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Rat cytotoxic T lymphocytes against human T-lymphotropic virus type 1-infected cells recognize gag gene and env gene encoded antigens.

T cell immune responses in syngeneic WKA/H rats were analyzed by using lymphoid cell lines, TARS-1, TART-1, and TARL-2, infected with human T-lymphotropic virus type 1 (HTLV-1). Spleen cells of rats in which these cell lines had been rejected were sensitized in vitro with the same cell lines, and cells cytotoxic to these HTLV-1+ cell lines, and cells cytotoxic to these HTLV-1+ cell lines were generated. The effector cells were CTL of the CD5+ CD8+ phenotype and showed restriction of MHC class I Ag. Direct tests as well as cold target cell inhibition tests with an array of cell populations showed that these CTL reacted only with syngeneic HTLV-1+ cell lines. When xenogeneic HTLV-1+ cell lines were similarly utilized for in vitro sensitization, rat CTL specific for syngeneic HTLV-1+ cells were generated. They were not, however, reactive with xenogeneic HTLV-1+ cells used for sensitization. Syngeneic rat cells selectively expressing gag, env, or pX gene coded Ag were prepared by infection of recombinant vaccinia viruses. In cold target cell inhibition tests of anti-HTLV-1 CTL with thus prepared cells, cytotoxicity against the syngeneic HTLV-1+ cells line, TARS-1, was inhibited by syngeneic cells expressing gag gene or env gene coded Ag. Inhibition was, however, more consistent and more dominant by cells with gag gene than those with env gene. Syngeneic cells with pX gene and MHC class I incompatible cells with gag, env, or pX gene did not inhibit cytotoxicity.

Animals

Protease gene structure and env gene variability of the AIDS virus.

The protease gene structure and the env gene variability have been precisely compared between the AIDS virus and members of the HTLV/BLV family. The conserved amino acid sequence (LVDT) which is repeated in the proteases of the HTLV/BLV family is not repeated in AIDS virus. Comparative analysis of the env gene sequences reveals the striking fact that the env gene of AIDS virus is 8-12-times more variable than those of the HTLV/BLV family. Within the AIDS virus env gene, the surface glycoprotein region is more liable to vary than is the transmembrane region; unexpectedly, however, this liability is not a characteristic feature of the AIDS virus because it is more prominent in other retroviruses including members of the HTLV/BLV family.

Acquired Immunodeficiency Syndrome

Purification of DNA complementary to the env gene of avian sarcoma virus and analysis of relationships among the env genes of avian leukosis-sarcoma viruses.

The env gene of avian leukosis-sarcoma viruses encodes a glycoprotein that determines the host range and surface antigenicitiy of virions. We have purified radioactive DNA (cDNAgp) complementary to at least a portion of the env gene for viral subgroups A and C; complementary DNA was synthesized with purified virions of wild-type avian sarcoma virus, and RNA from a mutant with a deletion in env was used to select DNA specific to env by molecular hybridization. The genetic complexity of cDNAgp for subgroup A (ca. 2,000 nucleotides) was sufficient to represent the entire deletion and most or all of the env cistron. The deletions in env in two independently isolated strains of virus (Bryan and rdNY8SR) overlap, and cDNAgp represents nucleotide sequences common to both deletions. By contrast, we could detect no overlap between deletions in env and deletions in the adjacent viral gene src. Laboratory stocks of viral subgroups A, B, C, D and E do not contain detectable amounts of env deletions when tested by molecular hybridization; hence, segregation of deletions in env is a less frequent event that the segregation of deletions in the viral transforming gene src (Vogt, 1971). We found extensive homology among the nucleotide sequences encoding the env genes of virus strains indigenous to chickens (subgroups A, B, C, D, and E) although subgorups B, D and E appear to differ slightly from subgroups A and C at the env locus. By contrast, viruses obtained from pheasant cells (subgroups F and G) have env genes with little or no relationship to env genes of chikcen viruses. According to available data, viruses of subgroup F arose by recombination between an avarian sarcoma virus and viral genes in the genome of ring-necked pheasants, whereas subgroup G viruses may be entirely endogenous to golden pheasants.

