Characterization of Determinants for Envelope Binding and Infection in Tva, the Subgroup A Avian Sarcoma and Leukosis Virus Receptor

Tva is the cellular receptor for subgroup A avian leukosis and sarcoma virus (ALSV-A). The viral interaction domain of Tva is determined by a 40-residue, cysteine-rich module closely related to the ligand binding domain of the human low-density lipoprotein receptor (LDLR). In this report, we examined the role of the LDLR-like module of Tva in envelope binding and viral infection by mutational analysis. We found that the entire LDLR module in Tva is essential for efficient binding to the viral envelope protein. However, the 17 N-terminal residues of this module can be deleted without affecting receptor function, suggesting that the major determinants for viral entry are located at the C terminus of the module. The effect on viral infection of many amino acid substitutions and deletions in the LDLR module is context dependent, suggesting that the residues important for viral entry are dispersed throughout the LDLR module. In addition, we found that all 27 mutations at residues D46, E47, and W48 greatly reduced envelope binding. These results are discussed in relation to a recently elucidated structure for an LDLR module.     

The spacing between cysteines two and three of the LDL-A module of Tva is important for subgroup A avian sarcoma and leukosis virus entry

Rong et al. have demonstrated previously that with a few substitutions, the fourth repeat of human low-density lipoprotein (hLDL-A4) receptor can functionally replace the LDL-A module of Tva, the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), in viral entry (L. Rong, K. Gendron, and P. Bates, Proc. Natl. Acad. Sci. USA 95:8467-8472, 1998). Here we have shown that swapping the amino terminus of hLDL repeat 5 (hLDL-A5) with that of Tva, in addition to the corresponding substitutions made in human LDL-A4, was required to convert hLDL-A5 into an efficient ASLV-A receptor. These results substantiated our previous findings regarding the role of the specific residues in the viral interaction domain of Tva and demonstrated the critical role of the amino terminus of the Tva LDL-A module in ASLV-A infection. Furthermore, we have shown that the residues between cysteines 2 and 3 of the Tva LDL-A module in a Tva/LDL-A5 chimeric protein can be functionally replaced by the corresponding region of another LDL-A module, human LDL receptor-related protein repeat 22 (LDL-A22), to mediate efficient ASLV-A entry. Since the only conserved feature between the C2-C3 region of LDL-A22 and the Tva LDL-A module is that both contain nine amino acids of which none are conserved, we conclude that the spacing between C2 and C3 of the LDL-A module of Tva is an important determinant for ASLV-A entry. Thus, the present study provides strong evidence to support our hypothesis that one role of the N terminus of the LDL-A module of Tva is to allow proper folding and conformation of the protein for optimal interaction with the viral glycoprotein EnvA in ASLV-A entry.     

Role of the HIV gp120 conserved domain 1 in processing and viral entry

The importance of the N-terminal region of HIV gp120 conserved domain 1 (gp120-C1) to envelope function has been examined by alanine-scanning mutagenesis and subsequent characterization of the mutagenic effects on viral entry; envelope expression, processing, and incorporation; and gp120 association with gp41. With respect to the wild-type gp120, mutational effects on viral entry fall into two classes: functional, as defined by >20% entry with respect to wild type, and impaired, as defined by <20% entry with respect to wild type. Based on Western blot analyses of cell lysates and virions, the entry impairment of W35A, V38A, Y39A, Y40A, G41A, V42A, and I52A is due primarily to disruption of envelope processing. The entry impairment of P43A and W45A is apparently due to a combination of effects on processing and incorporation into virions. In contrast, the entry impairment of V44A and F53A is primarily due to disruption of the gp120-gp41 interaction, which results in dissociation of gp120 from the virion. We present a model for gp120-C1 interactions with gp120-C5 and the gp41 disulfide loop in unprocessed gp160 and processed gp120/gp41.     

Role of basic residues in the subgroup-determining region of the subgroup A avian sarcoma and leukosis virus envelope in receptor binding and infection.

