Primate retroviruses: envelope glycoproteins of endogenous type C and type D viruses possess common interspecies antigenic determinants.

The major 70,000- to 80,000-molecular-weight envelope glycoproteins of the squirrel monkey retrovirus, Mason-Pfizer monkey virus, and M7 baboon virus and the related endogenous feline virus, RD114, were isolated and immunologically characterized. Immunoprecipitation and competition immunoassay analysis revealed these viral envelope glycoproteins to possess several distinct classes of immunological determinants. These include species-specific determinants, group-specific antigenic determinants unique to endogenous primate type C viruses, and group-specific determinants for type D viruses such as Mason-Pfizer monkey virus and squirrel monkey retrovirus. In addition, a class of broadly reactive antigenic determinants shared by envelope glycoproteins of both type C viruses of the baboon/RD114 group and type D viruses of the Mason-Pfizer monkey virus/squirrel monkey virus group are described. Other mammalian oncornaviruses tested, including isolates of nonprimate origin and representative type B viruses, lacked these determinants. The demonstration of antigenic determinants specific to envelope glycoproteins of type C and type D primate viruses indicates either that these viruses are evolutionarily related or that genetic recombination occurred between their progenitors. Alternatively, endogenous type D oncornaviruses may be replication defective, and acquisition of endogenous type C viral genetic sequences coding for envelope glycoprotein determinants may be necessary for their isolation as infectious virus.     

Acquisition of oncogenicity by endogenous mouse type C viruses: effects of variations in env and gag genes.

Several dual-tropic isolates derived from the thymuses of preleukemic or leukemic AKR mice and a more recrnt group of viruses generated by in vitro or in vivo passage of a poorly infectious endogenous virus of C3H mouse cells have been shown to be highly oncogenic. By analysis of the immunological properties of their gag gene-coded structural proteins, each of the AKR-derived isolates and two dual-tropic C3H-derived isolates were found to closely resemble AKR murine leukemia virus. In contrast, gag gene-coded proteins of two other leukemogenic isolates of C3H origin, including one ecotropic and one dual-tropic virus, were indistinguishable from those of Moloney murine leukemia virus. All of the oncogenic isolates, including those of AKR and C3H origin, were found to possess common envelope glycoprotein determinants of a unique class not shared by the nononcogenic ecotropic viruses from which they were derived. These findings support the possibility that oncogenic variants of endogenous ecotropic mouse type C viruses are derived by genetic recombination. This recombinational event appears to involve the acquisition, by different ecotropic viruses, of a common class of endogenous virus-coded envelope glycoprotein determinants which are presumably required, but not necessarily sufficient, for oncogenicity.     

Contribution of intrinsic reactivity of the HIV-1 envelope glycoproteins to CD4-independent infection and global inhibitor sensitivity

Human immunodeficiency virus (HIV-1) enters cells following sequential activation of the high-potential-energy viral envelope glycoprotein trimer by target cell CD4 and coreceptor. HIV-1 variants differ in their requirements for CD4; viruses that can infect coreceptor-expressing cells that lack CD4 have been generated in the laboratory. These CD4-independent HIV-1 variants are sensitive to neutralization by multiple antibodies that recognize different envelope glycoprotein epitopes. The mechanisms underlying CD4 independence, global sensitivity to neutralization and the association between them are still unclear. By studying HIV-1 variants that differ in requirements for CD4, we investigated the contribution of CD4 binding to virus entry. CD4 engagement exposes the coreceptor-binding site and increases the "intrinsic reactivity" of the envelope glycoproteins; intrinsic reactivity describes the propensity of the envelope glycoproteins to negotiate transitions to lower-energy states upon stimulation. Coreceptor-binding site exposure and increased intrinsic reactivity promote formation/exposure of the HR1 coiled coil on the gp41 transmembrane glycoprotein and allow virus entry upon coreceptor binding. Intrinsic reactivity also dictates the global sensitivity of HIV-1 to perturbations such as exposure to cold and the binding of antibodies and small molecules. Accordingly, CD4 independence of HIV-1 was accompanied by increased susceptibility to inactivation by these factors. We investigated the role of intrinsic reactivity in determining the sensitivity of primary HIV-1 isolates to inhibition. Relative to the more common neutralization-resistant ("Tier 2-like") viruses, globally sensitive ("Tier 1") viruses exhibited increased intrinsic reactivity, i.e., were inactivated more efficiently by cold exposure or by a given level of antibody binding to the envelope glycoprotein trimer. Virus sensitivity to neutralization was dictated both by the efficiency of inhibitor/antibody binding to the envelope glycoprotein trimer and by envelope glycoprotein reactivity to the inhibitor/antibody binding event. Quantitative differences in intrinsic reactivity contribute to HIV-1 strain variability in global susceptibility to neutralization and explain the long-observed relationship between increased inhibitor sensitivity and decreased entry requirements for target cell CD4.     

