Virus-specific precursor polypeptides in cells infected with Rauscher leukemia virus: Synthesis,identification, and processing

The synthesis of virus-specific polypeptides in JLS-V9 and JLS-V5 cells infected with Rauscher leukemia virus (R-MuLV) was studied in pulse-chase experiments, followed by radioimmunoprecipitation analysis with polyvalent and monospecific antisera against R-MuLV proteins. Two glycosylated polypeptides with molecular weights of about 8 (env-pr82) were identified as precursors of the virion envelope polypeptides gp69/71 and p15(E). On the other hand, virion polypeptides p30 and pl5 are derived from a 75,000-(gag-pr75) and a 65,000-dalton (gag-pr65) precursor polypeptide. These precursor-product relations were confirmed by analysis of chymotryptic digests of virion polypeptides and their precursors. In the presence of the arginine analog canavanine two polypeptides with molecular weights of 82,000 and 72,000 (gag-pr82 and gag-pr72, respectively) were synthesized instead of gag-pr75 and gag-pr65. Processing of precursor polypeptides is reduced in the presence of canavanine. From these results, we conclude that gag-pr82 is possibly the primary gag-gene product and is cleaved into gag-pr75. These studies provided the following additional information: First, we established that immediately after cleavage of their precursors, gp69/71 is found on the outer surface of the cell and p30, p15, and p12 leave the cell as components of budding virions. Therefore, these polypeptides were detected intracellularly in very small amounts only. Polypeptide p15(E) was present within the cell as well as on its outer surface. Second, despite a great similarity in virus-specific (precursor) polypeptides detected in JLS-V9 and JLS-V5 cells, small differences in molecular weights of some of these polypeptides were observed after SDS-PAGE.     

Virus-specific polypeptides of human parainfluenza virus type 4 and their synthesis in infected cells

We have studied the structural components of human parainfluenza virus type 4A (PIV-4A) and identified some virus-specific polypeptides by immunoprecipitation with polyclonal and monoclonal antibodies followed by one- or two-dimensional SDS-PAGE. HN polypeptides existed as monomer, disulfide-linked dimer, and disulfide-linked larger oligomer in cells infected with PIV-4A. Interestingly, the nonreduced NP, the nonreduced fusion, and the reduced F1 proteins migrated as doublets. Two F1 polypeptides were derived from different F1 + 2 proteins which migrated separately under nonreducing condition. In Vero cells infected with two strains of PIV-4A, two lower-molecular-weight proteins related to NP were detected. Oligopeptide patterns of the lower-molecular-weight protein were similar to those of NP protein synthesized in primary monkey kidney cells. The NP-related low-molecular-weight protein(s) was immunoprecipitated by 1 of 11 monoclonal antibodies against mumps virus NP protein. The MAb also reacted with NP proteins of PIV-2 and SV5. Thus, the epitope recognized by the MAb was common among PIV-2, PIV-4, mumps virus, and SV5, suggesting that the epitope might have an important biological function. However, the MAb did not react with the intact NP protein from cells infected with PIV-4, indicating that the epitope of PIV-4A was presented only when NP was cleaved. Phosphorylation was demonstrated for NP and P proteins.     

Early events of C-type virus budding in cells infected with a Rauscher leukemia virus temperature-sensitive mutant

Summary Surface replicas ofts25-infected cells reveal the organization of virus-specific knobs prior to and during the early stages of budding, and antibody-mediated ferritin labeling suggests a transmembrane association of viral envelope and core components.With 6 Figures     

The synthesis of Sendai virus polypeptides in infected cells. III. Phosphorylation of polypeptides.

Phosphorylated and unphosphorylated forms of the membrane polypeptide (M) and the nucleocapsid polypeptide (NP) of Sendai virus have been identified in both infected cells and virions. Polypeptide B, found previously in infected cells, has been shown to be a phosphorylated form of M by peptide mapping, by conversion of M to B by phosphorylation in both pulse-chase experiments in infected cells and in vitro by a virion-associated protein kinase and, conversely, by conversion of B to M through the loss of phosphate in cell lysates. Although very little of the phosphorylated form was found in virions, B and M were found in similar proportions in infected cells after a 30-min pulse, suggesting that the nonphosphorylated form is preferentially incorporated into virions or that phosphate is removed during the maturation process. The finding of phosphorylation of M and NP in cells suggests that this may play a role in virus replication or assembly. Two phosphorylated forms of the nucleocapsid polypeptide (NP_P1 and NP_P2) have been found, but the unphosphorylated NP is the predominant form in both cells and virions. The similarity of these three polypeptides, except for their phosphate content, has been shown by peptide mapping. The membrane polypeptides B and M were separated by electrophoresis on polyacrylamide gels in the absence of urea, but in the presence of 4 M urea they comigrated. In contrast to these results with polypeptides B and M, polypeptides NP, NP_P1 and NP_P2 were resolved in gels in the presence of 4 M urea but comigrated in its absence. Thus, the behavior of viral phosphopolypeptides in different polyacrylamide-gel systems varies depending on the polypeptides. Possible biological roles of phosphorylation of the virus polypeptides are discussed.     