Receptor specificity in avian sarcoma and leukosis viruses (ASLV) maps to the central region of the envelope surface protein, SU. Two hypervariable regions, hr1 and hr2, within this region of SU are the principal determinants of receptor specificity. The cellular receptor for subgroup A ASLV, Tva, utilizes a 40-residue, acidic, cysteine-rich sequence for viral binding and entry. This domain in Tva is closely related to the ligand-binding domain of the low-density lipoprotein receptor (LDLR). Ligands bind to LDLR via the interaction of clustered basic residues in the ligand with the acidic cysteine-rich domains of the receptor. Analysis of the ASLV envelope sequences revealed a cluster of basic residues within hr2 that is unique to the subgroup A viruses, suggesting a possible role for these residues in receptor recognition. Therefore, the effects of altering these basic residues on subgroup A envelope expression, receptor binding, and infectivity were examined. Most of the mutant proteins were transported to the cell surface and processed normally. Receptor binding was diminished approximately 50% by alanine substitution at amino acid R213 or K227, whereas substitution by alanine at R210, R223, or R224 had no effect. However, when coupled with mutations at R213 or K227, changes at R223,R224 reduced envelope binding by 90%. Mutation of all five basic residues abrogated receptor binding. The effect of the hr2 mutations on ASLV envelope-mediated infection did not parallel the effect on receptor binding. Residues 210, 213, 223, and 224 were important for efficient infection, while mutations at residue 227 had little effect on infectivity. These results demonstrate that the basic residues in the ASLV envelope have roles in both receptor recognition and post-receptor binding events during viral entry.     

Alanine scanning mutants of the HIV gp41 loop

Based on mutagenesis and structural studies of human immunodeficiency virus (HIV) envelope proteins, the loop region of gp41 is thought to directly interact with gp120. The importance of the HIV gp41 loop region to envelope function has been systematically examined by alanine scanning of all gp41 loop residues and the subsequent characterization of the mutagenic effects on viral entry, envelope expression, envelope processing, and gp120 association with gp41. With respect to the wild-type gp41, mutational effects on viral entry fall into four classes as follows: 1) little or no effect (G594A, S599A, G600A, K601A, N611A, S615A, N616A, and L619A); 2) significantly reduced entry (I595A, L602A, I603A, V608A, and K617A); 3) abolished entry (L593A, W596A, G597A, T606A, W610A, W614A, S618A, and I622A); and 4) enhanced entry (T605A, P609A, S613A, E620A, and Q621A). The reduced functionality of many mutants was apparently due to either disruption of envelope processing (L593A and T606A), viral incorporation of the envelope (W610A, W614A, and I662A), or increased dissociation of gp120 (W596A, G597A, and S618A). The extreme sensitivity of the gp120-gp41 interaction to alanine substitutions (e.g. the G597A and S618A mutants are relatively conservative substitutions) suggests that this association is an attractive and novel target for future drug discovery efforts.     

Evolutionary pressure of a receptor competitor selects different subgroup a avian leukosis virus escape variants with altered receptor interactions

A complex interaction between the retroviral envelope glycoproteins and a specific cell surface protein initiates viral entry into cells. The avian leukosis-sarcoma virus (ALV) group of retroviruses provides a useful experimental system for studying the retroviral entry process and the evolution of receptor usage. In this study, we demonstrate that evolutionary pressure on subgroup A ALV [ALV(A)] entry exerted by the presence of a competitive inhibitor, a soluble form of the ALV(A) Tva receptor linked to a mouse immunoglobulin G tag (quail sTva-mIgG), can select different populations of escape variants. This escape population contained three abundant ALV(A) variant viruses, all with mutations in the surface glycoprotein hypervariable regions: a previously identified variant containing the Y142N mutation in the hr1 region; a new variant with two mutations, W141G in hr1 and K261E in vr3; and another new variant with two mutations, W145R in hr1 and K261E. The W141G K261E and W145R K261E viruses escape primarily by lowering their binding affinities for the quail Tva receptor competitive inhibitor while retaining wild-type levels of binding affinity for the chicken Tva receptor. A secondary phenotype of the new variants was an alteration in receptor interference patterns from that of wild-type ALV(A), indicating that the mutant glycoproteins are possibly interacting with other cellular proteins. One result of these altered interactions was that the variants caused a transient period of cytotoxicity. We could also directly demonstrate that the W141G K261E variant glycoproteins bound significant levels of a soluble form of the Tvb(S3) ALV receptor in a binding assay. Alterations in the normally extreme specificity of the ALV(A) glycoproteins for Tva may represent an evolutionary first step toward expanding viral receptor usage in response to inefficient viral entry.     

