Two base changes restore infectivity to a noninfectious molecular clone of Moloney murine leukemia virus (pMLV-1).

The complete nucleotide sequence of a molecular clone of Moloney murine leukemia virus (pMLV-1) has previously been reported (Shinnick et al., Nature [London] 293:543-548, 1981). However, pMLV-1 does not generate infectious virus after transfection into cells (Berns et al., J. Virol. 36:254-263, 1980). The lesion in pMLV-1 has been localized by determining the biological activity of recombinants containing DNA from an infectious clone of Moloney murine leukemia virus (pMLV-48) and pMLV-1. Replacement of a 1.0-kilobase pair region which spans the gag-pol junction of pMLV-1 with the corresponding DNA fragment from the infectious clone restores its infectivity. Nucleotide sequence analysis of this fragment obtained from the infectious clone (pMLV-48) and pMLV-1 reveals two single base pair changes, one in the p30gag gene and the other in the 5' end of the pol gene. The mutation in the pol gene does not affect the production of infectious virus but renders them XC negative, whereas the mutation in the gag gene appears to be lethal. The complete nucleotide sequence of an infectious clone of Moloney murine leukemia virus can now be deduced.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Provirus of M7 baboon endogenous virus: nucleotide sequence of the gag-pol region

A 3,023-base nucleotide sequence of the M7 baboon endogenous virus genome, spanning the 5' noncoding region as well as the entire gag gene and part of the pol gene, is reported. Within the 562-base 5' noncoding region, a 21-base sequence complementary to the OH terminus of tRNApro is located immediately downstream from the long terminal repeat. Amino acid sequences were deduced from the 1,596 nucleotides comprising the gag gene, and the four structural gag polypeptides, p12, p15, p30, and p10, appeared to be coded contiguously. Only one termination codon interrupted the M7 gag and pol genes. The data suggest that 55 additional amino acids may be attached to the NH2 terminus of the gag precursor protein. However, such a sequence was not detected in virions or in virus-infected cells. With the exception of the p15 region, nucleotide and amino acid sequences of the gag and pol regions of M7 virus exhibited strong homologies to those of Moloney leukemia virus.     

Pseudoknot-dependent read-through of retroviral gag termination codons: importance of sequences in the spacer and loop 2.

Retroviruses whose gag and pol genes are in the same reading frame depend upon approximately 5% read-through of the gag UAG termination codon to make the gag-pol polyprotein. For murine leukemia virus, this read-through is dependent on a pseudoknot located eight nucleotides 3' of the UAG. Other retroviruses whose gag and pol genes are in the same frame can potentially form similar pseudoknots 3' of their UAG codons. Beyond the similar secondary structures, there is strong sequence conservation in the spacer region and in loop 2 of the pseudoknots. The detrimental effects of substitutions of several of these conserved spacer and loop 2 nucleotides in the murine leukemia virus sequence show their importance for the read-through process. The importance of specific nucleotides in loop 2 of the pseudoknot contrasts with the flexibility of sequence in loop 2 of the most intensively studied frameshift-promoting pseudoknot which occurs in infectious bronchitis virus. Two nucleotides in loop 2 of the murine leukemia virus pseudoknot, which were shown to be important by mutagenic analysis, display hypersensitivity to the single-strand specific nuclease, S1. They are likely to be particularly accessible or are in an unusually reactive conformation.     

Structure of glycosylated and unglycosylated gag and gag-pol precursor proteins of Moloney murine leukemia virus

