Receptor-binding domain of murine leukemia virus envelope glycoproteins.

The surface glycoprotein (SU) of murine leukemia viruses (MuLVs) comprises two domains connected by a proline-rich hinge. The interaction of MuLV particles with subgroup-specific cell surface receptors depends primarily on two variable regions (VRA and VRB) located in the amino-terminal domain. To delineate the minimal receptor-binding domains, we examined the capacity of soluble envelope fragments to compete with the entry of virus particles. Amphotropic, ecotropic, polytropic, and xenotropic truncated SUs were produced by inserting stop codons in the env gene of the 4070A, Friend, MCF247 and NZB MuLVs, respectively. These fragments, as well as full-length envelope glycoproteins, were stably expressed in cells bearing the corresponding receptor. Synthesis, posttranslational modifications, transport, and secretion of the env gene products were monitored by immunoprecipitation. Cells expressing the modified SUs or naive cells preincubated with SU-containing conditioned media were infected with different pseudotypes of a retroviral vector carrying a beta-galactosidase marker gene. Reduction of cell susceptibility to infection in the presence of SU was used as a measure of receptor occupancy. The results indicated that the amphotropic and ecotropic envelope amino-terminal domains contain all of the determinants required for receptor binding. In contrast, additional sequences in the proline-rich region were needed for efficient interaction of the polytropic and xenotropic amino-terminal domains with the receptors.     

An amino-terminal fragment of the Friend murine leukemia virus envelope glycoprotein binds the ecotropic receptor.

Retrovirus entry into cells is mediated by specific binding of the envelope glycoprotein to a cell membrane receptor. Constitutive envelope gene expression prevents infection by interfering with the binding of viruses which recognize the same receptor. We have used this property to investigate the receptor binding capacities of deleted or truncated murine leukemia virus ecotropic envelope glycoproteins. Friend murine leukemia virus envelope glycoproteins bearing internal amino-terminal deletions, or a soluble 245-amino-acid gp70 amino-terminal fragment, were expressed in NIH 3T3 cells. The susceptibility of these cells to ecotropic and amphotropic virus infection was determined. We observed that both membrane-bound and soluble forms of the gp70 245-amino-acid amino-terminal domain induced resistance to ecotropic virus, indicating that this fragment binds the ecotropic receptor. Binding occurs both at the cell surface and in the endoplasmic reticulum, as shown by the use of soluble envelope fragments either secreted in the culture supernatants or retained in the endoplasmic reticulum lumen by a KDEL sequence. These results suggest that the gp70 amino-terminal domain folds into a structure which recognizes the ecotropic receptor regardless of the carboxy-terminal part of the molecule.     

Receptor co-operation in retrovirus entry: recruitment of an auxiliary entry mechanism after retargeted binding.

We have constructed Moloney murine leukemia virus (MoMLV)-derived envelope glycoproteins (AMO) displaying an amino-terminal Ram-1-binding domain in which a variety of different amino acid spacers have been inserted between the displayed domain and the MoMLV surface (SU) subunit. Titres of retroviruses generated with these chimeric envelopes were enhanced on cells expressing both Ram-1 and Rec-1 receptors compared with the titres on cells expressing only one or other receptor type. The absolute viral titres and the degree of titre enhancement due to receptor cooperativity were highly variable between the different chimeric envelopes and were determined primarily by the properties of the interdomain spacer. An extreme example of receptor co-operativity was encountered when testing Ram-1-targeted AMOPRO envelopes with specific proline-rich interdomain spacers. AMOPRO viruses could not enter cells expressing only Rec-1 or only Ram-1 but could efficiently infect cells co-expressing both receptors. The data are consistent with a model for receptor co-operativity in which binding to the targeted (Ram-1) receptor triggers conformational rearrangements of the envelope that lead to complete unmasking of the hidden Rec-1-binding domain, thereby facilitating its interaction with the viral (Rec-1) receptor which leads to optimal fusion triggering.     

In vitro binding of purified murine ecotropic retrovirus envelope surface protein to its receptor, MCAT-1.