Alpharetrovirus

[The expression of a fragment of the HIV-1 env gene in the cells of Escherichia coli bacteria].

A recombinant plasmid pEK6 determining the synthesis of a hybrid protein the N-terminus of which was represented by full-size beta-galactosidase and C-terminus by HIV-1 gene env virus-specific sequence was constructed. The analysis of lysates of E. coli HB101/pEK6 bacteria in 6% PAAGE revealed additional proteins with molecular weights from 185 to 130 kDa. These proteins interacted with blood serum antibodies of a virus carrier but formed no specific bands with sera from normal donors. Densitometric analysis of polyacrylamide gels stained with Coomassi R250 demonstrated that the level of production of recombinant protein was at least 15% of the total cell protein. Hybrid polypeptides formed poorly soluble inclusion bodies in the bacterial cells. Study of the immunological properties of the recombinant polypeptides showed that immunization of rabbits with these proteins induced antibodies specifically reacting with viral polypeptides with molecular weights of about 82 and 140 kDa. Such features as a high level of synthesis, technologically feasible purification of inclusion bodies, and adequate antigenic properties recommend this preparation for use in the development of diagnostic test systems.

Amino Acid Sequence

Expression of the Rous sarcoma virus env gene from a simian virus 40 late-region replacement vector: effects of upstream initiation codons.

Expression of the Rous sarcoma virus envelope gene (env) from a simian virus 40 (SV40) late-region replacement vector is dependent on the position of env within the SV40 late-region sequences. The difference in expression levels appeared to be due to differences in the efficiency with which the env-specific transcripts were translated, because transcription levels from different constructions were similar. Deletion of the nucleotides encoding the agnoprotein initiator codon, located upstream of the env sequences in the poorly expressed construct, resulted in high levels of env expression. The agnoprotein initiator codon and overlapping open reading frame thus act as strong barriers to further ribosome scanning and prevent initiation at the env AUG codon. We conclude that AUG codons present in the late region of SV40 can reduce expression of inserted genes positioned downstream. Nevertheless, intrinsic properties of the gene may determine its ultimate level of expression.

Animals

Biochemical characterization of cell-associated and extracellular products of the Friend spleen focus-forming virus env gene.

The mature product of the env gene of Friend spleen focus-forming viruses (F-SFFV) is efficiently released from both leukemia cells and infected fibroblasts. Analyses of the kinetics of env protein synthesis and secretion in NRK cells infected with the Lilly-Steeves strain of SFFVp indicated that this product, gp65, was formed rapidly and remained stably associated with cells for up to 4 hr, at which point it was first detected in supernatant medium. By 12 hr after synthesis, greater than 95% of gp65 was found extracellularly. The release of this component was effectively blocked by 10 mM 1-deoxynojirimycin, an inhibitor of oligosaccharide processing, demonstrating a requirement for processing of high mannose precursor oligosaccharides in the secretion of gp65. Similar oligosaccharide substituents were found on cell-associated and extracellular forms of gp65. Enzymatic deglycosylation experiments demonstrated that in addition to the predicted four N-linked oligosaccharides, gp65 contains O-linked carbohydrates which are resistant to the action of peptide N-Glycanase F, but sensitive to neuraminidase and O-Glycanase. These structures may be related to O-linked oligosaccharides previously found on the env gene products of murine leukemia viruses. Comparison of the sizes of the deglycosylated forms of cell-associated and supernatant gp65 demonstrated that the extracellular molecules are approximately 3 kDa smaller than the cell-associated components. These data suggest the involvement of proteolysis at a C-terminal site in the release of gp65 from the plasma membrane.

Cell Membrane

Construction and characterization of expression systems for the env gene of ts1, a mutant of Moloney murine leukemia virus-TB.