Identification of key residues in subgroup A avian leukosis virus envelope determining receptor binding affinity and infectivity of cells expressing chicken or quail Tva receptor

To better understand retroviral entry, we have characterized the interactions between subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and Tva, the receptor for ALV(A), that result in receptor interference. We have recently shown that soluble forms of the chicken and quail Tva receptor (sTva), expressed from genes delivered by retroviral vectors, block ALV(A) infection of cultured chicken cells ( approximately 200-fold antiviral effect) and chickens (>98% of the birds were not infected). We hypothesized that inhibition of viral replication by sTva would select virus variants with mutations in the surface glycoprotein (SU) that altered the binding affinity of the subgroup A SU for the sTva protein and/or altered the normal receptor usage of the virus. Virus propagation in the presence of quail sTva-mIgG, the quail Tva extracellular region fused to the constant region of the mouse immunoglobulin G (IgG) protein, identified viruses with three mutations in the subgroup A hr1 region of SU, E149K, Y142N, and Y142N/E149K. These mutations reduced the binding affinity of the subgroup A envelope glycoproteins for quail sTva-mIgG (32-, 324-, and 4,739-fold, respectively) but did not alter their binding affinity for chicken sTva-mIgG. The ALV(A) mutants efficiently infected cells expressing the chicken Tva receptor but were 2-fold (E149K), 10-fold (Y142N), and 600-fold (Y142N/E149K) less efficient at infecting cells expressing the quail Tva receptor. These mutations identify key determinants of the interaction between the ALV(A) glycoproteins and the Tva receptor. We also conclude from these results that, at least for the wild-type and variant ALV(A)s tested, the receptor binding affinity was directly related to infection efficiency.     

Mutualistic interactions between vitamin B(12) -dependent algae and heterotrophic bacteria exhibit regulation

Many algae are auxotrophs for vitamin B₁₂ (cobalamin), which they need as a cofactor for B₁₂‐dependent methionine synthase (METH). Because only prokaryotes can synthesize the cobalamin, they must be the ultimate source of the vitamin. In the laboratory, a direct interaction between algae and heterotrophic bacteria has been shown, with bacteria supplying cobalamin in exchange for fixed carbon. Here we establish a system to study this interaction at the molecular level. In a culture of a B₁₂‐dependent green alga Chlamydomonas nivalis, we found a contaminating bacterium, identified by 16S rRNA analysis as Mesorhizobium sp. Using the sequenced strain of M. loti (MAFF303099), we found that it was able to support the growth of B₁₂‐dependent Lobomonas rostrata, another green alga, in return for fixed carbon. The two organisms form a stable equilibrium in terms of population numbers, which is maintained over many generations in semi‐continuous culture, indicating a degree of regulation. However, addition of either vitamin B₁₂ or a carbon source for the bacteria perturbs the equilibrium, demonstrating that the symbiosis is mutualistic and facultative. Chlamydomonas reinhardtii does not require B₁₂ for growth because it encodes a B₁₂‐independent methionine synthase, METE, the gene for which is suppressed by addition of exogenous B₁₂. Co‐culturing C. reinhardtii with M. loti also results in reduction of METE expression, demonstrating that the bacterium can deliver the vitamin to this B₁₂‐independent alga. We discuss the implications of this for the widespread distribution of cobalamin auxotrophy in the algal kingdom.     

Role of calcium in protein folding and function of Tva, the receptor of subgroup A avian sarcoma and leukosis virus

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 microM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.     