Evidence for genetic recombination between endogenous and exogenous mouse RNA type C viruses

Two viruses isolated following prolonged growth of serologically distinct mouse type C RNA viruses in human cells have previously been shown to have acquired common envelope properties distinct from those of either parental virus. Virus neutralization tests show that the viruses selected in human cells possess envelope antigens identical to those of endogenous mouse type C viruses of cells in which the parental viruses had been propagated. In contrast, the p12 polypeptide of each virus selected in human cells is antigenically indistinguishable from that of its respective parental virus and different from those of known endogenous mouse type C viruses. Molecular hybridization indicates significant differences in the genetic sequences of one virus and its parent, excluding the possibility that it arose from a point mutation. These findings indicate that the viruses selected in human cells represent genetic recombinants between exogenous and endogenous mouse type C viruses.     

Vector infection determinants of Venezuelan equine encephalitis virus reside within the E2 envelope glycoprotein

Epizootic subtype IAB and IC Venezuelan equine encephalitis viruses (VEEV) readily infect the epizootic mosquito vector Aedes taeniorhynchus. The inability of enzootic subtype IE viruses to infect this mosquito species provides a model system for the identification of natural viral determinants of vector infectivity. To map mosquito infection determinants, reciprocal chimeric viruses generated from epizootic subtype IAB and enzootic IE VEEV were tested for mosquito infectivity. Chimeras containing the IAB epizootic structural gene region and, more specifically, the IAB PE2 envelope glycoprotein E2 precursor gene demonstrated an efficient infection phenotype. Introduction of the PE2 gene from an enzootic subtype ID virus into an epizootic IAB or IC genetic backbone resulted in lower infection rates than those of the epizootic parent. The finding that the E2 envelope glycoprotein, the site of epitopes that define the enzootic and epizootic subtypes, also encodes mosquito infection determinants suggests that selection for efficient infection of epizootic mosquito vectors may mediate VEE emergence.     

Embryonic infection with the endogenous avian leukosis virus Rous-associated virus-0 alters responses to exogenous avian leukosis virus infection.

We inoculated susceptible chicken embryos with the endogenous avian leukosis virus Rous-associated virus-0 (RAV-0) on day 6 of incubation. At 1 week after hatching, RAV-0-infected and control chickens were inoculated with either RAV-1 or RAV-2, exogenous viruses belonging to subgroups A and B, respectively. The chickens injected with RAV-0 as embryos remained viremic with exogenous virus longer and either failed to develop type-specific humoral immunity to exogenous virus or developed it later than the control chickens not inoculated with RAV-0. The RAV-0-injected chickens also developed neoplasms at a much higher frequency than did the control chickens. We suggest that the lower immune responses of the RAV-0-injected chickens were due to an immunological tolerance to envelope group-specific glycoproteins shared among endogenous and exogenous viruses.     

Antigenic characterization of flavivirus structural proteins separated by isoelectric focusing.

Isoelectrofocusing of nonionic-detergent-disrupted flaviviruses separated the envelope glycoprotein of 53,000 to 58,000 daltons and the nucleocapsid protein of 14,000 daltons. The envelope protein and nucleocapsid protein were isolated at isoelectric points of pI 7.8 and 10.3, respectively. The antigenic determinants of St. Louis encephalitis, Japanese encephalitis, and dengue virus envelope and nucleocapsid proteins were examined by solid-phase competition radioimmunoassay. By the appropriate selection of antiserum and competing proteins, it was possible to distinguish type-specific, complex-reactive and flavivirus group-reactive antigenic determinants. The envelope glycoproteins of St. Louis encephalitis, Japanese encephalitis, and dengue viruses were found to contain each of these three classes of antigenic determinants. Most of the determinants on the envelope protein were type specific, some were complex reactive, and a small fraction were flavivirus group reactive. The nucleocapsid protein contained only flavivirus group-reactive antigenic determinants.     

Induction of neoplasms by subgroup E recombinants of exogenous and endogenous avian retroviruses (Rous-associated virus type 60).