A temperature-sensitive mutant of the Moloney murine leukemia virus-TB, ts1, causes hindlimb paralysis and immunodeficiency in mice. At the restrictive temperature, the envelope precursor polyprotein, gPr80env, is inefficiently processed intracellularly, and this is associated with the neurovirulence of ts1. To test the hypothesis that expression of the envelope proteins of ts1 alone without infectious virus production can induce paralysis, it is necessary to use either transmissible retroviral expression vectors or microinjection of eukaryotic gene expression plasmid to introduce the env gene of ts1 into germlines of mice. In this study, we have constructed three retrovirus vectors and three gene expression plasmids, all of which contain the env gene of ts1. By comparing the different expression systems, we found that one construct, pts1-env(F) can express the envelope proteins at a level comparable to the level expressed in ts1-infected cells. Furthermore, the expressed envelope proteins of pts1-env(F)-transfected cells possess the phenotypes of the proteins expressed by the env gene of ts1.

Animals

Lentivirus genomic organization: the complete nucleotide sequence of the env gene region of equine infectious anemia virus.

The nucleotide sequence of the envelope (env) gene region of equine infectious anemia virus (EIAV), a member of the lentivirus subfamily of retroviruses, has been determined from a clone of integrated proviral DNA for which the gag and pol sequences have been reported previously. The env gene is 859 codons in length and the sequence reported here is consistent with the published biochemical properties of EIAV glycoproteins. The env gene region of EIAV shares considerable structural similarities but negligible sequence homologies with the env genes of other members of the lentivirus subfamily, visna virus, and human T-lymphotropic virus (HTLV-III) or lymphadenopathy virus (LAV). As in visna virus and HTLV-III, the polymerase (pol) and env genes of EIAV do not overlap. EIAV contains two short open reading frames (orf) of 50 and 66 codons in the pol-env intergenic region. However, unlike the orf Q regions reported for visna virus and HTLV-III, neither EIAV orf overlaps the 3' terminus of the adjacent pol gene. The EIAV genome also contains a third short open reading frame of 135 codons which is contained completely within the env gene, in contrast to the 3'-orf/orf F gene reported for HTLV-III/LAV which extends beyond the env gene terminus. These results provide a detailed description of the env gene region of EIAV and describe a number of characteristic features of genomic organization in lentiviruses which contrast with the genomic organization of oncogenic retroviruses.

Amino Acid Sequence

Xenogenization of tumor cells by transfection with plasmid containing env gene of Friend leukemia virus.

A rat hepatocellular carcinoma cell line (cKDH-8 cl-11) showed decreased tumorigenicity after transfection with an envelope gene derived from a Friend leukemia virus (FV-env gene). FV-env gene product was found by indirect immunofluorescence staining to be expressed on the cell surface of the FV-env gene-transfected cells. The FV-env-transfected cells (FV-env cKDH-8), however, grew well in X-irradiated immunosuppressed rats, indicating that the reduction in tumorigenicity of the transfected cells is based on immunological reaction in the host. The rats which rejected FV-env cKDH-8 cells showed resistance to rechallenge with the parent cKDH-8 cl-11 tumor cells. These results suggest that the FV-env gene product may elicit antitumor immunity against FV-env cKDH-8 cells in a host with a resultant reduction in the tumorigenicity of these cells.

Animals

Mutations in the env gene of friend spleen focus-forming virus overcome Fv-2r-mediated resistance to Friend virus-induced erythroleukemia.

