Free and membrane-bound polyribosomes in BHK cells infected with Sindbis virus.

The data presented in the paper demonstrate that in BHK cells infected with Sindbis virus virtually all the 42S mRNA not in nucleocapsid is associated with free polyribosomes, whereas the 26S mRNA is distributed between free and membrane-bound polyribosomes. We suggest that the 26S RNA polyribosomes are bound to the membranes through the nascent chains of the B1 protein and that a large percentage of 26S RNA polyribosomes free in the cytoplasm may be due to the small amount of rough endoplasmic reticulum in BHK cells. In addition, we found that intracellular nucleocapsid is in the nonmembrane fraction of the cytoplasm of infected cells.     

Evolution of virus and defective-interfering RNAs in BHK cells persistently infected with Sindbis virus.

We analyzed a BHK cell line persistently infected with Sindbis virus for 16 months and a virus (Sin-16) cloned from these cells. Sin-16 virus was resistant to the defective interfering particles present in the original infection. We found that (i) cells infected with Sin-16 were impaired in the processing of a viral precursor glycoprotein, (ii) high-multiplicity passaging of Sin-16 gave rise to a variant that was able to generate and be inhibited by defective-interfering particles to which the original Sin-16 virus was resistant, and (iii) the persistently infected culture contained a heterogeneous mixture of defective Sindbis virus RNAs which were not packaged into extracellular particles. To determine whether these intracellular RNAs could interfere with the replication of Sin-16, we analyzed cells that were cloned from the persistently infected culture. One clone (A3) synthesized a single defective viral RNA which was lost with continued passaging in culture. Infection of A3 cells with Sin-16 showed that the presence of the defective RNA greatly enhanced cell survival and led to enrichment of this RNA. In contrast, cured cells were highly susceptible to killing by Sin-16, and survivors did not synthesize this RNA. Thus, A3 cells were not genetically altered in their response to Sin-16, but were protected from the cytopathic effects of infection by an RNA with the characteristics of a defective-interfering RNA.     

Specific secretion of polypeptides from cells infected with vaccinia virus.

The pattern of polypeptides specifically secreted by cells after infection with vaccinia virus has been analyzed. A complex pattern of apparently virus-specified polypeptides exhibiting temporal control of the type seen with intracellular polypeptides after virus infection was observed. Some of the specifically secreted polypeptides were shown to be modified by glycosylation and sulfation. The possible significance of these results is discussed.     

Temperature-sensitive virus derived from BHK cells persistently infected with HVJ (Sendai virus).

BHK-HVJ cells, a cell line of baby hamster kidney cells persistantly infected with HVJ (Sendai virus), started to produce infectious virus by shifting down the incubation temperature from 38 to 32 C. The virus derived from BHK-HVJ cells, designated as HJV-pB, was effectively neutralized with antibody against wild-type virus (HVJ-W) which was used for the establishment of BHK-HVJ cells. HVJ-pB replicated in eggs at 32 C, but not at 38 C, while HVJ-W grew equally well at both temperatures. When BHK cells infected with HVJ-PB were incubated at 38 C, production of infectious virus, hemagglutinin, and neuraminidase was markedly restrained, whereas a considerable amount of viral nucleocapisid and envelope antigens was detected in the cells by complement fixation tests. These viral activities became detectable immediately after temperature shift-down from 38 to 32 C even at the later stage of infection. HVJ-pB was indistinguishable from HJV-W with respect to particle size, density, and morphological characteristics, but appeared to possess a higher neuraminidase activity and was inactivated more rapidly at 50 C than HVJ-W. HVJ-pB was less cytocidal and could easily cause latent infection in BHK and mouse L cells.     

Studies on the polypeptides of varicella-zoster (V-Z) virus. 1. Detection of varicella-zoster virus polypeptides in infected cells.