An amino-terminal portion of the Friend murine leukemia virus (MLV) envelope surface protein [SU, residues 1 to 236 [SU:(1-236)]] and its receptor, MCAT-1, were each purified from insect cells after expression by using recombinant baculoviruses. Friend SU:(1-236) bound specifically to Xenopus oocytes that expressed MCAT-1 with an affinity (Kd, 55 nM) similar to that of viral SU binding to permissive cells. Direct binding of Friend SU:(1-236) to purified MCAT-1 was observed in detergent and after reconstitution into liposomes. Analysis of binding demonstrated that MCAT-1 and Friend SU:(1-236) interact with a stoichiometry of near 1:1. These findings demonstrate that the amino-terminal domain from the SU of ecotropic murine retroviruses contains an MCAT-1 binding domain.     

Role of Variable Regions A and B in Receptor Binding Domain of Amphotropic Murine Leukemia Virus Envelope Protein

For the amphotropic murine leukemia virus (MuLV), a 208-amino-acid amino-terminal fragment of the surface unit (SU) of the envelope glycoprotein is sufficient to bind to its receptor, Pit2. Within this binding domain, two hypervariable regions, VRA and VRB, have been proposed to be important for receptor recognition. In order to specifically locate residues that are important for the interaction with Pit2, we generated a number of site-specific mutations in both VRA and VRB and analyzed the resulting envelope proteins when expressed on retroviral vectors. Concurrently, we substituted portions of the amphotropic SU with homologous regions from the polytropic MuLV envelope protein. The amphotropic SU was unaffected by most of the point mutations we introduced. In addition, the deletion of eight residues in a region of VRA that was previously suggested to be essential for Pit2 utilization only decreased titer on NIH 3T3 cells by 1 order of magnitude. Although the replacement of the amino-terminal two-thirds of VRA with the polytropic sequence abolished receptor binding, smaller nonoverlapping substitutions did not affect the function of the protein. We were not able to identify a single critical receptor contact point within VRA, and we suggest that the amphotropic receptor binding domain probably makes multiple contacts with the receptor and that the loss of some of these contacts can be tolerated.     

Structural elements in glycoprotein 70 from polytropic Friend mink cell focus-inducing virus and glycoprotein 71 from ecotropic Friend murine leukemia virus, as defined by disulfide-bonding pattern and limited proteolysis.

The disulfide-bonding pattern of glycoprotein 70 (gp70), the surface glycoprotein (SU) encoded by the envelope gene of polytropic Friend milk cell focus-inducing virus, was elucidated and compared with that of glycoprotein 71 (gp71), the corresponding glycoprotein of the ecotropic Friend murine leukemia virus, which had previously been determined (M. Linder, D. Linder, J. Hahnen, H.-H. Schott, and Stirm, Eur. J. Biochem. 203:65-73, 1992). In the carboxy-terminal constant domain, in which these glycoproteins have about 97% sequence homology, the location of the four disulfide bonds was found to be analogous. In the amino-terminal differential domain, with about 37% sequence homology, 8 of the 12 cysteine residues of the ecotropic SU are conserved in the polytropic SU. In this domain, a similar clustering of disulfide bonds was detected, which led to the identification of three distinct disulfide-bonded regions in both glycoproteins. However, because of deletions and sequence deviations, the glycoproteins must have significantly different three-dimensional structures in these regions. Since the receptor-binding functions of both glycoproteins have been attributed to their amino-terminal domains and since each binds to a different receptor, these disulfide-bonded structures are likely candidates for receptor-binding functions. Limited proteolysis of both glycoproteins with various endoproteinases led to the identification of preferential proteolytic sites between disulfide-bonded regions, at the beginning of the hypervariable proline-rich region, and between differential and constant domains, further confirming the structural organization of the folded glycoproteins.     