Although Fv-2r homozygous mice are resistant to leukemias induced either by an erythropoietin-encoding virus or by wild-type Friend virus (FV) (M. E. Hoatlin, S. L. Kozak, F. Lilly, A. Chakraborti, C. A. Kozak, and D. Kabat, Proc. Natl. Acad. Sci. USA 87:9985-9989, 1990), they are susceptible to some variants of FV (R. A. Steeves, E. A. Mirand, A. Bulba, and P. J. Trudel, Int. J. Cancer 5:349-356, 1970; R. W. Geib, M. B. Seaward, M. L. Stevens, C.-L. Cho, and M. Majumdar, Virus Res. 14:161-174, 1989). To localize the virus gene involved in influencing the host range, we cloned and sequenced the env gene of the BB6 variant of FV (Steeves et al., Int. J. Cancer 5:349-356, 1970). In comparison with the wild-type env gene, the BB6 variant contains a 159-bp deletion that eliminates the membrane-proximal portion of the extracellular domain and 58 point mutations resulting in 13 amino acid changes. Substitution of the variant env gene for the wild-type env gene resulted in a recombinant virus that produced a Friend virus-like disease in Fv-2r homozygotes. Our results identify the spleen focus-forming virus env gene as the viral gene involved in this virus-host interaction. Additionally, they suggest that the product of the Fv-2r gene modifies the interaction between the spleen focus-forming virus envelope protein and the erythropoietin receptor.

Amino Acid Sequence

Biological and molecular analysis of LCV, an endogenous retrovirus with defective env gene.

Retrovirus infectivity is the result of a cooperative interaction of three structural genes, gag, pol, and env. Since the L-cell endogenous retrovirus (LCV) lacks the env gene translation product, our aim was to study the biological and molecular basis of its non-infectiousness. Fusion experiments between LCV and homologous or heterologous cells demonstrated that virus production could be obtained only after LCV artificial penetration in murine cells and that the new progeny was still noninfectious. Northern blot analysis and heteroduplex mapping of the genomic RNA revealed a 0.99 kb deletion including the 3' region of the pol reading frame, the whole xenotropic and part of the ecotropic domain of the env gene. The results suggest that the observed deletion is responsible for the absence of the gp 70 and the gp 15 E molecules in the virion and seems therefore to be the molecular basis for the non-infectiousness of this retrovirus.

Blotting, Northern

Avian endogenous provirus (ev-3) env gene sequencing: implication for pathogenic retrovirus origination.

The avian endogenous env gene product blocks the surface receptor and, as a result, cells become immune to related exogenous retroviruses. On the other hand, the same sequence can be included in the pathogenic retrovirus genome, as shown by oligonucleotide mapping. However, since the complete env gene sequence was not known, the comparison of genomic nucleotide sequences was not possible. Therefore an avian endogenous provirus with an intact env gene was cloned from a chicken gene bank and the regions coding for the C terminus of the gp85 and gp37 proteins were sequenced. Comparison of this sequence with those of other retroviruses proved that one of the pathogenic viruses associated with osteopetrosis is a cross between avian endogenous virus and Rous sarcoma virus. Retroviruses and, especially, endogenous retroviruses are traditionally of the most developed models of viral carcinogenesis. Many endogenous retroviruses are implicated in neoplastic transformation of the cell. For instance, endogenous mouse mammary tumor virus of some inbred lines appears to be the only causative agent in these mammary cancers. Other even nonpathogenic murine endogenous retroviruses are involved in the origination of MCF-type recombinant acute leukosis viruses. Some endogenous retroviruses are implicated in the transduction or activation of cellular protooncogenes. Our interest in endogenous viruses is based on their ability to make cells resistant to exogenous retroviruses. Expression of their major envelope glycoprotein leads to cellular surface receptor blockage and imparts immunity to infection by the related leukemia retroviruses. This problem is quite elaborated for chicken endogenous virus RAV-O (7-9).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

env genes of avian retroviruses: nucleotide sequence and molecular recombinants define host range determinants.

The env gene of avian sarcoma and leukosis retroviruses is allelic in the virus population permitting the virus to use different host cell receptors. This polymorphism has allowed the classification of these viruses into different subgroups. In order to understand further the role of viral sequences involved in determining this host range phenomenon, we constructed molecular recombinants between subgroup A, B, and E viruses and showed that the host range determinant defining subgroup specificity was located within a 1.1-kb region of the genome that included most of the coding region for the env gene product gp85. We also determined the nucleotide sequence of the region of the env gene encoding gp85 for virus isolates representing subgroup A and B viruses. We compared the predicted amino acid sequences of gp85 to themselves and to the previously published sequences of subgroup B, C, and E env genes. Based on these comparisons, we draw the following conclusions: Within the gp85 coding domain, there are four variable regions (VR-1 to VR-4) ranging in size from 9 to 52 amino acids. The variable regions are located in the same relative positions for each of the env gene alleles compared. The variable regions range in homology from 42% (A compared to B) to 57% (C compared to E) in pairwise comparisons; the flanking conserved domains are on average 95% homologous. The sequences of three different subgroup B virus isolates are highly homologous in both the conserved and variable regions. Secondary structure predictions suggest that gp85 is composed mostly of beta sheet topology. Hydrophilic loops within the variable regions may define sites of receptor interaction and binding sites for subgroup specific neutralizing antibodies.