Preparation,characterization and immunogenicity of HIV-1 related high-mannose oligosaccharides-CRM<Subscript>197</Subscript> glycoconjugates

The dense glycan shield on the surface of human immunodeficiency virus type 1 (HIV-1) gp120 masks conserved protein epitopes and facilitates virus entry via interaction to glycan binding proteins on susceptible host cells. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose oligosaccharides on the gp120 subunit of HIV-1 Env protein. This oligomannose epitope is currently being considered for the design of a synthetic vaccine. The cluster nature of the 2G12 epitope suggests that a multivalent antigen presentation is important to develop a carbohydrate-based vaccine candidate. In this work we describe the development of neoglycoconjugates displaying clustered HIV-1 related oligomannose carbohydrates. We exploited flexible polyamidoamine (PAMAM) scaffold to generate four- and eight-valent sugar clusters of HIV-1-related oligomannose antigens Man₄, Man₆ and Man₉. The multivalent presentation of oligomannoses increased the avidity of Man₄ and Man₉ to 2G12. The synthetic glycodendrons were then covalently coupled to the protein carrier CRM₁₉₇, formulated with the adjuvant MF59, and used to immunize two animal species. Oligomannose-specific IgG antibodies were generated; however, the antisera failed to recognize recombinant HIV-1 gp120 proteins. We conclude that further structural vaccinology work is needed to identify an antigen presentation that closely matches in vivo the structure of the epitope mapped by 2G12.     

Solution structure of the viral receptor domain of Tva and its implications in viral entry

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue, cysteine-rich motif called the LDL-A module. Here we report the solution structure of the LDL-A module of Tva, determined by nuclear magnetic resonance (NMR) spectroscopy. Although the carboxyl terminus of the Tva LDL-A module has a structure similar to those of other reported LDL-A modules, the amino terminus adopts a different conformation. The LDL-A module of Tva does not contain the signature antiparallel beta-sheet observed in other LDL-A modules, and it is more flexible than other reported LDL-A modules. The LDL-A structure of Tva provides mechanistic insights into how a simple viral receptor functions in retrovirus entry. The side chains of H38 and W48 of Tva, which have been identified as viral contact residues by mutational analysis, are solvent exposed, suggesting that they are directly involved in EnvA binding. However, the side chain of L34, another potential viral contact residue identified previously, is buried inside of the module and forms the hydrophobic core with other residues. Thus L34 likely stabilizes the Tva structure but is not a viral interaction determinant. In addition, we propose that the flexible amino-terminal region of Tva plays an important role in determining specificity in the Tva-EnvA interaction.     

Methylenetetrahydrofolate reductase and transcobalamin genetic polymorphisms in human spontaneous abortion: biological and clinical implications

The pathogenesis of human spontaneous abortion involves a complex interaction of several genetic and environmental factors. The firm association between increased homocysteine concentration and neural tube defects (NTD) has led to the hypothesis that high concentrations of homocysteine might be embryotoxic and lead to decreased fetal viability. There are several genetic polymorphisms that are associated with defects in folate- and vitamin B₁₂-dependent homocysteine metabolism. The methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C polymorphisms cause elevated homocysteine concentration and are associated with an increased risk of NTD. Additionally, low concentration of vitamin B₁₂ (cobalamin) or transcobalamin that delivers vitamin B₁₂ to the cells of the body leads to hyperhomocysteinemia and is associated with NTD. This effect involves the transcobalamin (TC) 776C>G polymorphism. Importantly, the biochemical consequences of these polymorphisms can be modified by folate and vitamin B₁₂ supplementation. In this review, I focus on recent studies on the role of hyperhomocysteinemia-associated polymorphisms in the pathogenesis of human spontaneous abortion and discuss the possibility that periconceptional supplementation with folate and vitamin B₁₂ might lower the incidence of miscarriage in women planning a pregnancy.     

Models for the binary complex of bacteriophage T4 gp59 helicase loading protein: gp32 single-stranded DNA-BINDING protein and ternary complex with pseudo-Y junction DNA

Bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596-18607).     

