Improvement of retroviral retargeting by using amino acid spacers between an additional binding domain and the N terminus of Moloney murine leukemia virus SU.

We previously reported a strategy to redirect the retroviral host range by expressing single-chain antibodies (S. J. Russell, R. E. Hawkins, and G. Winter, Nucleic Acids Res. 21:1081-1085, 1993) or ligands (F.-L. Cosset, F. J Morling, Y. Takeuchi, R. A. Weiss, M. K. L. Collins, and S. J. Russell, J. Virol. 69:6314-6322, 1995) at the N terminus of Moloney murine leukemia virus (MoMLV) surface proteins (SU). Although such chimeric envelopes were able to bind the new receptors, the transduction efficiency of retargeted viruses was generally low. We hypothesized that conformational rearrangements of envelope glycoproteins were not optimally triggered following binding, and to overcome these postbinding blocks, we have generated here a set of chimeric MoMLV-derived envelopes targeted to the Ram-1 phosphate transporter in which we have varied the spacing between the Ram-1-binding domain and the MoMLV SU. All of the recombinant envelopes were correctly expressed on virions, and all bound efficiently to Ram-1. However, the interdomain spacing greatly affected the efficiency of gene transfer by retroviral vectors that had bound to Ram-1 via their chimeric envelopes. Optimal interdomain spacing allowed a 100-fold-increased viral transduction via Ram-1 compared to our previous results.     

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.     

Characterization of chimeras between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A envelope proteins.

A series of 22 chimeric envelope (env) genes were generated between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A isolate. The chimeric envelopes were expressed within the complete, replication-competent provirus and tested for virus viability by transient expression assays. Eleven of the 22 viruses were viable. Five of these chimeric viruses showed an ecotropic host range, and six exhibited an amphotropic host range and viral interference. The host range determinants map to the first half of the surface (SU) protein. The N-terminal 72 amino acids of 4070A (42 of processed SU) are not required for amphotropic receptor usage. Ecotropic and amphotropic viruses differ in their ability to form large, multinucleated syncytia when cocultured with the rat XC cell line. Ecotropic murine leukemia virus forms large syncytia with XC cells, whereas no syncytia are reported for amphotropic virus. All chimeras which contained the N-terminal half of the ecotropic SU protein, encoding the receptor binding domain, formed the large multinucleated syncytia with XC cells.     

Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses.

The envelope glycoproteins (SU) of mammalian type C retroviruses possess an amino-terminal domain of about 200 residues, which is involved in binding a cell surface receptor. In this domain, highly conserved amino acid sequences are interrupted by two segments of variable length and sequence, VRA and VRB. We have studied the role of these variable regions in receptor recognition and binding by constructing chimeric molecules in which portions of the amino-terminal domains from amphotropic (4070A), xenotropic (NZB), and polytropic (MCF 247) murine leukemia virus SU proteins were permuted. These chimeras, which exchanged either one or two variable regions, were expressed at the surface of replication-defective viral particles by a pseudotyping assay. Wild-type or recombinant env genes were transfected into a cell line producing Moloney murine leukemia virus particles devoid of envelope glycoproteins in which a retrovirus vector genome carrying an Escherichia coli lacZ gene was packaged. The host range and sensitivity to interference of pseudotyped virions were assayed, and we observed which permutations resulted in receptor switch or loss of function. Our results indicate that the determinants of receptor choice are found within the just 120 amino acids of SU proteins. Downstream sequences contribute to the stabilization of the receptor-specific structure.     

Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry.

10A1 murine leukemia virus can enter cells by using either of two different cell surface phosphate transport proteins, the gibbon ape leukemia virus receptor Glvr-1 (Pit-1) or the amphotropic retrovirus receptor Ram-1 (Pit-2). Glvr-1 and Ram-1 are widely expressed in different tissues, but the relative amounts of each are highly variable. We have developed retrovirus packaging cell lines based on 10A1 virus to take advantage of this dual receptor utilization to improve gene transfer rates in somatic cells of animals and humans, in which the relative levels of the two receptors are not always known. Optimization of the Env expression vector allowed the generation of packaging lines that produce helper-free vector titers up to 10(7)/ml. By interference analysis, we found that a 10A1 pseudotype retroviral vector can utilize Ram-1 for efficient entry into mouse, rat, and human cells and can utilize Glvr-1 for entry into mouse and human cells but not for entry into rat cells. The 10A1 pseudotype vector efficiently enters mouse cells by using Glvr-1, while entry into human cells is much less efficient. Thus, the 10A1 pseudotype packaging cells may be advantageous compared with the standard amphotropic packaging cells because vectors produced by the cells can use an additional receptor for cell entry. These packaging cells will also be useful to further explore the complicated pattern of receptor usage conferred by the 10A1 viral surface protein.     