Chickens susceptible to infection with subgroup E viruses were inoculated with four independent isolates of Rous-associated virus type 60 (RAV-60) that are subgroup e recombinants of endogenous and exogenous virus. Neoplasms developed in each inoculated group. Therefore, nontransforming viruses of subgroup E can induce lymphoid leukosis at a moderate rate compared with RAV-0, a subgroup E endogenous virus, suggesting that oncogenicity is not a viral envelope (env)-related characteristic. Since the common (c) regions of the RAV-60s examined were of exogenous origin, we suggest that the c region rather than env is important for a high rate of induction of lymphoid leukosis and related neoplasms.     

Establishment of a C3Hf Mammary Tumor Cell Line Expressing Endogenous Mouse Mammary Tumor Virus: Antigenic and Genetic Relationships of This Virus with Highly Oncogenic Mouse Mammary Tumor Viruses

A single-cell clone of C3Hf mammary tumor cells (clone 14) was developed into a continuous cell line expressing high levels of endogenous mouse mammary tumor virus (MMTV) with less than 0.1% murine leukemia virus expression. Comparison of the C3Hf MMTV protein profile on sodium dodecyl sulfatepolyacrylamide gel electrophoresis with that of C3H MMTV revealed that the protein content of the two viruses was quite similar. However, oligonucleotide fingerprints obtained of MMTV 70S RNA revealed that approximately 20% of the large oligonucleotides examined were unique to each virus. The oligonucleotide fingerprint indicated that although the viruses were similar, they differed in their genetic content. The differences in the two viruses extended to immunological differences in the major envelope glycoprotein, gp52. C3Hf MMTV competed only partially in a homologous radioimmunoassay for gp52 of C3H MMTV, whereas C3H MMTV gave complete competition, indicating that gp52 of C3H MMTV contained type-specific determinants not present on gp52 of C3Hf MMTV. Comparison of C3Hf MMTV with highly oncogenic C3H, GR, and RIII MMTVs in a homologous C3H MMTV gp52 assay gave two patterns of reactivity: complete competition by GR and C3H MMTV and incomplete competition by C3Hf and RIII MMTV. Absorption of anti-C3H MMTV serum by either C3Hf MMTV or RIII MMTV removed all antibodies against both viruses but not against GR and C3H MMTVs. These results indicate that C3H and GR MMTVs are more closely related to each other than to RIII and C3Hf MMTVs.     

Search for mammalian type-C retrovirus polypeptides on infected animal cells and human tumor cell lines using interspecies-reacting antibodies

Cells producing endogenous and exogenous type-C retroviruses of murine, feline and primate origin were evaluated for expression of those virus-specific cell-surface antigens which cross-react with antibodies to interspecies determinants of mammalian type-C viral polypeptides. Surface polypeptides of cells replicating endogenous and exogenous type-C retroviruses were iodinated by the lactoperoxidase method. Labelled antigens were immunoprecipitated and analyzed in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This method detected gp71 and less frequently p15E/p12E on the surface of virus-producing cells; in addition, p27 was found on F422 cells replicating feline leukemia virus. Antibodies to the membrane protein p15E displayed the broadest cross-reactivity but only antibodies to gp71 mediated complement-dependent interspecies cell lysis. The pattern of cross-reactivity reflected known genetic relationships among these mammalian viruses. Although antiserum to the simian sarcoma virus complex (SSV) was strongly cytotoxic to some human tumor cell lines, this reactivity could not be attributed to antibodies cross-reacting with SSV gp71.     

Replicative function and neutralization sensitivity of envelope glycoproteins from primary and T-cell line-passaged human immunodeficiency virus type 1 isolates.

The structure, replicative properties, and sensitivity to neutralization by soluble CD4 and monoclonal antibodies were examined for molecularly cloned envelope glycoproteins derived from human immunodeficiency virus type 1 (HIV-1) viruses either isolated directly from patients or passaged in T-cell lines. Complementation of virus entry into peripheral blood mononuclear cell targets by primary patient envelope glycoproteins exhibited efficiencies ranging from that observed for the HXBc2 envelope glycoproteins, which are derived from a T-cell line-passaged virus, to approximately fivefold-lower values. The ability of the envelope glycoproteins to complement virus entry roughly correlated with sensitivity to neutralization by soluble CD4. Laboratory-adapted viruses were sensitive to neutralization by monoclonal antibodies directed against the CD4-binding site and the third variable (V3) loop of the gp120 glycoprotein. By comparison, viruses with envelope glycoproteins from primary patient isolates exhibited decreased sensitivity to neutralization by these monoclonal antibodies; for these viruses, neutralization sensitivity correlated with replicative ability. Subinhibitory concentrations of soluble CD4 and a CD4-binding site-directed antibody significantly enhanced the entry of viruses containing envelope glycoproteins from some primary patient isolates. The sensitivity of viruses containing the different envelope glycoproteins to neutralization by soluble CD4 or monoclonal antibodies could be predicted by assays dependent on the binding of the inhibitory molecule to the oligomeric envelope glycoprotein complex but less well by assays measuring binding to the monomeric gp120 glycoprotein. These results indicate that the intrinsic structure of the oligomeric envelope glycoprotein complex of primary HIV-1 isolates, while often less than optimal with respect to the mediation of early events in virus replication, allows a relative degree of resistance to neutralizing antibodies. The interplay of selective forces for higher virus replication efficiency and resistance to neutralizing antibodies could explain the temporal course described for the in vivo emergence of HIV-1 isolates with differing phenotypes.     

