Intracellular transport and leukemogenicity of spleen focus-forming virus envelope glycoproteins with altered transmembrane domains.

Friend murine spleen focus-forming virus (SFFV) encodes a glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 lacks a cytoplasmic domain and is defective in its transport to the cell surface. We constructed a chimeric envelope gene which codes for a molecule with an external domain derived from the SFFV envelope gene and membrane-spanning and cytoplasmic domains derived from the Friend murine leukemia virus envelope gene. Like gp52, the chimeric protein was defective in its transport to the cell surface, indicating that the absence of a cytoplasmic tail is not responsible for the defective intracellular transport of SFFV gp52. However, unlike wild-type SFFV, the chimeric SFFV genome failed to induce erythroleukemia in adult mice. The results indicate that the altered membrane-spanning domain, lack of a detectable cytoplasmic tail in gp52, or both factors are prerequisites for the erythroleukemia-inducing properties of SFFV but are not responsible for the block in intracellular transport of the glycoprotein.     

The spleen focus-forming virus envelope glycoprotein is defective in oligomerization

The gp52 envelope glycoprotein of Friend spleen focus-forming virus (SFFV) is a recombinant molecule derived from Friend murine leukemia virus (MuLV) by various deletions, insertions, and substitutions. The SFFV gp52 glycoprotein, unlike MuLV envelope glycoproteins, is defective in transport to the cell surface. Only 3-5% of gp52 eventually reaches the cell surface as a processed form (gp65). Although gp52 lacks cytoplasmic tail residues found in MuLV glycoproteins, we have previously shown that this deletion is not responsible for its defective transport. In order to investigate the basis for the defective transport of gp52, we have examined the folding and assembly of gp52 molecules into oligomeric molecules. CV-1 cells infected with vaccinia virus recombinants expressing SFFV gp52 were pulse labeled and the cell extracts were fractionated by velocity centrifugation through sucrose gradients. Immediately after a 10-min pulse, gp52 was detected as a monomer in the upper part of the sucrose gradient (fractions 12 and 14) and it remained as such after a 2-h chase period. However, the processed form, gp65, was found in a lower part of the gradient (fraction 8) after a 2-h chase. The position of gp65 was found to correspond to the position of trimeric influenza hemagglutinin which was analyzed on a parallel sucrose gradient, suggesting that gp65 also exists as a trimer in this fraction. These results indicate that changes in the external domain of gp52 result in improper folding of the glycoprotein molecule, and suggest that this lack of oligomerization is responsible for the defective transport of the molecules. Only those molecules that do form oligomeric structures are transported to the Golgi complex and undergo further oligosaccharide processing, and transport to the cell surface.     

Membrane association and defective transport of spleen focus-forming virus glycoproteins

The gp52 glycoprotein of the spleen focus-forming virus found in the Friend and Rauscher complexes of murine leukemia viruses (MuLV) has been previously identified as a recombinant molecule involving substitutions and deletions of the MuLV env gene. Unlike the MuLV structural glycoproteins, gp52 is defective in its transport to the cell surface. We have studied aspects of the intracellular transport and membrane association of gp52 to investigate the possible mechanisms underlying the defective transport process. It was found that a panel of monoclonal antibodies to different epitopes of p 15E, as well as an antiserum to a synthetic peptide corresponding to the carboxy terminus of MuLV envelope precursors, failed to react with gp52. Despite the possible absence of membrane-anchoring regions of MuLV envelope proteins known to reside on p 15E, gp52 was not found to be secreted into the culture fluids. Detergent extraction studies indicated that gp52 is associated with the membranes and not the contents of microsomal vesicles in speen focus-forming virus-infected cells. gp65, the processed form of gp52, could be labeled with [3H]palmitic acid, suggesting a membrane association. To determine whether a spontaneous denaturation occurs leading to aggregation and defective transport of gp52, we studied the surface expression of gp52 in cells grown at different temperatures, as well as the solubility of gp52 in low concentrations of Triton X-100. No evidence of aggregation or of a temperature-dependent difference in transport was obtained. gp52 appears to be a monotopic integral membrane protein, unlike MuLV envelope proteins which are bitopic integral membrane proteins; proteolytic digestion of intact microsomal vesicles did not reveal a detectable cytoplasmic tail under conditions where this could be demonstrated on MuLV envelope precursors. We suggest that a loss of putative signals involved in mediating intracellular transport is a likely cause for the defective transport of the spleen focus-forming virus glycoproteins.     