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.     

pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry.

Murine leukemia virus ecotropic and amphotropic envelope expression vectors were genetically engineered to generate truncations of the p15E TM cytoplasmic tail. The ecotropic construct CEET has the entire cytoplasmic tail of TM deleted, while the CEETR construct has only the R peptide portion of the tail deleted, thereby producing a TM subunit (p12E) that is identical to the one found in mature virions. The analogous amphotropic constructs were called CAET and CAETR. These envelopes, as opposed to their p15E TM counterparts, mediate cell-to-cell fusion at neutral pH in both transformed and nontransformed cell lines. Though the TM cytoplasmic domain is not required, its presence appears to augment such cell-to-cell fusion. This envelope-dependent fusion requires the presence of the viral receptor on the cell surface. Ecotropic virions bearing the p12E TM contain wild-type levels of the envelope complex and have near-normal titers. In contrast, virions which lack the cytoplasmic domain of TM (e.g., CEET) have 10- to 100-fold-lower titers but contain normal amounts of envelope. Both of the corresponding amphotropic virions contain normal amounts of envelope but have 10- to 100-fold-lower titers. Using immunofluorescent detection of envelope to monitor the fate of receptor-bound virions, we found that ecotropic murine leukemia virus envelope disappears from the cell surface while amphotropic envelope persists on the cell surface after virus binding. This pattern of immunofluorescence is consistent with the proposed routes of cell entry for these viruses, i.e., by endocytosis and direct fusion, respectively. In this assay, ecotropic virions bearing the genetically engineered p12E TM also appear to be internalized despite the ability of their envelope to mediate fusion at neutral pH in the same target cells. Our results show that direct fusion at neutral pH is a natural consequence of the surface expression of the mature ecotropic envelope and its receptor. We propose that the processing of the R peptide from the envelope TM (p15E) to yield p12E, at the time of virus budding or within virions, renders the envelope competent to fuse.     

Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells

HIV-1 is an enveloped retrovirus that acquires its outer membrane as the virion exits the cell. Because of the association of apoptosis with the progression of AIDS, HIV-1-infected T cells or macrophages might be expected to express elevated levels of surface phosphatidylserine (PS), a hallmark of programmed cell death. Virions produced by these cells would also be predicted to have PS on the surface of their envelopes. In this study, data are presented that support this hypothesis and suggest that PS is required for macrophage infection. The PS-specific protein annexin V was used to enrich for virus particles and to inhibit HIV-1 replication in primary macrophages, but not T cells. HIV-1 replication was also significantly inhibited with vesicles consisting of PS, but not phosphatidylcholine. PS is specifically required for HIV-1 infection because viruses pseudotyped with vesicular stomatitis virus G and amphotropic murine leukemia virus envelopes were not inhibited by PS vesicles or annexin V. These data indicate that PS is an important cofactor for HIV-1 infection of macrophages.     

Disruption of the actin cytoskeleton can complement the ability of Nef to enhance human immunodeficiency virus type 1 infectivity

The human immunodeficiency virus (HIV) protein Nef has been shown to increase the infectivity of HIV at an early point during infection. Since Nef is known to interact with proteins involved in actin cytoskeleton rearrangements, we tested the possibility that Nef may enhance HIV infectivity via a mechanism that involves the actin cytoskeleton. We find that disruption of the actin cytoskeleton complements the Nef infectivity defect. The ability of disruption of the actin cytoskeleton to complement the Nef defect was specific to envelopes that fuse at the cell surface, including a variety of HIV envelopes and the murine leukemia virus amphotropic envelope. In contrast, the infectivity of HIV virions pseudotyped to enter cells via endocytosis, which is known to complement the HIV Nef infectivity defect and can naturally penetrate the cortical actin barrier, was not altered by actin cytoskeleton disruption. The results presented here suggest that Nef functions to allow the HIV genome to penetrate the cortical actin network, a known barrier for intracellular parasitic organisms.     

Independence of H-2 and viral antigens on the cell surface and absence of H-2 antigens on murine leukemia virus and mouse mammary tumor virus particles

By indirect immunoelectron microscopy we tested for the presence of H-2 antigens on murine mammary tumor virus (MMTV) and murine leukemia virus (MuLV) particles. The association of H-2 antigens and viral antigens on the virus-infected cell surface was investigated with antibody-induced redistribution. Mammary tumor cells and leukemia cell lines with different H-2 genotypes and carrying different MuMTV or MuLV were used. No H-2 antigens could be demonstrated on the envelope of MMTV and MuLV particles, even after the permeabilization of their envelopes with saponin. On the surface of virus-infected cells antibody-induced patching or capping of the viral antigens did not result in copatching or cocapping of the H-2 antigens. In the reciprocal tests no co-redistribution of viral antigens with H-2 antigens was seen. Our experiments failed to show any physical association between H-2 antigens and MMTV or MuLV antigens on the cell surface.Abbreviations used in this paper MMTV mammary tumor virus - MuLV murine leukemia virus - MHC major histocompatibility complex - IEM immunelectron microscopy     

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.     

