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.     

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55^gag) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55^gag expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55^gag sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5′-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7–8% TSP, equivalent to 312–363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55^gag polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55^gag polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. IGV publication no. 330     

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.     

Immunogenicity of DNA and recombinant Sendai virus vaccines expressing the HIV-1 gag gene

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens. Foundation item: National 863 project (2003AA219070)     

Scrambled duplications in the feline leukemia virusgag gene: A putative pattern for molecular evolution

Summary The present study is a detailed computer-assisted analysis of the feline leukemia virus gag gene nucleotide sequence together wit its flanking sequences (ST-FeLV GAG) that is compared with the aligned sectors of the Moloney strain of murine leukemia virus (Mo-MuLV GAG) and of three strains of feline sarcoma virus. It shows that perfectly matched repeated oligomers up to 13 nucleotides long are overrepresented and scattered throughout both ST-FeLV GAG and Mo-MuLV GAG, in noncoding and coding sectors, with no stringent correlation to codon usage in ST-FeLV gPr80^gag. Many repeated oligomers share a core consensus that is intriguingly part of the inverted repeat at the termini of the long terminal repeat. Local scrambled repetitions of nucleotide subsequences have been found; they suggest a model of molecular evolution byslippage-like mechanisms. Thus, viral genomes could be subject to the same evolutionary mechanisms that are now known to be operating extensively in eukaryotic genomes. The data are discussed in light of putative patterns of molecular evolution.     

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.     

Cloning of Two Genetically Transmitted Moloney Leukemia Proviral Genomes: Correlation Between Biological Activity of the Cloned DNA and Viral Genome Activation in the Animal

The Mov-7 and Mov-9 substrains of mice, carrying Moloney murine leukemia virus (M-MuLV) in their germ line at the Mov-7 locus and Mov-9 locus, respectively, are different with respect to virus activation. Infectious virus appears in all mice carrying the Mov-9 locus but is not activated in animals carrying the Mov-7 locus. Consequently, only Mov-9 mice develop viremia and subsequent leukemia. The endogenous M-MuLV provirus with flanking mouse sequences corresponding to the Mov-7 and Mov-9 loci was molecularly cloned. Detailed restriction maps obtained from the cloned DNAs revealed no detectable differences in the proviral genomes. The flanking mouse sequences, however, were different, confirming that the Mov-7 and Mov-9 loci represent different integration sites of M-MuLV. Both clones induced XC plaques in a transfection assay. The specific infectivity of the clones, however, was different. A total of 10⁻⁵ XC plaques per genome equivalent were induced by the Mov-9 clone, whereas only 10⁻⁹ XC plaques per genome equivalent were induced by the Mov-7 clone. Moreover, NIH 3T3 cells transfected with the Mov-9 clone produced NB-tropic M-MuLV, whereas cells transfected with the Mov-7 clone did not produce infectious virus. The results suggest that M-MuLV integrated at the Mov-7 locus carries a mutation which prevents synthesis of infectious virus but permits XC plaque induction by partial genome expression or synthesis of noninfectious particles. Thus, the pattern of virus expression in Mov-7 and Mov-9 mice correlates with the biological properties of the respective clones. Genomic DNA from Mov-9 mice was not infectious in the transfection assay (specific infectivity < 10⁻⁷ PFU per genome equivalent). As the only difference between the genomic and the cloned Mov-9 DNA appears to be the presence of 5-methylcytosine in CpG sequences, our results suggest that removal of methyl groups by molecular cloning in procaryotes permits genome expression in transfected eucaryotic cells. Our results support the hypothesis that DNA methylation is relevant not only in genome expression in the animal but also in expression of genes transfected into eucaryotic cells.     

The human immunodeficiency virus type 1 carboxyl-terminal third of capsid sequence in Gag-Pol is essential but not sufficient for efficient incorporation of Pr160<Superscript><Emphasis Type="Italic">gag-pol</Emphasis></Superscript> into virus particles

To elucidate the role of the C-terminal portion of Gag in the incorporation of human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virus particles, a series of HIV-1 Gag-Pol mutants with deletions in the C-terminalgag sequence was constructed and viral incorporation of the Gag-Pol deletion mutants was analyzed using cotransfecting 293T cells with a Pr55 ^gag expression plasmid. The biological function of the incorporated HIV-1pol gene product was tested using an infectivity assay of the released virus particles which were pseudotyped with the murine leukemia virus Env. Analysis indicated that Gag-Pol deletion mutants, with a removal of the matrix (MA) and/or nucleocapsid (NC) or of the N-terminal two thirds of thegag coding sequence, could be incorporated efficiently into virus particles and produce significant amounts of infectious virions when assayed in a single-cycle infection assay. In contrast, mutations involving a deletion of the major homology region and the adjacent C-terminal capsid sequence significantly affected Gag-Pol incorporation. However, incorporation into virus particles of a Gag-Pol deletion mutant retaining both the major homology region and the adjacent C-terminal capsid intact was still severely impaired. This suggests that the capsid major homology region and the adjacent C-terminal capsid sequence in Gag-Pol are necessary but not sufficient for the incorporation of HIV-1 Pr160 ^gag-pol into virus particles.     