Precursor polyproteins containing translational products of the gag gene of Moloney murine leukemia virus were purified by gel electrophoresis and cleaved into large fragments by hydroxylamine, mild acid hydrolysis, or cyanogen bromide. The hydroxylamine cleavage method (specific for asparagine-glycine bonds) was adapted especially for this study. The electrophoretic mobility and antigenicity of the fragments and, in some cases, the presence or absence of [35S]methionine revealed detailed information on the structure of Pr65gag, gPr80gag, and Pr75gag (the unglycosylated variant of gPr80gag formed in vivo in the presence of tunicamycin or in vitro in a reticulocyte cell-free system). When compared with Pr65gag, gPr80gag contains 7,000 daltons of additional amino acids, presumably as, or as part of, a leader sequence at or very close to its N terminus. We present evidence that this leader may have replaced part of the p15 sequence. Furthermore, gPr80gag contains three separate carbohydrate groups. One is attached to the presumed leader sequence or to the p15 domain, and two are attached to the p30 domain. Each of the Moloney murine leukemia virus gag precursor proteins Pr65gag, gPr80gag, and Pr75gag corresponds with a read-through product into the pol gene. We designated these products Pr180gag-pol, gPr200gag-pol, and Pr190gag-pol (the unglycosylated variant of gPr200gag-pol), respectively. gPr200gag-pol contains all of the extra amino acids and carbohydrate groups present in gPr80gag and at least one carbohydrate group in its pol sequences.     

Molecular properties of a gag- pol- env+ murine leukemia virus from cultured AKR lymphoma cells.

We have described the isolation of a replication-defective murine leukemia virus from a culture of AKR lymphoma cells [Rein et al., Nature (London) 282:753-754, 1979]. To facilitate the characterization of this murine leukemia virus, we transmitted it to mink cells and analyzed its genome by restriction mapping of the mink cellular DNA. This genome resembled the Akv genome quite closely, but it had an additional KpnI cleavage site at 1.3 kilobase pairs from the 5' end of the provirus and a small (approximately 50-base-pair) deletion between 1.8 and 3.0 kilobase pairs from the 5' end. When we tested these mink cells by immune precipitation or by competition radioimmunoassay, we found that they synthesized gPr82env, but contained no detectable gag or pol proteins. It seems likely that the KpnI cleavage site at 1.3 kilobase pairs reflects an abnormal sequence at or near the beginning of the gag gene, which prevents gag or pol translation by introducing a frameshift or termination codon into this region.     

Retroviral gag gene amber codon suppression is caused by an intrinsic cis-acting component of the viral mRNA.

In some type C retroviruses, translation of the pol gene appears to require translational suppression of the proximal gag amber codon. To identify the region of the viral nucleic acid responsible for synthesis of the pol gene products, a 300-base-pair DNA fragment containing the stop codon from a type C murine virus (AK virus) was inserted into the Escherichia coli lacZ gene such that the translational reading frame was maintained. Introduction of the resulting fusion gene into cells resulted in the suppression of the viral stop codon. As measured by beta-galactosidase production, suppression occurred at a frequency of approximately 10%. Suppression could occur in at least several vertebrate cell types and was not augmented by virus replication or the expression of viral gene products. This indicates that gag amber codon suppression does not require augmented levels of suppressor tRNA species.     

Translational readthrough of an amber termination codon during synthesis of feline leukemia virus protease.

Feline leukemia virus contains a protease which apparently has the same specificity as murine leukemia virus protease. It cleaves in vitro the Pr65gag of Gazdar-mouse sarcoma virus into the constituent p15, p12, p30, and p10 proteins. We purified the protease and determined its NH2-terminal amino acid sequence (the first 15 residues). Alignment of this amino acid sequence with the nucleotide sequence (I. Laprevotte, A. Hampe, C. H. Sherr, and F. Galibert, J. Virol. 50:884-894, 1984) reveals that the protease is a viral-coded enzyme and is located at the 5' end of the pol gene. As previously found for murine leukemia virus (Y. Yoshinaka, I. Katoh, T. D. Copeland, and S. Oroszlan, Proc. Natl. Acad. Sci. U.S.A. 82:1618-1622, 1985), feline leukemia virus protease is synthesized through in-frame suppression of the gag amber termination codon by insertion of a glutamine in the fifth position, and the first four amino acids are derived from the gag gene.     

Characterization of intracellular precursor polyproteins of Moloney murine leukemia virus.