Glycoprotein encoded by the Friend spleen focus-forming virus.

The Friend spleen focus-forming virus (F-SFFV) released from cultured erythroleukemia cells (cell line F4-6/K) was cloned free of its helper lymphatic leukemia virus (F-MuLV). After allowing adsorption to Sc-1 fibroblasts at a low multiplicity of infection, the cells were seeded individually into wells of a microtitier test plate and the resulting colonies were grown into large cultures. Among 14 of these cell cultures that have been analyzed thoroughly, 6 contained F-SFFV alone, 1 contained F-MuLV plus F-SFFV, and 7 were uninfected. Each of the Sc-1 cell lines which had been infected with cloned F-SFFV contained a glycoprotein with an apparent molecular weight of 55,000 (gp55) that was absent from the cell lines that lacked F-SFFV. gp55 was also present in Friend erythroleukemia cells and in fibroblasts infected with an F-SFFV that had been doubly cloned in another laboratory. These results indicate that gp55 is encoded by the F-SFFV genome. gp55 has the following additional properties. It can be immunoprecipitated with antiserum made to the F-MuLV virion envelope glycoprotein (gp75). Its unglycosylated polypeptide, formed in cells treated with 2-deoxy-D-glucose, has a molecular weight of approximately 45,000. Its tryptic peptide map contains peptides in common with F-MuLV gp75 but it also contains unique peptides. It appears to be absent or present in only low concentrations in erythroleukemia cell plasma membranes as determined by lactoperoxidase-catalyzed iodination, and it accumulates intracellularly in large amounts. In addition, it is absent from released virions. The majority of the cellular gp55 has an isoelectric point of 8.5 to 9.0. These results are consistent with the idea that an env gene recombination event was involved in the origin of F-SFFV.     

The hydrophobic membrane-spanning sequences of the gp52 glycoprotein are required for the pathogenicity of Friend spleen focus-forming virus.

Friend spleen focus-forming virus (SFFV) codes for a transport-defective envelope glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 is a monotopic integral membrane protein anchored in the membrane by a stretch of hydrophobic amino acid residues located near the carboxy terminus of the molecule. We have constructed a mutant SFFV envelope gene in which the sequences that code for the hydrophobic membrane-spanning domain have been deleted, and we expressed this gene by using recombinant vaccinia virus vectors or retroviral vectors. The mutant SFFV envelope gene was found to encode a truncated glycoprotein (gp52t) which was also transport defective; a majority of gp52t remained cell associated, while a small proportion of the molecules underwent oligosaccharide processing. The processed form of gp52t was secreted from the cells. Retroviral vectors carrying the mutant SFFV envelope gene were found to be nonpathogenic in adult mice. These results indicate that the hydrophobic membrane-spanning region of gp52 is required for pathogenicity of SFFV and suggest that these sequences may play a role in signal transduction. The results also indicate that the transport defect of SFFV gp52 is due to structural features of the ectodomain of the molecule.     

Nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells

Vaccinia virus recombinants were generated which express the intact gp70/p15E of Friend mink cell focus inducing virus (F-MCFV) or truncated forms of the glycoprotein that lack the transmembrane and cytoplasmic domains. The transport of the intact and truncated envelope glycoproteins to apical or basolateral surfaces was studied in the polarized epithelial MDCK cell line. Infection of MDCK cells with the recombinant expressing the intact F-MCFV envelope glycoprotein resulted in transport exclusively to the basolateral surfaces, whereas the recombinant expressing the truncated glycoprotein was found to be secreted from both the apical and basolateral surfaces. Thus removal of the transmembrane and cytoplasmic domains of the p15E protein results in a loss of directional transport to the basolateral membrane of polarized epithelial cells.     

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.     

Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions.