Alleles

Construction and characterization of the recombinant Moloney murine leukemia viruses bearing the mouse Fv-4 env gene.

A nucleotide sequence of the mouse Fv-4 env gene was completed. Structural comparison revealed a close relationship of Fv-4 to the ecotropic Cas-Br-E murine leukemia virus isolated from a wild mouse in southern California. Various portions of the env gene of Moloney murine leukemia virus were replaced by the corresponding Fv-4 env sequence to construct recombinant murine leukemia virus clones. Infectivity of these recombinants was checked by the S+L- cell focus induction assay and the XC cell syncytium formation assay. Recombinants bearing the following Fv-4 env sequence retained ecotropic infectivity; the AccI-BamHI and BamHI-BalI regions coding for the N- and C-terminal halves of Fv-4 gp70SU, respectively; and the BalI-NcoI region encoding the cleavage site between gp70SU and p15(E)TM of the Fv-4 env. However, when the Fv-4 sequence was substituted for the p15(E)TM-coding NcoI-EcoRV region or the AccI-EcoRV region covering almost the entire env gene, infectivity was undetectable in our assays. The recombinant clone containing the Fv-4 AccI-EcoRV region, i.e., almost the entire Fv-4 env sequence, was introduced with pSV2neo into NIH 3T3 cells, and a G418r cell line named NIH(Fv4)-2 was isolated. The NIH(Fv4)-2 cell released viral particles that contained reverse transcriptase, Fv-4 env molecules as well as the other viral proteins, and viral genomic RNA. However, proviral DNA synthesis was not detected upon inoculation of this virus in NIH 3T3 cells. The loss of infectivity of the recombinant virus bearing the Fv-4 AccI-EcoRV region appeared to be caused by failure in an early step of replication.

Amino Acid Sequence

A 585-bp deletion found in the spleen focus-forming virus (SFFV) env gene is responsible for the defective intracellular transport of SFFV gp52.

The Friend spleen focus-forming virus (F-SFFV) codes for a transport defective, leukemogenic envelope glycoprotein designated as gp52. Gp52 closely resembles the envelope glycoproteins (gp70-p15E) encoded by the mink cell focus-forming viruses (MCFV). The major differences between SFFV and MCFV include a 585-bp deletion and a frame-shift mutation near the 3' end of the SFFV env gene. We have constructed a mutant MCFV env gene, which contains a 585-bp deletion like that found in the SFFV env gene, and expressed this gene using recombinant vaccinia vectors or retroviral vectors. The mutant MCFV env gene expressed a truncated, transport defective glycoprotein (gp57). Only a small proportion of gp57 underwent further oligosaccharide processing. Intracellular gp57 remained predominantly monomeric and only a small proportion of gp57 (and its processed forms) formed disulfide-linked dimers and trimers which resembled those formed by SFFV gp52. Processed forms of gp57 were found on the cell surfaces and in culture fluids. The extracellular forms had a faster electrophoretic mobility than the intracellular-processed forms of gp57. These results indicate that the 585-bp deletion found in SFFV env gene is responsible for the folding, transport, and secretion of gp52. Retroviral vectors carrying the mutant MCFV env gene were nonpathogenic (or weakly pathogenic) in adult mice. The results indicate that the 585-bp deletion, although essential, is not the sole determinant of SFFV-induced disease in adult mice.

