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.     

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.     

Selective inhibition of cellular protein synthesis by amphotericin B in EMC virus-infected cells

The addition of amphotericin B to EMC-infected cells induces a selective inhibition of cellular as compared to viral protein synthesis. A concentration of 5 μM amphotericin B in the culture medium produced 80% inhibition of actin synthesis, whereas the synthesis of the viral protein γ was inhibited by only 20%. The selective inhibition of cellular mRNA translation induced by this polyene antibiotic depends on the extracellular concentration of NaCl: In the absence of NaCl in the culture medium, the inhibition of actin synthesis was 25% in the presence of 10 μM amphotericin B, whereas, in hypertonic media, the inhibition of cellular protein synthesis was complete. These results indicate that the modification of membrane permeability by amphotericin B has a selective effect on the inhibition of cellular protein synthesis, and that the concentration of NaCl in the culture medium strongly influences the relative synthesis of cellular and viral proteins in EMC-infected cells.     

Protein synthesis in Rift Valley fever virus-infected cells

The complete sequence of the neuraminidase (NA) gene of the influenza A strain A/parrot/ Ulster /73 ( H7N1 ) has been determined after reverse transcribing and cloning it into the pBR322 plasmid, followed by subcloning into M13 vectors and sequencing with dideoxynucleotide chain terminators. The gene consists of 1458 nucleotides and codes for a protein of 469 amino acids. The neuraminidase has seven potential glycosylation sites. According to the molecular weight as determined by electrophoretic migration in polyacrylamide gel all of these sites might carry a carbohydrate side chain. When the parrot Ulster NA was compared with two other N1 neuraminidases, those of the human PR8 and WSN strains, deletions in the stalk region of 15 amino acids for PR8 NA and of 16 amino acids for WSN NA were apparent. No further rearrangements were found within N1 neuraminidases. Although the parrot Ulster strain was isolated 40 years after the two human strains, the base sequence homology of their NA genes is still 83 or 82%, respectively.     

Relation between the levels of mRNA abundance and kinetics of protein synthesis in pseudorabies virus-infected cells

Virus proteins synthesized by pseudorabies virus-infected cells can be classified into five groups on the basis of the kinetics of their synthesis at various stages of the infective process; virus mRNAs can similarly be classified into four groups. To determine whether the kinetics of synthesis of specific proteins are determined solely by the level of abundance in the cells of their mRNAs, we have compared at various times after infection the relative synthesis of these proteins with the relative abundance of their mRNAs. We have focused on two proteins: the 142K major capsid protein, an early-late protein, and the 136K major DNA binding protein, an early protein. The mRNAs encoding these proteins were identified. The relative abundances of these mRNAs in the cytoplasms of the infected cells were found to be the same as those associated with the polysome fractions. The relative amount of the proteins synthesized by the infected cells at a given stage of the infective process closely reflected the relative amount of the mRNA encoding these proteins that was present in the cells at that stage of the infective process. Most virus mRNA species that are present in the cytoplasm of infected cells were represented on polysomes to approximately an equal extent. Some RNA species were, however, significantly underrepresented under certain conditions in the polysomal fractions. We conclude that whereas the amount of many virus proteins synthesized by the infected cells is determined mainly by the level of the abundance of their mRNAs, additional controls operate in the cells that determine the relative rates of synthesis of some other virus proteins.     

The polypeptides of canine distemper virus: Synthesis in infected cells and relatedness to the polypeptides of other morbilliviruses

The biochemical and immunological properties of the polypeptides of canine distemper virus (CDV), their synthesis and processing in infected cells, and their relatedness to the polypeptides of other morbilliviruses have been studied. CDV virions contain six major polypeptides which are analogous to those of measles virus (MV). These polypeptides with their estimated molecular weights (m_r) are: L (200,000); H (76,000); P (66,000); NP (58,000); F (62,000), which consists of two disulfide-linked polypeptides, F₁ (40,000) and F₂ (23,000); and M (34,000). The H, F₁, and F₂ polypeptides of CDV are glycosylated; the presence of carbohydrate on F₁ is in contrast to its absence on the F₁ of MV. The CDV F₂ has a larger apparent M_r than the MV F₂ (23,000 vs 12,000). The NP and P polypeptides of CDV are phosphorylated, and in pulse-chase experiments in CDV-labe;ed cells the P polypeptide was rapidly lost. In addition to the structural polypeptides, a putative nonstructural protein, NS (M_r 18,000), was found in CDV-infected cells. The polypeptide also turned over rapidly in pulse-chase experiments.The immunological relatedness of the polypeptides of MV and CDV and of two other morbilliviruses, rinderpest (RV) and a bovine encephalitis virus (107) was shown by immuno-precipitation of the viral polypeptides from CDV- and MV-infected cells with antisera against each of the four viruses. The only failure to exhibit reciprocal reactivity between CDV and MV was found with the H polypeptides, where only a one-way cross was found, i.e., MV antiserum precipitated all of the CDV polypeptides, whereas CDV antiserum precipitated all of the MV polypeptides except H. RV antiserum resembled that of MV; it precipitated all of the polypeptides of both MV and CDV, whereas 107 antiserum, like that of CDV, precipitated all of the CDV polypeptides and all of the MV polypeptides except H. These results indicate that these four morbilliviruses with different host ranges are antigenically closely related, with MV apparently more closely related to RV, and CDV to 107 virus. In spite of their antigenic similarities, the individual polypeptides of CDV and MV could be easily distinguished by peptide mapping. Some similarities were found in the internal polypeptides P, NP, and M, but very little in the surface glycoproteins, H and F₁.     