Identification of two residues within the LDL-A module of Tva that dictate the altered receptor specificity of mutant subgroup A avian sarcoma and leukosis viruses

Avian sarcoma and leukosis virus subgroup A (ASLV-A) entry is mediated by interactions between the viral glycoprotein EnvA and its cognate receptor Tva. Previously, some interesting mutants of ASLV-A have been selected by others which can use chicken Tva, but not quail Tva, for efficient entry. The mutant phenotypes are caused by two point mutations within the surface subunit of EnvA (S. L. Holmen, D. C. Melder, and M. J. Federspiel, J. Virol. 75:726-737, 2001). In this study, we have shown that the altered receptor specificity maps to the LDL-A module of Tva. Further, we have identified two residues in the chicken LDL-A module that allow more efficient viral entry by the mutant viruses. These results demonstrate that the altered receptor specificity of the mutant viruses is determined by specific interactions with residues in the LDL-A module of Tva.     

Characterization of the tertiary structure of soluble CD4 bound to glycosylated full-length HIVgp120 by chemical modification of arginine residues and mass spectrometric analysis

The initial step of infection of blood cells with the human immunodeficiency virus, HIV, is the formation of a complex of the viral envelope protein gp120 and its human receptor CD4. We have examined structural features of recombinant soluble CD4 (sCD4) by chemical modification of arginine residues with hydroxyphenylglyoxal and subsequent analysis by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. As R58, R59, R131, R134, R219, R240, R293, and R329 could be derivatized free in solution, these arginine residues were exposed on the surface of the protein. In the noncovalent complex of sCD4 with HIV(SF2)gp120, only R58, R131, R134, R219, R240, R293, and R329 were accessible for the derivatizing agent. R59 was shielded from hydroxyphenylglyoxal and was, therefore, considered to be part of the interaction site with gp120. This indicates that the carbohydrate moieties and the flexible variable loops of the glycosylated full-length gp120 from HIV strain SF2 do not induce a reorganization of CD4 in its binding to gp120 and, therefore, do not appear to significantly affect the structural orientation of the primary receptor in complex with the HIV envelope protein as compared to the binding observed in the crystal structure of CD4 with truncated deglycosylated gp120.     

Development and application of an α-face-specific radioimmunoassay for vitamin B12

The first development of an α-face-specific radioimmunoassay for vitamin B₁₂ is described. Sheep, fed a cobalt-deficient diet, and immunized with a conjugate between Co-β carboxypropyl cobalamin and keyhole limpet hemocyanin, were used to produce antisera. The antisera crossreacted with Co-β derivatives of vitamin B₁₂, but did not crossreact with the α-face vitamin B₁₂ analog cobinamide. The antisera were used to develop a sensitive and reproducible radioimmunoassay that was free from contamination with the nonspecific vitamin B₁₂ binding protein, R-protein. Both the radioimmunoassay and measurements of plasma concentrations of methylmalonic acid were applied to the diagnosis of cobalt/vitamin B₁₂ deficiency in sheep. The assay correlated well with a commercially available radioassay and did not falsely detect normal vitamin B₁₂ concentration in plasma samples containing elevated concentrations of methylmalonic acid.     

Pathogenicity of a Mutant Friend Spleen Focus-Forming Virus Encoding an Env-Like Membrane Glycoprotein (gp55) with Substitution by a Xenotropic Murine Leukemia Virus Env gp70 Sequence

Friend spleen focus-forming virus (F-SFFV) is a replication-defective acutely leukemogenic mouse retrovirus and encodes an envelope protein (Env)-like membrane glycoprotein (gp55) in its defective env gene, which is responsible for the early stage of the viral leukemogenesis. Gp55 is a modified Env protein and contains a polytropic mink cell focus-inducing (MCF) murine leukemia virus (MuLV) Env gp70-derived sequence in its amino-terminal region. To evaluate the possibility that the presumed binding of gp55 to an MCF MuLV receptor protein has some role in leukemogenesis, we examined the biological activities of a mutant gp55 (XE gp55), which has a xenotropic MuLV Env gp70 amino-terminal region. XE gp55 displayed almost the same biological activities as the wild-type gp55, excluding the above possibility.     