3T3 Cells

Identification of simian immunodeficiency virus SIVMAC env gene products.

A monoclonal antibody recognizing an antigenic determinant on the env transmembrane protein, gp32 of simian immunodeficiency virus SIVMAC has been developed and designated SF8/5E11. The reactivity of this antibody was found to be type specific, since it did not cross-react with either SIVSMM or SIVMNe transmembrane proteins. The availability of both this antibody and the complete nucleotide sequence of SIVMAC allowed us to define the organization of the env gene products of this virus. Radiolabel sequencing of the amino termini of both gp160 and gp32 confirmed the positions of both cleavage sites predicted by alignment of the inferred amino acid sequences of the SIVMAC and human immunodeficiency virus type 1 env genes. The cleavage site between the signal peptide and the external env glycoprotein resides between the cysteine residue at position 21 and the threonine residue at position 22, starting from the first residue after the env gene initiator methionine. The env precursor polyprotein gp160 is cleaved between arginine 526 and glycine 527 to give rise to the external glycoprotein and the transmembrane of SIVMAC.

Animals

Phenotype-associated env gene variation among eight related human immunodeficiency virus type 1 clones: evidence for in vivo recombination and determinants of cytotropism outside the V3 domain.

The nucleotide sequences of the env genes of eight phenotypically heterogeneous human immunodeficiency virus type 1 (HIV-1) clones recovered from a single individual within a 3-week period were compared. In addition, the accessory gene sequences for four of these clones were obtained. Variation among most accessory genes was limited. In contrast, pronounced phenotype-associated sequence variation was observed in the env gene. At least three of these clones most likely resulted from genetic recombination events in vivo, indicating that this phenomenon may account for the emergence of proviruses with novel phenotypic properties. Within the env genes of the eight clones, four domains could be defined, the sequence of each of which clustered in two groups with high internal homology but 11 to 30% cluster variation. The extensive env gene variation among these eight clones could largely be explained by the unique manner in which the alleles of these four domains were combined in each clone. Experiments with chimeric proviruses demonstrated that the HIV-1 env gene determined the capacity to induce syncytia and tropism for T-cell lines. Amino acids previously shown to be involved in gp120-CD4 and gp120-gp41 interaction were completely conserved among these eight clones. The finding of identical V3 sequences in clones differing in tropism for primary monocytes and T-cell lines demonstrated the existence of determinants of tropism outside the env V3 region.

Amino Acid Sequence

env Gene of chicken RNA tumor viruses: extent of conservation in cellular and viral genomes.

The env gene of avian sarcoma-leukosis viruses codes for envelope glycoproteins that determine viral host range, antigenic specificity, and interference patterns. We used molecular hybridization to analyze the natural distribution and possible origins of the nucleotide sequences that encode env; our work exploited the availability of radioactive DNA (cDNA(gp)) complementary to most or all of env. env sequences were detectable in the DNAs of chickens which synthesized an env gene product (chick helper factor positive) encoded by an endogenous viral gene and also in the DNAs of chickens which synthesized little or no env gene product (chick helper factor negative). env sequences were not detectable in DNAs from Japanese quail, ring-necked pheasant, golden pheasant, duck, squab, salmon sperm, or calf thymus. The detection of sequences closely related to viral env only in chicken DNA contrasts sharply with the demonstration that the transforming gene (src) of avian sarcoma viruses has readily detectable homologues in the DNAs of all avian species tested [D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature (London) 260: 170-173, 1976] and in the DNAs of other vertebrates (D. Spector, personal communication). Thermal denaturation studies on duplexes formed between cDNA(gp) and chicken DNA and also between cDNA(gp) and RNAs of subgroup A to E viruses derived from chickens indicated that these duplexes were well matched. In contrast, cDNA(gp) did not form stable hybrids with RNAs of viruses which were isolated from ring-necked and golden pheasants. We conclude that substantial portions of nucleotide sequences within the env genes of viruses of subgroups A to E are closely related and that these genes probably have a common, perhaps cellular, evolutionary origin.

Alpharetrovirus