The Unique Envelope Gene of the Subgroup J Avian Leukosis Virus Derives from ev/J Proviruses, a Novel Family of Avian Endogenous Viruses

A new subgroup of avian leukosis virus (ALV), designated subgroup J, was identified recently. Viruses of this subgroup do not cross-interfere with viruses of the avian A, B, C, D, and E subgroups, are not neutralized by antisera raised against the other virus subgroups, and have a broader host range than the A to E subgroups. Sequence comparisons reveal that while the subgroup J envelope gene includes some regions that are related to those found in env genes of the A to E subgroups, the majority of the subgroup J gene is composed of sequences either that are more similar to those of a member (E51) of the ancient endogenous avian virus (EAV) family of proviruses or that appear unique to subgroup J viruses. These data led to the suggestion that the ALV-J env gene might have arisen by multiple recombination events between one or more endogenous and exogenous viruses. We initiated studies to investigate the origin of the subgroup J envelope gene and in particular to determine the identity of endogenous sequences that may have contributed to its generation. Here we report the identification of a novel family of avian endogenous viruses that include env coding sequences that are over 95% identical to both the gp85 and gp37 coding regions of subgroup J viruses. We call these viruses the ev/J family. We also report the isolation of ev/J-encoded cDNAs, indicating that at least some members of this family are expressed. These data support the hypothesis that the subgroup J envelope gene was acquired by recombination with expressed endogenous sequences and are consistent with acquisition of this gene by only one recombination event.     

Determinants of thymotropism in Kaplan radiation leukemia virus and nucleotide sequence of its envelope region.

Radiation leukemia viruses (RadLVs) are a group of murine leukemia viruses which are induced by radiation and cause T-cell leukemia. Viral clones isolated from the BL/VL3 lymphoid cell line derived from a thymoma show variable tropism and leukemogenic potential. We have constructed chimeric viruses by in vitro recombination between two viruses, a RadLV that is thymotropic and an endogenous ecotropic virus that is nonthymotropic. We show here that, in contrast to thymotropism determinants identified previously, which lie in the long terminal repeat (LTR), it is the envelope region that is responsible for the thymotropism of BL/VL3 RadLV. The nonthymotropic virus which we have rendered thymotropic by transfer of the env region of RadLV in the present study has been shown previously to become thymotropic when the LTR of another thymotropic virus is inserted in its genome. Thus, the LTR and envelope gene may be involved in complementary action to lead to thymotropism.     

Antigenic determinants of the 70,000 molecular weight glycoprotein of woolly monkey type C RNA virus.

The 70,000 molecular weight glycoprotein (gp70) of a type-C RNA virus originally isolated from a woolly monkey has been partially purified and immunologically characterized. Evidence that this viral protein is viral coded was derived from studies showing its antigenic properties to be unaltered by virus passage in cells of different species. A broadly reactive competition immunoassay was developed utilizing antiserum prepared against feline leukemia virus to precipitate 125I-labeled woolly monkey virus gp70. Gibbon and woolly viruses, as well as feline and several mouse type-C viruses, all reacted with equal efficiency in this assay. In contrast, an endogenous virus of the baboon failed to cross-react, suggesting that viruses of this latter group are less immunologically related to the others. In a homologous competition immunoassay for the woolly viral glycoprotein, the woolly virus was readily distingusihed from otherwise colsely related viruses of gibbon apes. These findings demonstrate the pronounced type-specific antigenic dterminants possessed by this viral protein. The antigenic determinants of gp70 responsible for neutralization have also been investigated.     

Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein.