The envelope glycoprotein complex of Rous sarcoma virus consists of a knoblike, receptor-binding gp85 polypeptide that is linked through disulfide bonds to a membrane-spanning gp37 spike. We used oligonucleotide-directed mutagenesis to assess the role of the hydrophobic transmembrane region and hydrophilic cytoplasmic domain of gp37 in intracellular transport and assembly into virions. Early termination codons were introduced on either side of the hydrophobic transmembrane region, and the mutated env genes were expressed from the late promoter of simian virus 40. This resulted in the synthesis of glycoprotein complexes composed of a normal gp85 and a truncated gp37 molecule that lacked the cytoplasmic domain alone or both the cytoplasmic and transmembrane domains. The biosynthesis and intracellular transport of the truncated proteins were not significantly different from those of the wild-type glycoproteins, suggesting that any protein signals for biosynthesis and intracellular transport of this viral glycoprotein complex must reside in its extracellular domain. The glycoprotein complex lacking the cytoplasmic domain of gp37 is stably expressed on the cell surface in a manner similar to that of the wild type. In contrast, the complex lacking both the transmembrane and cytoplasmic domains is secreted as a soluble molecule into the media. It can be concluded, therefore, that the transmembrane domain alone is essential for anchoring the RSV env complex in the cell membrane and that the cytoplasmic domain is not required for anchor function. Insertion of the mutated genes into an infectious proviral genome allowed us to assess the ability of the truncated gene products to be assembled into virions and to determine whether such virions were infectious. Viral genomes encoding the secreted glycoprotein were noninfectious, whereas those encoding a glycoprotein complex lacking only the cytoplasmic domain of gp37 were infectious. Virions produced from these mutant-infected cells contained normal levels of glycoprotein. The cytoplasmic tail of gp37 is thus not required for the assembly of envelope glycoproteins into virions. It is unlikely, therefore, that this region of gp37 interacts with viral core proteins during the selective incorporation of viral glycoproteins into the viral envelope.     

Subcellular localization of the env-related glycoproteins in Friend erythroleukemia cells.

A scheme was developed for the subcellular fractionation of murine erythroleukemia cells transformed by Friend leukemia virus. The subcellular localization of the env-related glycoproteins was determined by immune precipitation with antiserum against gp70, the envelope glycoprotein of the helper virus, followed by gel electrophoresis. In cells labeled for 2 h with [35S]methionine, the glycoprotein encoded by the defective spleen focus-forming virus, gp55SFFV, was found primarily in the nuclear fraction and in fractions containing dense cytoplasmic membranes such as endoplasmic reticulum. A similar distribution was noted for gp85env, the precursor to gp70. The concentration of viral glycoproteins in the nuclear fraction could not be accounted for by contamination with endoplasmic reticulum. In pulse-chase experiments, neither glycoprotein underwent major redistribution. However, labeled gp85env disappeared from intracellular membranes with a half-time of 30 min to 1 h, whereas labeled gp55SFFV was stable during a 2-h chase. In plasma membrane preparations with very low levels of contamination with endoplasmic reticulum, gp70 was the major viral env-related glycoprotein detected; a minor amount of gp55SFFV and no gp85env could be detected. The unexpected result of these experiments is the amount of viral glycoproteins found in the nuclear fraction. Presence of viral proteins in the nucleus could be relevant to the mechanism of viral leukemogenesis.     

Conversion of Friend mink cell focus-forming virus to Friend spleen focus-forming virus by modification of the 3'' half of the env gene.

The 3' half of the env gene of the dualtropic Friend mink cell focus-forming virus was modified by replacing the restriction enzyme fragment of the genome DNA with the corresponding fragment of the acutely leukemogenic, polycythemia-inducing strain of Friend spleen focus-forming virus (F-SFFVP) genome DNA. Replacement with the fragment of F-SFFVP env containing the 585-bp deletion, the 6-bp duplication, and the single-base insertion converted the resulting chimeric genome so that the mutant had a pathogenic activity like that of F-SFFVP. Replacement with the fragment containing only the 585-bp deletion did not result in a pathogenic virus. However, when this virus pseudotyped by Friend murine leukemia virus was passaged in newborn DBA/2 mice, we could recover weakly pathogenic viruses with a high frequency. Molecular analysis of the genome of the recovered virus revealed the presence of a single-base insertion in the same T5 stretch where the wild-type F-SFFV env has the single-base insertion. These results provided evidence that the unique genomic structures present in the 3' half of F-SFFV env are the sole determinants that distinguish the pathogenicity of F-SFFV from that of Friend mink cell focus-forming virus. The importance of the dualtropic env-specific sequence present in the 5' half of F-SFFV env for the pathogenic activity was evaluated by constructing a mutant F-SFFV genome in which this sequence was replaced by the ecotropic env sequence of Friend murine leukemia virus and by examining its pathogenicity. The results indicated that the dualtropic env-specific sequence was essential to pathogenic activity.     