Activation of a cell entry pathway common to type C mammalian retroviruses by soluble envelope fragments

Mutations that negatively or positively affect the fusion properties of murine leukemia viruses (MLVs) have been found within all subdomains of their SU (surface) and TM (transmembrane) envelope units. Yet, the interrelations between these different regions of the envelope complex during the cell entry process are still elusive. Deletion of the histidine residue of the conserved PHQV motif at the amino terminus of the amphotropic or the ecotropic MLV SU resulted in the AdelH or the MOdelH fusion-defective mutant envelope, respectively. These delH mutant envelopes are incorporated on retroviral particles at normal densities and normally mediate virion binding to cells expressing the retroviral receptors. However, both their cell-cell and virus-cell fusogenicities were fully prevented at an early postbinding stage. We show here that the fusion defect of AdelH or MOdelH envelopes was also almost completely reverted by providing either soluble SU or a polypeptide encompassing the receptor-binding domain (RBD) to the target cells, provided that the integrity of the amino-terminal end of either polypeptide was preserved. Restoration of delH envelope fusogenicity was caused by activation of the target cells via specific interaction of the latter polypeptides with the retrovirus receptor rather than by their association with the delH envelope complexes. Moreover crossactivation of the target cells, leading to fusion activation of AdelH or MOdelH envelopes, was achieved by polypeptides containing various type C mammalian retrovirus RBDs, irrespective of the type of entry-defective glycoprotein that was used for infection. Our results indicate that although they recognize different receptors for binding to the cell surface, type C mammalian retroviruses use a common entry pathway which is activated by a conserved feature of their envelope glycoproteins.     

Definition of a 14-Amino-Acid Peptide Essential for the Interaction between the Murine Leukemia Virus Amphotropic Envelope Glycoprotein and Its Receptor

Hydrophilic loops in the receptor binding domain of the amphotropic murine leukemia virus (MLV) envelope glycoprotein (SU) are predicted and may participate in SU-receptor interactions. We have replaced five segments of 6 to 15 amino acids located in each of these regions with an 11-amino-acid tag from the vesicular stomatitis virus glycoprotein (VSV-G). Substitution was compatible with envelope processing, transport, and incorporation into virions. However, three substitution mutants showed a temperature-dependent phenotype, suggesting structural unstability. Accessibility of the tagging epitope for a monoclonal anti-VSV-G antibody was greater in oligomeric than in monomeric SUs when insertion was done in VRA, a domain essential for receptor recognition. In contrast, accessibility was independent of structural constraints when insertion was done in VRB, a domain playing an accessory role in receptor binding. Interaction with the amphotropic receptor was investigated by interference assay and study of binding and infection of target cells with MLV particles coated with the substituted envelopes. Envelope-receptor interaction was abolished when substitution was performed in a potential loop-forming segment located at the N-terminal half of VRA. Although interaction was affected to variable extents, depending on the substituted segment, other mutants conserved the ability to interact with the amphotropic receptor. These experiments indicate the 14-amino-acid segment between positions 50 and 64 of SU as an essential determinant of amphotropic-receptor recognition. They also show that a foreign linear epitope can be tolerated in several locations of the amphotropic SU receptor binding site, and this result has implications for the design of targeted retroviral vectors.     

Identification of Envelope Determinants of Feline Leukemia Virus Subgroup B That Permit Infection and Gene Transfer to Cells Expressing Human Pit1 or Pit2

The retroviral vector systems that are in common use for gene therapy are designed to infect cells expressing either of two widely expressed phosphate transporter proteins, Pit1 or Pit2. Subgroup B feline leukemia viruses (FeLV-Bs) use the gibbon ape leukemia virus receptor, Pit1, as a receptor for entry. Our previous studies showed that some chimeric envelope proteins encoding portions of FeLV-B could also enter cells by using a related receptor protein, Pit2, which serves as the amphotropic murine leukemia virus receptor (S. Boomer, M. Eiden, C. C. Burns, and J. Overbaugh, J. Virol. 71:8116--8123, 1997). Here we show that an arginine at position 73 within variable region A (VRA) of the FeLV-B envelope surface unit (SU) is necessary for viral entry into cells via the human Pit2 receptor. However, C-terminal SU sequences have a dominant effect in determining human Pit2 entry, even though this portion of the protein is outside known receptor binding domains. This suggests that a combination of specific VRA sequences and C-terminal sequences may influence interactions between FeLV-B SU and the human Pit2 receptor. Binding studies suggest that the C-terminal sequences may affect a postbinding step in viral entry via the Pit2 receptor, although in all cases, binding of FeLV-B SU to human Pit2 was weak. In contrast, neither the arginine 73 nor specific C-terminal sequences are required for efficient binding or infection with Pit1. Taken together, these data suggest that different residues in SU may interact with these two receptors. The specific FeLV-Bs described here, which can enter cells using either human Pit receptor, may be useful as envelope pseudotypes for viruses used in gene therapy.     