Insertion of four amino acids into various locations within the amino-terminal halves of the human immunodeficiency virus type 1 gp120 or gp41 envelope glycoprotein disrupts the noncovalent association of these two envelope subunits (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. A. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987). To localize the determinants on the gp120 envelope glycoprotein important for subunit association, amino acids conserved among primate immunodeficiency viruses were changed. Substitution mutations affecting either of two highly conserved regions located at the amino (residues 36 to 45) and carboxyl (residues 491 to 501) ends of the mature gp120 molecule resulted in nearly complete dissociation of the envelope glycoprotein subunits. Partial dissociation phenotypes were observed for some changes affecting residues in the third and fourth conserved gp120 regions. These results suggest that hydrophobic regions at both ends of the gp120 glycoprotein contribute to noncovalent association with the gp41 transmembrane glycoprotein.     

Comparisons of the immunological properties of two structural polypeptides of type C RNA viruses endogenous to old world monkeys.

Immunologically very closely related type C RNA viruses are endogenous to the domestic cat and to an old world primate, the baboon. In the present studies, radioimmunological techniques have been developed for detection of the 15,000 and 30,000 molecular weight (MW) polypeptides of each virus. The much more pronounced type-specific antigenic determinants of the lower MW polypeptides made it possible to readily differentiate these viruses from each other as well as from a type C virus isolate from a second baboon species. Normal rhesus monkey tissues were partially purified and shown to contain a reactivity with MW and immunological properties similar to that of the baboon virus 30,000 MW polypeptide. Despite a similar degree of purification, antigenic reactivity like that of the baboon virus 15,000 MW polypeptide was undetectable even in the brodest immunological tests available for this polypeptide. The present findings indicate that the immunological properties of two structural polypeptides of closely related viruses endogenous to primate and feline species have undergone different rates of antigenic change in the course of evolution within their respective host cell genome.     

Isolation and antigenic characterization of human immunodeficiency virus (HIV) in Brazil

A retrovirus infecting a Brazilian AIDS patient was isolated and characterized in terms of its reactivity with sera from individuals infected with human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2). The Western blot analysis revealed that the Brazilian isolate is very similar to the well characterized HIV-1 strain. The serum of the patient from whom the virus was isolated did not react with the 140 kDa envelope glycoprotein specific for HIV-2.     

囊膜病毒膜融合分子机制研究进展

囊膜病毒通过病毒与宿主细胞膜融合的方式感染宿主,病毒囊膜蛋白介导了膜融合过程。根据这些囊膜蛋白在病毒囊膜表面的排列、蛋白结构及其在融合肽中的位置不同,可将囊膜病毒分为三类,其利用这些囊膜特殊的蛋白分子与受体相互作用完成膜融合。在分子水平上研究这一过程有助于认识病毒侵染的本质和发现关键环节,达到预防与治疗病毒病的目的。     

Increased envelope spike density and stability are not required for the neutralization resistance of primary human immunodeficiency viruses.

Previous observations that the gp120 envelope glycoprotein contents of some primary, clade B human immunodeficiency virus type 1 (HIV-1) isolates were higher than those of laboratory-passaged HIV-1 isolates suggested the hypothesis that increased envelope glycoprotein spike density or stability contributes to the relative neutralization resistance of the primary viruses. To test this, the structural, replicative, and neutralization properties of a panel of recombinant viruses with HIV-1 envelope glycoproteins from divergent clades were examined in an env complementation assay. In this system, although the spike density and stability of envelope glycoproteins from primary HIV-1 isolates were not greater than those from a laboratory-adapted isolate, relative resistance to neutralizing antibodies and soluble CD4 was observed for the viruses with primary envelope glycoproteins. Thus, neither high envelope glycoprotein spike density nor stability is necessary for the relative neutralization resistance of primary HIV-1 viruses.     

Isolation from cats of an endogenous type C virus with a novel envelope glycoprotein.

A search for variant endogenous cat viruses led to a novel isolate. Although the major envelope glycoprotein of this virus was similar in size to that of an RD-114-like virus that was coisolated, it was unrelated to RD-114 or feline leukemia virus by immunological and biological criteria. This degree of dissimilarity suggests a different evolutionary progenitor from that for the RD-114 and feline leukemia virus viral envelopes. The novel virus did, however, code for gag gene polypeptides which are closely related to RD-114 virus. Neither the novel isolate nor the RD-114-like coisolate induced foci in S+L- cat cells which restrict focus induction by RD-114 virus. This suggests that the two viruses share a common genomic target of restriction which resides outside of the env region.