Structure of glycosylated and unglycosylated gag polyproteins of Rauscher murine leukemia virus: carbohydrate attachment sites.

The structural relationships among the gag polyproteins Pr65gag, Pr75gag, and gPr80gag of Rauscher murine leukemia virus were studied by endoglycosidase H digestion and formic acid cleavage. Fragments were identified by precipitation with specific antisera to constituent virion structural proteins followed by one-dimensional mapping. Endoglycosidase H reduced the size of gPr80gag to that of Pr75gag. By comparing fragments of gPr80gag and the apoprotein Pr75gag, the former was shown to contain two mannose-rich oligosaccharide units. By comparing fragments of Pr65gag and Pr75gag, the latter was shown to differ from Pr65gag at the amino terminus by the presence of a leader peptide approximately 7,000 daltons in size. The internal and carboxyl-terminal peptides of the two unglycosylated polyproteins were not detectably different. The location of the two N-linked carbohydrate chains in gPr80gag has been specified. One occurs in the carboxyl-terminal half of the polyprotein at asparagine177 of the p30 sequence and the other is found in a 23,000-dalton fragment located in the amino-terminal region of gPr80gag and containing the additional amino acid sequences not found in Pr65gag plus a substantial portion of p15.     

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200^gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65^gag and Moloney murine leukemia virus Pr63^gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70^gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70^gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200^gag-pol coincided closely with the molar loss of Pr63^gag. Enhancement of Pr200^gag-pol and Pr70^gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63^gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63^gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67^gag, appeared, whereas Pr63^gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67^gag which appeared, 1 mol of Pr63^gag was lost.     

Isolation and characterization of a mouse cell line containing a defective Moloney murine leukemia virus genome.

A culture of mouse cells containing a 1,000-nucleotide deletion mutant of Moloney murine leukemia virus has been isolated. The deletion did not affect the size or function of the 21S mRNA that encodes the env gene products. Both the deleted RNA and the 21S mRNA were recovered in polyribosomes. Cells containing the deleted virus made no detectable Pr180gag-pol. Pr65gag synthesis with also absent, but a 45,000-molecular-weight gag gene product was found that might be encoded by the deleted genome. Biosynthesis of Pr80env proceeded normally in these cells; the intracellular precursor was cleaved and migrated to the cell surface as gp70. The cells could not be superinfected by homologous Moloney murine leukemia virus presumably because of surface restriction due to the gp70. Although the cells express the Moloney murine leukemia virus gp70 on their surface, they will not make pseudotypes after infection with vesicular stomatitis virus implying that Pr65gag may play a critical role in pseudotype formation. Induction of endogenous virus expression in the cells carrying the deletion mutant generated an N-tropic murine leukemia virus that can fuse XC cells. This may represent a recombinant between the deletion mutant and an endogenous virus.     

Deletion mutants of Moloney murine leukemia virus which lack glycosylated gag protein are replication competent

A series of deletion mutations localized near the 5' end of the Moloney murine leukemia virus genome was generated by site-specific mutagenesis of cloned viral DNA. The mutants recovered from such deleted DNAs failed to synthesize the normal glycosylated gag protein gPr80gag. Two of the mutants made no detectable protein, and a third mutant, containing a 66-base pair deletion, synthesized an altered gag protein which was not glycosylated. All the mutants made normal amounts of the internal Pr65gag protein. The viruses were XC positive and replicated normally in NIH/3T3 cells as well as in lymphoid cell lines. These results indicate that the additional peptides of the glycosylated gag protein are encoded near the 5' end, that the glycosylated and internal gag proteins are synthesized independently, and that the glycosylated gag protein is not required during the normal replication cycle. In addition, the region deleted in these mutants apparently encodes no cis-acting function needed for replication. Thus, all essential sequences, including those for packaging viral RNA, must lie outside this area.     

Assembly and composition of intracellular particles formed by Moloney murine leukemia virus.