Functional characterization of the N termini of murine leukemia virus envelope proteins

The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry.     

A Proline-Rich Motif Downstream of the Receptor Binding Domain Modulates Conformation and Fusogenicity of Murine Retroviral Envelopes

The entry of retroviruses into cells depends on receptor recognition by the viral envelope surface subunit SU followed by membrane fusion, which is thought to be mediated by a fusion peptide located at the amino terminus of the envelope transmembrane subunit TM. Several fusion determinants have been previously identified in murine leukemia virus (MLV) envelopes, but their functional interrelationships as well as the processes involved in fusion activation upon retroviral receptor recognition remain unelucidated. Despite both structural and functional similarities of their envelope glycoproteins, ecotropic and amphotropic MLVs display two different postbinding properties: (i) while amphotropic MLVs fuse the cells at neutral pH, penetration of ecotropic MLVs is relatively acid pH dependent and (ii) ecotropic envelopes are more efficient than amphotropic envelopes in inducing cell-to-cell fusion and syncytium formation. By exploiting the latter characteristic in the analysis of chimeras of ecotropic and amphotropic MLV envelopes, we show here that substitution of the ecotropic MLV proline-rich region (PRR), located in the SU between the amino-terminal receptor binding domain and the TM-interacting SU carboxy-terminal domains, is sufficient to revert the amphotropic low-fusogenic phenotype into a high-fusogenic one. Furthermore, we have identified potential β-turns in the PRR that control the stability of SU-TM associations as well as the thresholds required to trigger either cell-to-cell or virus-to-cell fusion. These data, demonstrating that the PRR functions as a signal which induces envelope conformational changes leading to fusion, have enabled us to derive envelopes which can infect cells harboring low levels of available amphotropic receptors.     

Identification of a subdomain in the Moloney murine leukemia virus envelope protein involved in receptor binding.

We have mutated amino acids within the receptor-binding domain of Moloney murine leukemia virus envelope in order to identify residues involved in receptor binding. Analysis of mutations in the region of amino acids 81 to 88 indicates that this region is important for specific envelope-receptor interactions. None of the aspartate 84 (D-84) mutants studied bind measurably, although they are efficiently incorporated into particles. D-84 mutants have titers that correspond to the severity of the substitution. This observation suggests that D-84 may provide a direct receptor contact. Mutations in the other charged amino acids in this domain (R-83, E-86, and E-87) yield titers similar to those of wild-type envelope, but the affinity of the mutant envelope in the binding assay is decreased by nonconservative substitutions in parallel to the severity of the change. These other amino acids may either provide secondary receptor contacts or assist in maintaining a structure in the domain that favors efficient binding. We also studied other regions of high hydrophilicity. Our initial characterization indicates that amino acids 106 to 111 and 170 to 188 do not play a major role in receptor binding. Measurements of relative binding affinity and titer indicate that most mutations in the region of amino acids 120 to 131 did not significantly affect receptor binding. However, SU encoded by mutants H123V, R124L, and C131A as well as C81A could not be detected in particles and therefore did not bind measurably. Therefore, the region encompassed by amino acids 81 to 88 appears to be directly involved in receptor binding.     

Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein

The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.     

Bovine leukemia virus SU protein interacts with zinc,and mutations within two interacting regions differently affect viral fusion and infectivity in vivo