Suppression of a fusion defect by second site mutations in the ecotropic murine leukemia virus surface protein

Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein-histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.     

Human immunodeficiency virus type 1 Vif- mutant particles from restrictive cells: role of Vif in correct particle assembly and infectivity.

Disruption of the vif gene of human immunodeficiency virus (HIV) type 1 affects virus infectivity to various degrees, depending on the T-cell line used. We have concentrated our studies on true phenotypic Vif- mutant particles produced from CEMx174 or H9 cells. In a single round of infection, Vif- virus is approximately 25 (from CEMx174 cells) to 100 (from H9 cells) times less infectious than wild-type virus produced from these cells or than the Vif- mutant produced from HeLa cells. Vif- virions recovered from restrictive cells, but not from permissive cells, are abnormal both in terms of morphology and viral protein content. Notably, they contain much reduced quantities of envelope proteins and altered quantities of Gag and Pol proteins. Although wild-type and Vif- virions from restrictive cells contain similar quantities of viral RNA, no viral DNA synthesis was detectable after acute infection of target cells with phenotypically Vif- virions. To examine the possible role of Vif in viral entry, attempts were made to rescue the Vif- defect in H9 cells by pseudotyping Vif+ and Vif- HIV particles with amphotropic murine leukemia virus envelope. Vif- particles produced in the presence of HIV envelope could not be propagated when pseudotyped. In contrast, when only the murine leukemia virus envelope was present, significant propagation of Vif- HIV particles could be detected. These results demonstrate that Vif is required for proper assembly of the viral particle and for efficient HIV Env-mediated infection of target cells.     

Developmental-stage-specific expression and regulation of an amphotropic retroviral receptor in hematopoietic cells.

Expression of the transmembrane receptor protein Ram-1 may be critical to optimizing retroviral gene transfer. Ram-1 acts as both a sodium-dependent phosphate transporter and a receptor for amphotropic retroviruses. We previously reported detectable Ram-1 in murine hematopoietic fetal liver cells (FLC) despite resistance of these cells to amphotropic retroviral transduction (infection). We document here that Ram-1 expression is completely absent in murine yolk sac cells from days 9.5 through 13.5 of ontogeny and first appears at low levels in midgestational FLC between days 13.5 and 14.5. In addition, Ram-1 expression is detected only in more differentiated populations within FLC, day 14.5, and not in those highly enriched for stem cells, indicating developmental regulation of Ram-1 during murine hematopoiesis. Others have reported the in vitro use of phosphate-free medium as a stimulus to increase levels of Ram-1 mRNA in nonhematopoietic cells. We now demonstrate that Ram-1 poly(A)+ mRNA increases significantly following culture of FLC in phosphate-free medium. Further, transduction of FLC in phosphate-free medium with an amphotropic retrovirus containing the multiple drug resistance gene leads to gene transfer not observed previously. These data demonstrate that (i) the normal resistance of FLC to amphotropic transduction is most likely due to an insufficient number of Ram-1 molecules for efficient retroviral recognition and binding, and (ii) Ram-1 can be upregulated by increasing the need for phosphate transport across the cell membrane.     

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.     

Initial Binding of Murine Leukemia Virus Particles to Cells Does Not Require Specific Env-Receptor Interaction

The initial step of virus-cell interaction was studied by immunofluorescence microscopy. Single particles of murine leukemia virus (MLV) vectors and human immunodeficiency virus (HIV) were visualized by immunofluorescence. Fluorescent dots representing single virions could be localized by staining of capsid proteins (CA) or surface envelope proteins (SU) after fixation of virus supernatants. This technique can be used to determine particle concentration in viral supernatants and also to study virus-cell interaction. We investigated the role of the Env-receptor interaction for the initial binding event between the cell and the viral particles. Ecotropic MLV vector particles were shown to bind to human cells which do not express the specific viral receptor. In addition, MLV particles defective for Env were shown to bind the cells similarly to infectious MLV. Time course experiments of virus-cell binding and dissociation showed identical profiles for infectious and Env-defective MLV particles and suggested that MLV Env is not involved in the early phases of attachment of virus to cells. The possible implication of cellular factors in enhancing viral binding and infectivity is discussed.     