Assembly of type C retroviruses such as Moloney murine leukemia virus (M-MuLV) ordinarily occurs at the plasma membranes of infected cells and absolutely requires the particle core precursor protein, Pr65gag. Previously we have shown that Pr65gag is membrane associated and that at least a portion of intracellular Pr65gag protein appears to be routed to the plasma membrane by a vesicular transport pathway. Here we show that intracellular particle formation can occur in M-MuLV-infected cells. M-MuLV immature particles were observed by electron microscopy budding into and within rough endoplasmic reticulum, Golgi, and vacuolar compartments. Biochemical fractionation studies indicated that intracellular Pr65gag was present in nonionic detergent-resistant complexes of greater than 150S. Additionally, viral RNA and polymerase functions appeared to be associated with intracellular particles, as were Gag-beta-galactosidase fusion proteins which have the capacity to be incorporated into virions. Immature intracellular particles in postnuclear lysates could be proteolytically processed in vitro to mature forms, while extracellular immature M-MuLV particles remained immature as long as 10 h during incubations. The occurrence of M-MuLV-derived intracellular particles demonstrates that Pr65gag can associate with intracellular membranes and indicates that if a plasma membrane Pr65gag receptor exists, it also can be found in other membrane compartments. These results support the hypothesis that intracellular particles may serve as a virus reservoir during in vivo infections.     

Composition, arrangement and cleavage of the mouse mammary tumor virus polyprotein precursor Pr77gag and p110gag.

The amino acid sequence relationship between the nonglycosylated structural proteins of murine mammary tumor virus and the polyproteins from infected cells immunoprecipitated with an anti-p27 serum were examined using two-dimensional tryptic peptide mapping procedures. The proteins were labeled with ¹⁴C-lysine and ¹⁴C-arginine so that all but one of the tryptic peptides released from a protein could be detected. Previous studies have shown that immunoprecipitation of mammary tumor cells with anti-p27 serum results in the isolation of seven proteins in the molecular weight range of 34,000–160,000 daltons; and that cell-free translation using viral genomic RNA yields three p27-related proteins of 160,000, 110,000 and 77,000 daltons, similar to the three high molecular weight proteins detected in vivo. The proteins of lower molecular weight were thought to be cleavage intermediates of Pr77^gag. As judged from the peptide maps, Pr77^gag contained the complete sequences of the four major internal proteins of the virion (p27, pp21, p14 and p10) and possibly a fifth highly basic protein (p8) also found in virions. The putative cleavage intermediates, as expected, lacked some tryptic peptides that could be assigned to one or more of the major virion proteins and thus allow a scheme for the cleavage events to be constructed. p110^gag contained all the tryptic peptides found in Pr77^gag, plus some additional peptides. A minor virion protein p30 was found to include the peptides of p14 as well as some of the additional peptides present in p110^gag, suggesting a precursor-product relationship between the pr110^gag and p30. The data obtained from these studies lead us to propose that there are three protein precursors which include, at least in part, the gag gene region of the virion—p160 (potentially a gag/pol precursor), p110^gag and Pr77^gag—and that the arrangement of the virion proteins within the gag gene (pr77^gag) is p10-pp21-p27-p14.     

A new RNA vaccine platform based on MS2 virus-like particles produced in Saccharomyces cerevisiae

mRNA vaccines are potentially attractive alternatives to DNA vaccines more often discussed, as they are generally considered safer than their DNA counterparts. The major limitations on the potency of RNA vaccines are their instability and inability to spread in vivo. Virus-like particles (VLPs) based on the bacteriophage MS2 have demonstrated remarkably high stability and may provide an improved platform for RNA-based genetic vaccination. However, no in vivo study of an MS2 VLP-mediated RNA vaccine has been reported. Therefore, we developed a model vaccine wherein MS2 VLPs packaging HIV-1 gag mRNAs (1544 bases) were produced in Saccharomyces cerevisiae, and then, used to immunize BALB/c mice. Serological analyses showed that antigen-specific antibody responses were elicited by immunization. These findings suggest that MS2 VLPs can be used in the design and construction of novel and safe phage-based mRNA delivery vectors.     

Tunicamycin inhibits glycosylation of precursor polyprotein encoded by env gene of Rauscher murine leukemia virus.

The molecular weight of the precursor polyprotein to the envelope proteins of Rauscher murine leukemia virus is reduced from 85,000 to 68,000 daltons when synthesized in the presence of tunicamycin, a specific inhibitor of the synthesis of oligosaccharides that attach to glycoproteins via asparagine residues. The unglycosylated precursor protein (Pr68^$\text{env}$) is synthesized at a rate comparable to that of the normal carbohydrate-containing envelope precursor (gPr85^$\text{env}$). Pr68^$\text{env}$ is not proteolytically processed and remains undegraded in the cell. Thus, most if not all of the carbohydrate content of gPr85^$\text{env}$ is N-linked, and glycosylation appears to be necessary for normal processing of precursor proteins into viral particles.