Bovine leukemia virus (BLV) and human T-cell lymphotropic virus type 1 (HTLV-1) belong to the genus of deltaretroviruses. Their entry into the host cell is supposed to be mediated by interactions of the extracellular (SU) envelope glycoproteins with cellular receptors. To gain insight into the mechanisms governing this process, we investigated the ability of SU proteins to interact with specific ligands. In particular, by affinity chromatography, we have shown that BLV SU protein specifically interacted with zinc ions. To identify the protein domains involved in binding, 16 peptides distributed along the sequence were tested. Two of them appeared to be able to interact with zinc. To unravel the role of these SU regions in the biology of the virus, mutations were introduced into the env gene of a BLV molecular clone in order to modify residues potentially interacting with zinc. The fusogenic capacity of envelope mutated within the first zinc-binding region (104 to 123) was completely abolished. Furthermore, the integrity of this domain was also required for in vivo infectivity. In contrast, mutations within the second zinc-binding region (218 to 237) did not hamper the fusogenic capacity; indeed, the syncytia were even larger. In sheep, mutations in region 218 to 237 did not alter infectivity or viral spread. Finally, we demonstrated that the envelope of the related HTLV-1 was also able to bind zinc. Interestingly, zinc ions were found to be associated with the receptor-binding domain (RBD) of Friend murine leukemia virus (Fr-MLV) SU glycoprotein, further supporting their relevance in SU structure. Based on the sequence similarities shared with the Fr-MLV RBD, whose three-dimensional structure has been experimentally determined, we located the BLV zinc-binding peptide 104-123 on the opposite side of the potential receptor-binding surface. This observation supports the hypothesis that zinc ions could mediate interactions of the SU RBD either with the C-terminal part of SU, thereby contributing to the SU structural integrity, or with a partner(s) different from the receptor.     

Variable regions A and B in the envelope glycoproteins of feline leukemia virus subgroup B and amphotropic murine leukemia virus interact with discrete receptor domains.

The surface (SU) envelope glycoproteins of feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) are highly related, even in the variable regions VRA and VRB that have been shown to be required for receptor recognition. However, FeLV-B and A-MLV use different sodium-dependent phosphate symporters, Pit1 and Pit2, respectively, as receptors for infection. Pit1 and Pit2 are predicted to have 10 membrane-spanning domains and five extracellular loops. The close relationship of the retroviral envelopes enabled us to generate pseudotype virions carrying chimeric FeLV-B/A-MLV envelope glycoproteins. We found that some of the pseudotype viruses could not use Pit1 or Pit2 proteins but could efficiently utilize specific chimeric Pit1/Pit2 proteins as receptors. By studying Mus dunni tail fibroblasts expressing chimeric Pit1/Pit2 proteins and pseudotype virions carrying chimeric FeLV-B/A-MLV envelopes, we show that FeLV-B and A-MLV VRA and VRB interact in a modular manner with specific receptor domains. Our results suggest that FeLV-B VRA interacts with Pit1 extracellular loops 4 and 5 and that residues Phe-60 and Pro-61 of FeLV-B VRA are essential for receptor choice. However, this interaction is insufficient for infection, and an additional interaction between FeLV-B VRB and Pit1 loop 2 is essential. Similarly, A-MLV infection requires interaction of A-MLV VRA with Pit2 loops 4 and 5 and VRB with Pit2 loop 2, with residues Tyr-60 and Val-61 of A-MLV VRA being critical for receptor recognition. Together, our results suggest that FeLV-B and A-MLV infections require two major discrete interactions between the viral SU envelope glycoproteins and their respective receptors. We propose a common two-step mechanism for interaction between retroviral envelope glycoproteins and cell surface receptors.     

The Receptor Binding Site of Feline Leukemia Virus Surface Glycoprotein Is Distinct from the Site Involved in Virus Neutralization

The external surface glycoprotein (SU) of feline leukemia virus (FeLV) contains sites which define the viral subgroup and induce virus-neutralizing antibodies. The subgroup phenotypic determinants have been located to a small variable region, VR1, towards the amino terminus of SU. The sites which function as neutralizing epitopes in vivo are unknown. Recombinant SU proteins were produced by using baculoviruses that contained sequences encoding the SUs of FeLV subgroup A (FeLV-A), FeLV-C, and two chimeric FeLVs (FeLV-215 and FeLV-VC) in which the VR1 domain of FeLV-A had been replaced by the corresponding regions of FeLV-C isolates. The recombinant glycoproteins, designated Bgp70-A, -C, -215, and -VC, respectively, were similar to their wild-type counterparts in several immunoblots and inhibited infection of susceptible cell lines in a subgroup-specific manner. Thus, Bgp70-A interfered with infection by FeLV-A, whereas Bgp70-C, -VC, and -215 did not. Conversely, Bgp70-C, -VC, and -215 blocked infection with FeLV-C, while Bgp70-A had no effect. These results indicate that the site on SU which binds to the FeLV cell surface receptor was preserved in the recombinant glycoproteins. It was also found that the recombinant proteins were able to bind naturally occurring neutralizing antibodies. Bgp70-A, -VC, and -215 interfered with the action of anti-FeLV-A neutralizing antibodies, whereas Bgp70-C did not. Furthermore, Bgp70-C interfered with the action of anti-FeLV-C neutralizing antibodies, while the other proteins did not. These results indicate that the neutralizing epitope(s) of FeLV SU lies outside the subgroup-determining VR1 domain.     