Immunological and physicochemical studies of influenza matrix (M) polypeptides.

Immunologically active preparations of M polypeptides were recovered from influenza A viruses and had a sedimentation coefficient of 3.3 s. The type-specific antigenic determinants of the M polypeptide were resistant to denaturation by heat at 100° for 2 min in the presence of detergents and to treatment with acidic-chloroform methanol. Mild proteolysis with trypsin or caseinase C produced fragments of 13,000 and 6,000 daltons. The 13,000-dalton fragments possessed some identical antigenic determinants to the whole 25,000-dalton M molecule and partially absorbed antibody to the 25,000-dalton polypeptide from a specific antiserum. Immunization of ferrets with M polypeptide or M polypeptide fragments had no preventative or enhancing effect on influenza virus infection, whereas immunization with homologous bromelain-released HA polypeptides conferred absolute protection to virus challenge.     

Immunoprecipitation of virus-specific immediate-early and early polypeptides from cells lytically infected with human cytomegalovirus strain AD 169

By immunoprecipitation of human cytomegalovirus-infected cell-specific polypeptides (ICPs) with a variety of human cytomegalovirus-positive sera and analysis by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, we can identify at least 20 ICSP bands from lytic infections by 6 hr postinfection (pi). Three of these polypeptide bands (78K, 73K, and 68K) may represent more than one species of polypeptide. Four polypeptide bands (78K, 77K, 73K, and 31K) can be identified as immediate-early based on their synthesis in the presence of actinomycin-D after removal from a protein synthesis block mediated by cycloheximide (CH). An immediate-early 78K polypeptide and an early 49K polypeptide are synthesized only transiently during the first 4 hr pi. Most immediate-early polypeptide synthesis is enhanced after removal of a 5 hr CH block. Taken together, these results identify many previously undetected immediate-early and early ICSPs and suggest that several regulatory events are occurring during the early phase of the lytic cycle.     

Cyclic synthesis of human cytomegalovirus-induced proteins in infected cells.

Synthesis of most human embryo lung cellular proteins is more sensitive to inhibition by a hypertonic condition than is synthesis of cytomegalovirus (CMV)-induced proteins. Results using the selective suppression of host cell protein synthesis by the hypertonic condition indicate that virus-induced proteins are synthesized in a cyclic manner.     

Alterations in monovalent cation transport in Sindbis virus-infected chick cells

Influx experiments using the potassium tracer 86Rb+ indicated that the activity of the Na+K+ ATPase, or sodium pump, was reduced 40-50% as a consequence of Sindbis virus infection of avian fibroblasts. The inhibition of this ouabain-sensitive, active transport system temporally correlated with a decrease in the intracellular K+ concentration and the termination of cellular protein synthesis. By contrast, the rate of influx facilitated by the furosemide-sensitive (Na+K+Cl-) cotransport system was only slightly depressed. Efflux experiments indicated that no alterations in the relative rate of nonspecific permeability or "leakage" of K+ could be detected in chick cells infected by Sindbis virus. The amount of [3H]ouabain bound to Sindbis virus-infected cells paralleled the reduction in Na+K+ ATPase activity. These binding studies revealed no difference in the number of Na+ pump sites. The Km of ouabain binding, however, increased approximately 3.5-fold in the virus-infected cells. No change in the apparent affinity of the Na+ pump for K+ could be detected, yet the Vmax for ouabain-sensitive K+ transport was decreased. These experiments suggest that a reduction in Na+K+ ATPase turnover results in the altered intracellular monovalent cation levels found in Sindbis virus-infected chick cells.     

Induction of a deoxyuridine triphosphate nucleotidohydrolase activity in Epstein-Barr virus-infected cells

Superinfection of Raji cells with Epstein-Barr virus (EBV) or chemical induction of HR-1 cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) results in the induction of a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) which is not observed in mock-treated cells or TPA-treated EBV genome-negative BJAB cells. The EBV-induced dUTPase could be distinguished from the host dUTPase based upon differences in their migration in polyacrylamide gels and sensitivity to the 5-mercurithioguanosine derivitive of dUTP. The expression of the EBV-specified dUTPase is prevented by phosphonoacetic acid indicating that its expression is dependent upon EBV-DNA replication.     

Properties of herpesvirus-induced "immediate early" polypeptides

It has been shown that both HSV1- and HSV2-induced “immediate early” polypeptides bind, with a range of affinities, to native calf thymus DNA in vitro. The biological relevance of this observation is reflected in cell fractionation studies, which demonstrate that HSV1-and HSV2-induced immediate early polypeptides migrate to the nucleus and there bind strongly to chromatin from infected cells.     

