immunodeficiency virus envelope protein

In an attempt to generate broadly cross-reactive, neutralizing monoclonal antibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two immunization protocols using different preparations of oligomeric SIV envelope (Env) glycoproteins. In the first protocol, mice were immunized with soluble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzyme-linked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generated. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of Env-expressing cells, and preferentially recognized monomeric Env protein. A subset of these antibodies directed toward the V1/V2 loop, the V3 loop, or nonlinear epitopes were capable of neutralizing CP-MAC, a closely related isolate (SIVmac1A11), and/or two more divergent strains (SIVsmDeltaB670 CL3 and SIVsm543-3E). In the second protocol, mice were immunized with unfixed CP-MAC-infected cells and MAbs were screened for the ability to inhibit cell-cell fusion. In contrast to MAbs generated against sgp140, the seven MAbs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferentially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsmDeltaB670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both groups. MAbs to the V3 loop and one MAb reactive with the V1/V2 loop interfered with CCR5 binding, indicating that these regions of Env play similar roles for SIV and human immunodeficiency virus. Remarkably, several of the MAbs generated against infected cells blocked CCR5 binding in a V3-independent manner, suggesting that they may recognize a region analogous to the conserved coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently than infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.