The Hypervariable Domain of the Murine Leukemia Virus Surface Protein Tolerates Large Insertions and Deletions, Enabling Development of a Retroviral Particle Display System

The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.     

Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix.

Incorporation of envelope glycoproteins into a budding retrovirus is an essential step in the formation of an infectious virus particle. By using site-directed mutagenesis, we identified specific amino acid residues in the matrix domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein that are critical to the incorporation of HIV-1 envelope glycoproteins into virus particles. Pseudotyping analyses were used to demonstrate that two heterologous envelope glycoproteins with short cytoplasmic tails (the envelope of the amphotropic murine leukemia virus and a naturally truncated HIV-2 envelope) are efficiently incorporated into HIV-1 particles bearing the matrix mutations. Furthermore, deletion of the cytoplasmic tail of HIV-1 transmembrane envelope glycoprotein gp41 from 150 to 7 or 47 residues reversed the incorporation block imposed by the matrix mutations. These results suggest the existence of a specific functional interaction between the HIV-1 matrix and the gp41 cytoplasmic tail.     

Mapping of receptor binding domains in the envelope protein of spleen necrosis virus.

Spleen necrosis virus (SNV) is an amphotropic retrovirus originally isolated from a duck. Although of avian origin, it also replicates on some mammalian cells. SNV-derived retroviral vectors work with high efficiency and have a high potential for various gene transfer applications. However, little is known about the envelope-receptor interactions of this virus. We constructed a series of recombinant envelope proteins to characterize the SU peptide of SNV. We found that, in contrast to the envelope proteins of other retroviruses, truncated envelope proteins of SNV are transported to the cell surface. Surprisingly, particles displaying truncated envelope proteins can still infect cells, although at reduced efficiencies. Furthermore, these proteins can confer partial superinfection interference. Our data suggest that peptides throughout SU are involved in envelope-receptor interactions. To more precisely determine the localization of the main receptor binding domain, point mutations were introduced at certain regions of the SNV SU which are highly conserved among retroviruses belonging to the same receptor interference group. We identified one point mutation in the middle of SU (position 192) which drastically reduced infectivity and strongly reduced the ability to confer superinfection interference. The level of expression was not abolished, and translocation to the cell membrane of the mutant envelope occurred efficiently. This indicates that amino acid 192 may be directly involved in receptor binding.     

Functional Domains in the Retroviral Transmembrane Protein

The envelope glycoproteins of the mammalian type C retroviruses consist of two subunits, a surface (SU) protein and a transmembrane (TM) protein. SU binds to the viral receptor and is thought to trigger conformational changes in the associated TM protein that ultimately lead to the fusion of viral and host cell membranes. For Moloney murine leukemia virus (MoMuLV), the envelope protein probably exists as a trimer. We have previously demonstrated that the coexpression of envelope proteins that are individually defective in either the SU or TM subunits can lead to functional complementation (Y. Zhao et al., J. Virol. 71:6967–6972, 1997). We have now extended these studies to investigate the abilities of a panel of fusion-defective TM mutants to complement each other. This analysis identified distinct complementation groups within TM, with implications for interactions between different regions of TM in the fusion process. In viral particles, the C-terminal 16 amino acids of the MoMuLV TM (the R peptide) are cleaved by the viral protease, resulting in an increased fusogenicity of the envelope protein. We have examined the consequences of R peptide cleavage for the different TM fusion mutants and have found that this enhancement of fusogenicity can only occur in cis to certain of the TM mutants. These results suggest that R peptide cleavage enhances the fusogenicity of the envelope protein by influencing the interaction of two distinct regions in the TM ectodomain.