Intracellular Moloney murine leukemia viral precursor polyproteins were compared with mature viral proteins by immunoprecipitation and tryptic peptide mapping experiments. The results were consistent with precursor roles for Pr65gag, Pr200gag-pol, Pr135pol, and gPr83env. The glycosylated gag gene product gPr85gag, although containing sequences characteristic of all four core proteins plus additional sequences not found in Pr65gag, lacked a major tyrosine-containing p30 tryptic peptide, suggesting that gPr85gag is not processed to p30.     

UAG readthrough is not increased in vivo by Moloney murine leukemia virus infection.

Expression of the pol gene of the murine leukemia viruses is subject to translational control at the UAG termination codon of the upstream gene gag. Previous experiments have suggested that: i) Moloney murine leukemia virus infection induces a tRNA(Gln)iii) in an in vitro system using the tobacco mosaic virus as template, this tRNA is able to increase readthrough at the UAG codon [1]. Here we demonstrate that, in vivo, Moloney murine leukemia virus infection does not increase translational readthrough at either the tobacco mosaic virus or the Moloney murine leukemia virus UAG stop codons.     

Spleen necrosis virus gag polyprotein is necessary for particle assembly and release but not for proteolytic processing.

The nature of spleen necrosis virus pol gene expression and the role of gag and gag-pol polyproteins in virion assembly was investigated. The DNA sequence of the gag-pol junction revealed that the two genes occupy the same open reading frame but are separated by an in-frame amber stop codon. Biochemical analysis of gag-pol translational readthrough in vitro and in Escherichia coli suggests that, in a manner similar to that in other mammalian type C retroviruses, amber stop codon suppression is required for pol gene expression. Removal of the gag stop codon had little or no effect on synthesis or cleavage of the polyprotein but interrupted particle assembly. This block could be overcome by complementation with wild-type gag protein.     

Fluidity of a retrovirus genome.

Comparison of the genomic sequences of the Friend spleen focus-forming virus with other murine retroviral sequences indicated that the spleen focus-forming virus was derived from at least three retroviruses. The 5' end of the virus, from the primer binding site through most of gag, was derived from AKV. The rest of gag and all of pol were of uncertain origin, but were probably derived from the same xenotropic virus that gave rise to the 5' half of env. The remainder was derived from Friend murine leukemia virus. The positions of a 585-base deletion, a 6-base duplication, and a point insertion that leads to a frame shift and premature protein termination in the ecotropic 3' end of env were invariant between three spleen focus-forming virus strains, indicating that they had a single common ancestor. However, the point of crossover between xenotropic viral sequences and Friend murine leukemia virus was different in each strain, and two strains were much more closely related to each other than to the third in the xenotropic region, indicating that these strains had diverged by multiple recombinations. Furthermore, a different nucleotide comprised the single point insertion near the 3' end of env, suggesting that these viruses have an extremely high transition and transversion rate.     

Nucleotide sequences of feline sarcoma virus long terminal repeats and 5' leaders show extensive homology to those of other mammalian retroviruses.

The nucleotide sequences of the Gardner-Arnstein feline sarcoma virus (FeSV) long terminal repeat and the adjacent leader sequences 5' to the viral gag gene were determined. These were compared with homologous portions of Synder-Theilen FeSV and with previously published sequences for Moloney murine sarcoma virus and simian sarcoma virus proviral DNA. More than 75% of the residues in the FeSV R and U5 regions were homologous to sequences within the same regions of the other viral long terminal repeats. Unexpectedly, alignment of the FeSV sequences with those of the Moloney murine sarcoma and simian sarcoma viruses showed similar extents of homology within U3. The homologous U3 regions included the inverted repeats, a single set of putative enhancer sequences, corresponding to a "72-base-pair" repeat, and sequences, including the CAT and TATA boxes, characteristic of eucaryotic promotors. The 5' leader sequences of both FeSV strains included a binding site for prolyl tRNA and a putative splice donor sequence. In addition, the FeSV leader contained a long open reading frame which was adjacent to and in phase with the ATG codon at the 5' end of the FeSV gag gene. The open reading frame could code for a signal peptide of about 7.4 kilodaltons. Our results support the concept that the virogenic portions of both FeSV and simian sarcoma virus were ancestrally derived from viruses of rodent origin, with conservation of regulatory sequences as well as the viral structural genes.     