Both the changes of six amino acids and the C-terminal truncation caused by a one-base insertion in the defective env gene of Friend spleen focus-forming virus significantly affect the pathogenic activity of the encoded leukemogenic membrane glycoprotein (gp55).

Friend spleen focus-forming virus (F-SFFV) causes acute erythroleukemia in mice and encodes in its defective env gene an Env-like membrane glycoprotein (gp55). The F-SFFV env gene has three characteristic structures compared with that of ecotropic murine leukemia viruses (MuLVs): substitution by the polytropic MuLV env sequence, a 585-bp deletion, and a 1-bp insertion. All of these characteristic structures are essential for the leukemogenic potential of gp55 of polycythemia-inducing isolates of F-SFFV (F-SFFVp). The 1-bp insertion causes changes of six amino acids and truncation by 34 amino acids at the C terminus. In this study, we constructed 12 mutant F-SFFV genomes starting from the wild-type F-SFFVp and examined the effect of the C-terminal truncation and the six altered amino acids on the pathogenic activity of gp55. The results indicated that at least 18 to 24 amino acids must be deleted from the C terminus for the env product to be pathogenically active. We also found that the six altered amino acids contributed significantly to the pathogenic activity of gp55. Analyses of the cellular processing of these mutant gp55s supported a correlation between the pathogenic activity of gp55 and its efficiency in overall cellular processing.     

Human immunodeficiency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domains of CD4: role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic reticulum.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is a transmembrane phosphoprotein which induces rapid degradation of CD4 in the endoplasmic reticulum (ER). To identify sequences in CD4 for Vpu-induced degradation, we generated four chimeric envelope glycoproteins having the ectodomain of HIV-1 gp160, the anchor domain of CD4, and 38, 25, 24, and 18 amino acids (aa) of the CD4 cytoplasmic domain. Using the vaccinia virus-T7 RNA polymerase expression system, we analyzed the expression of chimeric proteins in the presence and absence of Vpu. In singly transfected cells, the chimeric envelope glycoproteins having 38, 24, and 18 aa of the CD4 cytoplasmic domain were endoproteolytically cleaved and biologically active in the fusion of HeLa CD4+ cells. However, one of the chimeras having 25 aa of the CD4 cytoplasmic tail was retained in the ER using the transmembrane ER retention signal and was defective in membrane fusion. Furthermore, biochemical analyses of the coexpressing cells revealed that the Vpu protein induced degradation of the envelope glycoproteins having 38, 25, and 24 aa of the CD4 cytoplasmic tail and degradation occurred in the ER. Consequently, the fusion-competent glycoproteins did not induce the formation of syncytia in HeLa CD4+ cells expressing Vpu. However, the HIV-1 gp160 and chimeric envelope glycoprotein having the membrane-proximal 18 aa of the CD4 cytoplasmic tail were stable and fusion competent in cells expressing Vpu. In addition, we examined the stability of CD4 molecules in the presence of Vpu. Coexpression analyses revealed that the Vpu protein induced degradation of CD4 whereas mutant CD4 having the membrane-proximal 18 aa of the cytoplasmic domain was relatively stable in the presence of Vpu. Taken together, these studies have elucidated that the Vpu protein requires sequences or sequence determinants in the cytoplasmic domain of CD4 to induce degradation of the glycoproteins in the cell.     

Monoclonal antibody to spleen focus-forming virus-encoded gp52 provides a probe for the amino-terminal region of retroviral envelope proteins that confers dual tropism and xenotropism.