Biosynthesis of Rauscher leukemia viral proteins: presence of p30 and envelope p15 sequences in precursor polypeptides.

Viral structural polypeptides p30 and a 17,000-dalton polypeptide, termed envelope p15, are formed in Rauscher leukemia virus (RLV)-infected N.I.H. Swiss mouse embryo fibroblasts by cleavage of high molecular weight precursor polypeptides. The evidence for this conclusion is based on the analysis of polypeptides precipitated from RLV-infected cells by antiserum directed against RLV structural proteins. High resolution sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE) of such immune precipitates from infected cells pulse-labeled with [³⁵S]methionine or pulse-labeled and then chased in unlabeled medium provides evidence that three size classes of unstable polypeptides are precursors to virion p30. They are: two polypeptides with an approximate molecular weight of 200,000 (termed Pr1a and b), an 80,000-dalton polypeptide (Pr3) and a 65,000-dalton polypeptide (Pr4). Ion-exchange chromatography of tryptic digests showed that methionine-containing tryptic peptides of p30 are present in these precursor polypeptides. Methionine-labeled tryptic peptide sequences of envelope p15 were present in a 90,000-dalton peptide fraction containing two components (Pr2a and b). The latter polypeptides comigrated with viral specific fucose-free glycoproteins not present in virions or uninfected cells.     

The synthesis of Sendai virus polypeptides in infected cells. II. Intracellular distribution of polypeptides.

The intracellular distribution and the kinetics of association of Sendai virus polypeptides with cytoplasmic fractions and plasma membranes have been studied. The viral surface glycoproteins HN and F₀ have been found in pulse-chase experiments to migrate from rough to smooth membranes and to the plasma membrane. The M protein was found in varying amounts in most cell fractions but was predominantly associated with smooth membranes; there was no evidence of its migration from rough to smooth membranes. The L, P, and NP polypeptides were found with the rough membrane and free ribosome fractions. Polypeptides B and C, which were previously found in extracts of whole infected cells, were found to be unstable during cell fractionation. In the presence of protease inhibitors, polypeptide C, which is thought to be a virus-specific nonstructural protein, was found with the rough membrane fraction. Thus its instability in cell fractions appears to be due to proteolytic digestion. Polypeptide B was not found in cell fractions even in the presence of protease inhibitors. Evidence reported in the following paper has indicated that B is a phosphorylated form of polypeptide M and that its instability is presumably due to loss of phosphate. Polypeptides I–IV, which the available evidence suggests are cellular polypeptides whose synthesis may be enhanced in infected cells, were found in significant amount in soluble form after cell fractionation, although IV was also associated with most membrane fractions.     

Macromolecular synthesis in cells infected with frog virus 3. III. Virus-specific protein synthesis by temperature-sensitive mutants.

Six temperature-sensitive (ts) mutants of frog virus 3, (five DNA⁺ and one DNA^?) representing six separate complementation groups, were examined for their intracellular patterns of virus-specific protein synthesis both at permissive (23°) and nonpermissive (30°) temperature. At permissive temperature protein synthesis and its regulation by each mutant was similar to wild type. At nonpermissive temperature all proteins were detected but some had altered rates of synthesis, indicating defective regulation by three of the six ts mutants.The six mutants can be divided into three categories based upon the time and nature of the ts defect during the replication cycle. The first category includes three mutants, each representing a separate complementation group in which virus-specific protein synthesis and its regulation is apparently normal at nonpermissive temperature. These mutants have defects in the virus maturation and assembly process suggesting the participation of several frog virus 3 genes in the assembly process. The second category consists of a single mutant that has an early defect in the regulation of viral protein synthesis; consequently a late pattern of virus-specific protein (VSP) synthesis is absent in cells infected with this mutant at nonpermissive temperature. The third category includes two mutants; these mutants are unable to regulate the rate of synthesis of certain VSP but have some features of the late pattern of virus-specific protein synthesis.The data presented in this report confirm and extend the evidence for temporal control of the rate of viral protein synthesis during frog virus 3 infection. This control appears to be mediated through several viral regulatory proteins.     