Generation of a recombinant Moloney murine leukemia virus carrying the v-src gene of avian sarcoma virus: transformation in vitro and pathogenesis in vivo.

A Moloney murine leukemia virus (M-MuLV) recombinant carrying the v-src gene of avian sarcoma virus was generated by the introduction of a cloned portion of v-src from Schmidt-Ruppin A avian sarcoma virus into a molecular clone of M-MuLV provirus at the recombinant DNA level. The v-src sequences (lacking a portion of the 5' end of v-src) were inserted into the p30 region of the M-MulV gag gene so that M-MuLV gag and v-src were in the same reading frame. Transfection of this chimeric clone, pMLV(src), into NIH 3T3 cells which were constitutively producing M-MuLV gag and pol protein resulted in the formation of foci of transformed cells. Infectious and transforming virus could be recovered from the transformed cells. This virus was designated M-MuLV(src). M-MuLV(src)-transformed cells contained two novel proteins of 78 and 90 kilodaltons. The 78-kilodalton protein, p78gag-src, contained both gag and src determinants, exhibited kinase activity in an immune kinase assay, and is probably a fusion of Pr65gag and src. The 90-kilodalton protein, which is of the appropriate size to be the gPr80gag fused to src, contained gag determinants as well as a V8 protease cleavage fragment typical of the carboxy terminus of avian sarcoma virus pp60src. However, it could not be immunoprecipitated with an anti-v-src serum. M-MuLV(src)-transformed cells showed elevated levels of intracellular phosphotyrosine in proteins, although the elevation was intermediate compared with cells transformed with wild-type v-src. M-MuLV and amphotropic murine leukemia virus pseudotypes of M-MuLV(src) were inoculated into newborn NIH Swiss mice. Inoculated mice developed solid tumors at the site of inoculation after 3 to 6 weeks, with most animals dying by 14 weeks. Histopathological analysis indicated that the solid tumors were mesenchymally derived fibrosarcomas that were both invasive and metastatic.     

Mutation in the glycosylated gag protein of murine leukemia virus results in reduced in vivo infectivity and a novel defect in viral budding or release

All gammaretroviruses, including murine leukemia viruses (MuLVs), feline leukemia viruses, and gibbon-ape leukemia virus, encode an alternate, glycosylated form of Gag polyprotein (glyco-Gag or gPr80gag) in addition to the polyprotein precursor of the viral capsid proteins (Pr65gag). gPr80gag is translated from an upstream in-frame CUG initiation codon, in contrast to the AUG codon used for Pr65gag. The role of glyco-Gag in MuLV replication has been unclear, since gPr80gag-negative Moloney MuLV (M-MuLV) mutants are replication competent in vitro and pathogenic in vivo. However, reversion to the wild type is frequently observed in vivo. In these experiments, in vivo inoculation of a gPr80gag mutant, Ab-X-M-MuLV, showed substantially lower (2 log) initial infectivity in newborn NIH Swiss mice than that of wild-type virus, and revertants to the wild type could be detected by PCR cloning and DNA sequencing as early as 15 days postinfection. Atomic force microscopy of Ab-X-M-MuLV-infected producer cells or of the PA317 amphotropic MuLV-based vector packaging line (also gPr80gag negative) revealed the presence of tube-like viral structures on the cell surface. In contrast, wild-type virus-infected cells showed the typical spherical, 145-nm particles observed previously. Expression of gPr80gag in PA317 cells converted the tube-like structures to typical spherical particles. PA317 cells expressing gPr80gag produced 5- to 10-fold more infectious vector or viral particles as well. Metabolic labeling studies indicated that this reflected enhanced virus particle release rather than increased viral protein synthesis. These results indicate that gPr80gag is important for M-MuLV replication in vivo and in vitro and that the protein may be involved in a late step in viral budding or release.