Template-dependent RNA polymerase from black beetle virus-infected Drosophila melanogaster cells

Infection of cultured cells of Drosophila melanogaster with black beetle virus (BBV) induces an RNA polymerase that is bound to cellular particulate material in a complex with a template RNA. We have solubilized the polymerase by treatment of the relevant particulates with detergents such as dodecyl-beta-D-maltoside. The polymerase activity was made dependent upon exogenous RNA by destruction of the endogenous template RNA with micrococcal nuclease. Addition of BBV RNA1 or RNA2 induced synthesis of full-length negative-strand RNA isolated as a double-stranded complex with the added RNA. Newly synthesized plus strands were also detected in the RNA2 complexes. Certain other viral RNAs also induced synthesis of their negative strands.     

DNA synthesis in mouse embryo fibroblast cells infected with murine cytomegalovirus.

The overall rate of DNA synthesis in mouse embryo fibroblast (MEF) cells infected with murine cytomegalovirus (MCMV) decreased immediately after infection, reaching its minimum at 10–12 hr postinfection (PI), and then increased gradually. CsCl equilibrium centrifugation analysis of synthesis of both host and viral DNA in MEF cells infected with MCMV revealed the following: (1) Host cell DNA synthesis was inhibited by more than 95% by 10–12 hr PI. (2) Viral DNA synthesis began at 10–12 hr PI, and by 22–24 hr PI the rate was slightly higher than the rate of host cell DNA synthesis observed at 0–2 hr PI. (3) Viral DNA synthesis occurred without the concurrent synthesis of any significant amount of host cell DNA, indicating that there was preferential viral DNA synthesis. The inhibition of host DNA synthesis occurred when cells were infected with either uv- or heat-inactivated MCMV. This occurred in the absence of both cytopathic effects (CPE) and the synthesis of viral-induced proteins. In these instances, the suppression of host DNA synthesis was observed only during the first 12 hr PI. At 12–14 hr PI, host DNA synthesis began to increase, and finally reached or surpassed the level seen in mock-infected cells. Since the suppression of the host DNA synthesis occurred in the absence of viral-induced protein synthesis, the possibility that the suppression is caused by one or more of the structural proteins of the infecting viral particles is discussed.     

An analysis of type-C retrovirus polypeptides and their associations in the virion.

The polypeptide compositions of [³H]leucine-labeled Prague-Rous sarcoma virus-subgroup C (Pr-RSV-C) and Friend murine leukemia virus (FLV) were investigated using guanidine hydrochloride gel filtration and a high-resolution SDS-polyacrylamide-gel system. These techniques resolved seven major structural components in Pr-RSV-C (gp85, gp35, p27, p19, p12, p15, and p10), as reported previously for other avian leukosis-sarcoma viruses. However, in the case of FLV two previously unresolved proteins (p15E and p12E) were clearly demonstrated in addition to gp71, p30, p15C, p12, and p10. FLV p15E, p12E, and gp71 were removed when intact virions were digested with bromelain, whereas the remaining components were not affected. These and other studies support the notion that gp71, p15E, and p12E are situated on the surface of the virion. The linkages (covalent and noncovalent) between viral polypeptides were also examined. The results of these studies indicate that more than 90% of avian gp85 and gp35 are disulfide linked as a viral glycoprotein complex (VGP). The data also suggests that p19 exists as a network of disulfide-linked molecules, some of which may be further linked to VGP. In contrast to the avian system, only about 10–15% of FLV gp71 is disulfide linked to p15E in the VGP complex; the remaining gp71 is apparently loosely attached to the virus, perhaps by a noncovalent interaction with p12E. The implications of these associations to virus structure and assembly are discussed.     

Restriction of Moloney murine leukemia virus replication in Moloney murine sarcoma virus-infected cells

MuSV349 is a TB-cell line which produces infectious MuSV with little or no MuLV detectable by the XC assay. The apparent restriction of MuLV replication in MuSV349 cells was investigated. A replication-competent helper virus, with protein composition nearly identical to that of Mo-MuLV was isolated from the viruses produced by MuSV349 cells. This helper MuLV after it was separated from MuSV and upon infection of TB cells produced viral titer similar to that of Mo-MuLV-infected TB cells indicating that its replication might have been restricted in the MuSV349 cells. To find out whether the suppression of the helper virus replication is due to the genetic peculiarities of the virus or MuSV349 cells, the relative amounts of MuLV and MuSV produced by several distinct clonal MuSV isolates (derived from a common progenitor) upon superinfection with Mo-MuLV were determined. The results of these experiments showed that while both SV7 and SV15F on coinfection with Mo-MuLV produced MuSV in excess over MuLV; and ts110 and tsSV13 on coinfection with Mo-MuLV produced MuLV in excess over MuSV. Since the same Mo-MuLV is used in these experiments and since upon transfer to a different cell, SV7, SV15F, and ts110 retain the property to restrict or not restrict MuLV replication it appears that the above property is determined by the genetics of the MuSV.