Virus-specific immune response in the lungs of mice infected with influenza virus.

The time course of primary humoral immune response in NFS/N mice infected with the adapted influenza virus A/Aichi 2/68(H3N2) was followed by determination of the different class immunoglobulins in lungs, lung washings, and in blood serum. The quantity of antibody-producing cells (APC) was estimated by local haemolysis in gel. The presence of antibodies in serum and lavage fluid was tested by the methods of radial haemolysis and radial immunodiffusion. It was shown that the local immune response had developed earlier than systemic antibodies occurred in the serum.     

Virus-specific sequences within restriction DNA fragments of cells persistently infected with tick-born encephalitis virus

Summary Using the Southern blot technique and a³²P-labelled DNA copy of RNA TBEV as a probe, we studied the state of proviral DNA sequences in nuclear DNA of cells persistently infected (p.i.) with TBEV. Integration of virus-specific DNA sequences in DNA of p.i. was demonstrated. Discrete bands were observed, suggesting the existence of limited and specific intergration sites in host cellular DNA.With 1 Figure     

Induction of leukemia in Rauscher virus-resistant mice by mixed infection with M. arthritidis and Rauscher virus]

At 2-10 months after combined infection with Rauscher virus and M. arthritidis of mice (C57BL/6XA/He)F1 resistant to this virus 14 out of 23 animals developed leukemia morphologically identical to Rauscher leukemia induced in sensitive mice. In control groups of similar animals infected with virus alone or mycoplasma alone not a single case of leukemia developed. As a result of serial intraperitoneal passages in syngeneic mice of cells of leukemias primarily induced by mixed mycoplasma-virus infection 3 transplantable leukemia strains were obtained the cytological picture of which was similar with the original. Upon intraperitoneal and subcutaneous inoculations of leukemic cells generalized leukemia develops as well as a local transplant under the skin or in the abdominal wall at the site of needle puncture.     

Antigenic differences among multiply charged Moloney murine leukemia virus p30 polypeptides found inside infected cells

At least three Moloney murine leukemia virus (M-MuLV) p30 polypeptides (p30's), viz., a major species at pI 6.3 and two minor ones at pI 6.1 and pI 6.6, have previously been identified in purified virions by 2-dimensional gel electrophoresis and chromatofocusing (Katoh, I., Yoshinaka, Y. and Luftig, R.B. (1984) J. Gen. Virol. 65, 733-741). We have observed a similar, but distinctive pI pattern for [35S]methionine-labeled MuLV p30's in lysates from chronically infected (MuLV) cells. The variation in pI pattern of the intracellular MuLV p30's was dependent on the type of p30 reactive antibody used for immunoprecipitation. Specifically: a p30 spot with pI 6.3 was always precipitated as the major spot with three different antibodies, minor spots with pI 6.0 and 6.6 were variably seen dependent on the antibody used, and an intracellular p30 spot at pI 6.1 was only precipitated with a rat p30 monoclonal antibody but not with monospecific mouse or intact MuLV cross-reacting p30 sera. These results indicate that first, there are differences between the pI pattern of virion and intracellular MuLV p30's, and second, the antigenic determinants of intracellular p30's vary dependent on the antibody used for immunoprecipitation.     

Macromolecular synthesis in cells infected by frog virus 3. I. Virus-specific protein synthesis and its regulation

Heat-inactivated frog virus 3 inhibited protein synthesis in fathead minnow and baby hamster kidney cells but did not affect the replication of superinfecting infectious frog virus 3 in these cells. This method of controlling host-cell protein synthesis enabled us to identify 20 proteins induced by frog virus 3 infection. All detectable virus-specific proteins were synthesized within 2 hr after infection. Three viral structural proteins (VSP)—7, 9, and 13—were synthesized at maximum rates early in infection, and two others, VSP 5 and 11, reached their peak rate of synthesis late in infection. All structural and nonstructural proteins were made in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. In the absence of viral DNA replication, the kinetics of synthesis of VSP 5 and 11 were similar to those during normal infection, but there was no reduction in the synthesis of VSP 7, 9, and 13 late in the infection. These results suggest that synthesis of all viral structural proteins is an early event in the FV 3 replication cycle, with progeny viral DNA required to regulate the synthesis of certain viral proteins.