Retrovirus Targeting by Tropism Restriction to Melanoma Cells

Targeted vectors will be necessary for many gene therapy applications. To target retroviruses to melanomas, we fused a single-chain variable fragment antibody (scFv) directed against the surface glycoprotein high-molecular-weight melanoma-associated antigen (HMW-MAA) to the amphotropic murine leukemia virus envelope. A proline-rich hinge and matrix metalloprotease (MMP) cleavage site linked the two proteins. The modified viruses bound only to HMW-MAA-expressing cells, as inclusion of the proline-rich hinge prevented viral binding to the amphotropic viral receptor. Following attachment to HMW-MAA, MMP cleavage of the envelope at the melanoma cell surface removed the scFv and proline-rich hinge, allowing infection. Complexing of targeted retroviruses with 2, 3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]N, N-dimethyl-1-propanaminium trifluoroacetate-dioleoyl phosphatidylethanolamine liposomes greatly increased their efficiency without affecting their target cell specificity. In a cell mixture, 40% of HMW-MAA-positive cells but less than 0.01% of HMW-MAA-negative cells were infected. This approach can therefore produce efficient, targeted retroviruses suitable for in vivo gene delivery and should allow specific gene delivery to many human cell types by inclusion of different scFv and protease combinations.     

Use of virus-attached antibodies or insulin molecules to mediate fusion between Sendai virus envelopes and neuraminidase-treated cells

Anti-human erythrocyte antibodies or insulin molecules were covalently coupled to the glycoproteins (the hemagglutinin/neuraminidase and the fusion polypeptides) of Sendai virus envelopes with N-succinimidyl 3-(2-pyridyldithio)propionate and succinimidyl 4-(p-maleimidophenyl)butyrate as cross-linking reagents. Reconstituted Sendai virus envelopes, bearing covalently attached anti-human erythrocyte antibodies or insulin molecules, were able to bind to but not fuse with virus receptor depleted human erythrocytes (neuraminidase-treated human erythrocytes). Only coreconstitution of Sendai virus glycoproteins, bearing attached anti-human erythrocyte antibodies or insulin molecules with intact, untreated viral glycoproteins, led to the formation of fusogenic, targeted reconstituted Sendai virus envelopes. Binding and fusion of reconstituted Sendai virus envelopes, bearing anti-human erythrocyte antibodies or insulin molecules, with neuraminidase-treated human erythrocytes were blocked by the monovalent fraction, obtained after papain digestion of immunoglobulins, made of anti-human erythrocyte antibodies or free insulin molecules, respectively. The results of this work demonstrate an active role of the viral binding protein (hemagglutinin/neuraminidase polypeptide) in the virus membrane fusion process and show a novel and efficient method for the construction of targeted, fusogenic Sendai virus envelopes.     

Structural studies on Rauscher murine leukemia virus: isolation and characterization of viral envelopes.

A preparative method for isolating pure viral envelopes from a type-C RNA tumor virus, Rauscher murine leukemia virus, is described. Fractionation of virions of Rauscher murine leukemia virus was studied after disruption of the virions with the detergents sodium dodecyl sulfate of Nonidet P-40 in combination with ether. Fractionation was performed through flotation in a discontinuous sucrose gradient and, as appeared from electron microscopic examination, a pure viral envelope fraction was obtained in this way. By use of sensitive competition radioimmunoassays or sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera directed against Rauscher murine leukemia virus proteins, the amount of the gag and env gene-encoded structural polypeptides in the virions and the isolated envelope fraction was compared. The predominant viral structural polypeptides in the purified envelope fraction were the env gene-encoded polypeptides gp70, p15(E), and p12(E), whereas, except for p15, there was only a relatively small amount of the gag gene-encoded structural polypeptides in this fraction.     

Biochemical characterization of the amphotropic group of murine leukemia viruses.

The recently described amphotropic group of murine leukemia viruses constitutes a distinct biological group, differing from the ecotropic and xenotropic groups in host range, cross interference, and serological reactivity. Viruses of this group have been detected only in wild mice from certain areas in California. By using a [3H]DNA probe synthesized in an endogenous reaction from detergent-lysed amphotropic virus (strain 1504-A), it was demonstrated that the amphotropic murine leukemia viruses are distinct biochemically, in that 20% of the viral genome sequences are not shared by AKR-type ecotropic or nay of three types of xenotropic murine leukemia virus tested. A subset of these amphotropic unique sequences, comprising one half of them, is present in the genome of wild mouse ecotropic viruses and in Moloney and Rauscher viruses as well. Sequences homologous to the entire genome of 1504-A amphotropic virus are present in the cellular DNA of all eight inbred mouse strains tested, as well as in wild Mus in Asia, in amounts varying from three to six complete viral genomes per haploid cell genome. Evidence is presented that at least 20% of the DNA sequences in both mouse- and mink-grown murine leukemia virus probes are of host-cell origin.