The disulfide loop of gp41 is critical to the furin recognition site of HIV gp160

The importance of the HIV gp41 conserved disulfide loop to envelope function has been examined by mutational and functional analyses. Based on a luciferase-reporter entry assay, mutants gp41-CC/AA (C598A/C604A) and gp41-Delta (deletion of residues 596-606) result in a nonfunctional envelope protein. Western blot analysis shows both mutants to be properly expressed but not processed to form gp120 and gp41, which explains their nonfunctionality. The presence of mutant gp160 on the cell surface, as well as their ability to bind to sCD4, suggests that the mutations have disrupted processing at the furin recognition site encoded within the gp120 conserved domain 5, without resulting in an overall misfolding of the protein. With respect to the furin recognition site, the mutations are sequentially distant, which implies that the gp41 disulfide loop is interacting with gp120 C5 in gp160. In addition, we have modeled the gp120-gp41 interaction in unprocessed precursor gp160 using structural data available for gp120 and gp41 domains in isolation, supplemented by mutagenesis data. We suggest that the mutations have altered the interaction between gp120 C5 and the gp41 disulfide loop, resulting in decreased accessibility of the furin recognition site and implying that the interaction between the gp120 C5 and gp41 loop is a conformational requirement for gp160 processing. The sensitivity of this interaction could be exploited in future antivirals designed to disrupt HIV pathogenesis by disrupting gp160 processing.     

Soluble receptor-induced retroviral infection of receptor-deficient cells

Current models of retroviral entry hypothesize that interactions between the host cell receptor(s) and viral envelope protein induce structural changes in the envelope protein that convert it to an active conformation, allowing it to mediate fusion with the membrane. Recent evidence supporting this hypothesis is the demonstration that Tva, the receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), induces conformational changes in the viral envelope protein. These changes include conversion of the envelope protein to an active, membrane-binding state likely representing a fusogenic conformation. To determine whether binding of the soluble Tva (sTva) receptor was sufficient to activate fully the fusogenic potential of the ASLV-A envelope protein, we have evaluated the ability of ASLV-A to infect receptor-deficient cell lines in the presence of sTva. Soluble receptor efficiently mediated infection of cells devoid of endogenous Tva in a dose-dependent manner, and this infection was dependent absolutely on the addition of sTva. The infectivity of the virus was enhanced dramatically in the presence of the polycationic polymer Polybrene or when centrifugal forces were applied during inoculation, resulting in viral titers comparable to those achieved on cells expressing endogenous receptor. sTva functioned to mediate infection at low concentrations, approaching the estimated binding constant of the receptor and viral envelope protein. These results demonstrate that receptor binding can activate the ASLV-A envelope protein and convert it to a fusogenic conformation competent to mediate the fusion of the viral and cellular membranes.     

Nef can enhance the infectivity of receptor-pseudotyped human immunodeficiency virus type 1 particles

Nef is an accessory protein of human immunodeficiency virus type 1 (HIV-1) that enhances the infectivity of progeny virions when expressed in virus-producing cells. The requirement for Nef for optimal infectivity is, at least in part, determined by the envelope (Env) glycoprotein, because it can be eliminated by pseudotyping HIV-1 particles with pH-dependent Env proteins. To investigate the role of Env in the function of Nef, we have examined the effect of Nef on the infectivity of Env-deficient HIV-1 particles pseudotyped with viral receptors for cells expressing cognate Env proteins. We found that Nef significantly enhances the infectivity of CD4-chemokine receptor pseudotypes for cells expressing HIV-1 Env. Nef also increased the infectivity of HIV-1 particles pseudotyped with Tva, the receptor for subgroup A Rous sarcoma virus (RSV-A), even though Nef had no effect if the pH-dependent Env protein of RSV-A was used for pseudotyping. However, Nef does not always enhance viral infectivity if the normal orientation of the Env-receptor interaction is reversed, because the entry of Env-deficient HIV-1 into cells expressing the vesicular stomatitis virus G protein was unaffected by Nef. Together, our results demonstrate that the presence of a viral Env protein during virus production is not required for the ability of Nef to increase viral infectivity. Furthermore, since the infectivity of Tva pseudotypes was blocked by inhibitors of endosomal acidification, we conclude that low-pH-dependent entry does not always bypass the requirement for Nef.