Monoclonal antibodies which recognize a region common to Friend spleen focus-forming virus encoded gp52 and Friend mink cell focus-inducing viral gp70 were isolated. One such antibody from hybridoma 7C10 was tested extensively in immune precipitation and was found to react with a determinant on envelope gp70s of all mink cell focus-inducing, xenotropic, and amphotropic mouse retroviruses tested, but not with envelope gp70s of ecotropic viruses, including Friend, Moloney, and AKR murine leukemia viruses. Monoclonal antibody from hybridoma 7C10 precipitated a 23,000-molecular-weight fragment, derived by V8 protease digestion of Friend mink cell focus-inducing gp70. This 23,000-molecular-weight peptide was determined to derive from the amino terminus of the molecule. These results correlate well with other genetic data which indicate that endogenously acquired sequences of mink cell focus-inducing viruses are found at the 5' end of the envelope gene.     

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.     

The carboxy terminus of human immunodeficiency virus type 1 gp160 limits its proteolytic processing and transport in transfected cell lines.

Mutagenesis of the transmembrane domain and cytoplasmic tail of human immunodeficiency virus type 1 envelope glycoprotein gp160 revealed that its intracellular transport and processing in transfected cell lines were modulated by a functional domain included in the carboxy-terminal sequence consisting of residues 751 to 856.     

The transmembrane domain and cytoplasmic tail of herpes simplex virus type 1 glycoprotein H play a role in membrane fusion

Herpes simplex virus glycoprotein H (gH) is one of the four virion envelope proteins which are required for virus entry and for cell-cell fusion in a transient system. In this report, the role of the transmembrane and cytoplasmic tail domains of gH in membrane fusion was investigated by generating chimeric constructs in which these regions were replaced with analogous domains from other molecules and by introducing amino acid substitutions within the membrane-spanning sequence. gH molecules which lack the authentic transmembrane domain or cytoplasmic tail were unable to mediate cell-cell fusion when coexpressed with gB, gD, and gL and were unable to rescue the infectivity of a gH-null virus as efficiently as a wild-type gH molecule. Many amino acid substitutions of specific amino acid residues within the transmembrane domain also affected cell-cell fusion, in particular, those introduced at a conserved glycine residue. Some gH mutants that were impaired in cell-cell fusion were nevertheless able to rescue the infectivity of a gH-negative virus, but these pseudotyped virions entered cells more slowly than wild-type virions. These results indicate that the fusion event mediated by the coexpression of gHL, gB, and gD in cells shares common features with the fusion of the virus envelope with the plasma membrane, they point to a likely role for the membrane-spanning and cytoplasmic tail domains of gH in both processes, and they suggest that a conserved glycine residue in the membrane-spanning sequence is crucial for efficient fusion.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

A chimeric protein of simian immunodeficiency virus envelope glycoprotein gp140 and Escherichia coli aspartate transcarbamoylase

The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R(2)) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C(6)R(6) ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.     

Spleen focus-forming virus: specific neutralization by antisera to certain gag gene-encoded proteins.

Immunization of rats with syngeneic cells infected with spleen focus-forming virus (SFFV) but not with its helper, Friend murine leukemia virus (FMuLV), produces antisera which specifically neutralize SFFV, and not FMuLV, in the Friend virus complex. To determine which SFFV-encoded protein molecule bears the antigen recognized by these neutralizing antibodies, we studied different lots of rat anti-SFFV antiserum by immunoprecipitation and virus neutralization assays. The ability of these sera to neutralize SFFV correlated with the titer of antibodies to p45gag and not with the titer of those to gp52, suggesting that the neutralizing antibodies recognize the p45gag molecule. To verify this specificity for p45gag, we tested antisera to various MuLV gag gene-encoded proteins for neutralization of SFFV. Goat anti-Rauscher murine leukemia virus (RMuLV) p30 and goat anti-RMuLV p10 sera neither precipitated p45gag from SFFV-infected nonproducer cells nor neutralized SFFV. In contrast, goat anti-RMuLV Pr65gag and goat anti-RMuLV p12 sera precipitated p45gag from SFFV-infected cells and also specifically neutralized SFFV in the Friend virus complex. These findings suggest that, unlike the gag proteins coded for by FMuLV, the proteins coded for by defective SFFV are incorporated into the envelope of virions carrying the SFFV genome, but not into the envelope of those carrying the helper FMuLV genome.