Virus-induced polypeptides in cells infected with varicella-zoster virus (VZV) were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. When human embryonic lung (HEL) cells infected with the Oka strain of VZV were labelled with 35S-methionine or 14C-glucosamine from 40 hr to 46 hr after infection, at least 18 VZV-induced polypeptides and 10 glycoproteins could be identified in the infected cells. The molecular weights of the polypeptides and glycoproteins ranged from about 145,000 to 23,000, and from about 105,000 to 48,000, respectively. Lysates of VZV-infected cells were treated with specific antisera prepared in green monkeys or guinea-pigs, and analysed by SDS-PAGE and fluorography. In all, 33 polypeptides (with molecular weight of about 145,000 to 22,000) and 13 glycoproteins (molecular weight, about 105,000 to 38,000) were found in the immunoprecipitates. None of these polypeptides and glycoproteins were detected when infected cells cultured in the presence of phosphonoacetic acid (PAA) were treated in the same way.     

Rapid metabolism of moloney leukemia virus precursor polypeptides in virus infected Swiss mouse embryo cells.

Viral protein synthesis in Moloney murine leukemia virus infected high passage mouse embryo cells was studied utilizing monospecific antisera to the viral core protein p30 and envelope protein gp71. Pulse-chase analysis of [³⁵S]methionine-labeled polypeptides in combination with the demonstration of the presence of either gp71 or p30-specific antigenic determinants in them indicated a 84,000-dalton polypeptide as the precursor of viral glycoproteins and four metabolically unstable polypeptides of approximate molecular weights 88,000, 72,000, 62,000, and 39,000 as the precursors of viral core protein, p30. The p30-containing 88,000 and 72,000-dalton polypeptides were distinctly seen in this system under normal growth conditions. Further, the processing of p30 precursors was very rapid and was complete during a 40 min chase while only partial processing of glycoprotein precursor was observed during the same period.     

Viral proteins and RNAs in BHK cells persistently infected by lymphocytic choriomeningitis virus

Some Syrian hamster cell lines persistently infected with lymphocytic choriomeningitis virus (LCMV) do not produce extracellular virus particles but do contain intracytoplasmic infectious material. The proteins of these cells were labeled with [35S]methionine or with [3H]glucosamine and [3H]mannose, and immunoprecipitates were prepared with anti-LCMV sera. A substantial amount of the LCMV nucleocapsid protein (molecular weight about 58,000) was detected, along with GP-C, the precursor of the virion glycoproteins GP-1 and GP-2. GP-1 and GP-2 themselves were not detected. A new method of transferring proteins electrophoretically from sodium dodecyl sulfate-polyacrylamide gels to diazotized paper in high yield revealed several additional LCMV proteins present specifically in the persistently infected cells, at apparent molecular weights (X10(3] of 112, 107, 103, 89, 71 (probably GP-C), 58 (nucleocapsid protein), 42 to 47 (probably GP-1), and 40 (possibly GP-2). By iodinating intact cells with I3, GP-1 but not GP-2 or GP-C was revealed on the surfaces of the persistently infected cells, whereas both GP-1 and GP-C were found on the surfaces of acutely infected cells. The absence of GP-C from the plasma membrane of the persistently infected cells might be related to defective maturation of the virus in these cells. Cytoplasmic viral nucleoprotein complexes were labeled with [3H]uridine in the presence or absence of actinomycin D, purified partially by sedimentation in D2O-sucrose gradients, and adsorbed to fixed Staphylococus aureus cells in the presence of anti-LCMV immunoglobulin G. Several discrete species of viral RNA were released from the immune complexes with sodium dodecyl sulfate. Some were appreciably smaller than the 31S and 23S species of standard LCMV virions, indicating that defective interfering viral RNAs are probably present in the persistently infected cells. Ribosomal 28S and 18S RNAs, labeled only in the absence of actinomycin D, were coprecipitated with anti-LCMV serum but not with control serum, indicating their association with LCMV nucleoproteins in the cells.     

Subcellular distribution of newly synthesized virus-specific polypeptides in Moloney murine leukemia virus infected cells.

Immune precipitation analysis of pulse-labeled proteins present in subcellular fractions of mouse embryo cells infected with Moloney murine leukemia virus showed the presence of anti-gp70 serum-precipitable viral envelope gene products mainly in the microsomal fractions of these cells. In contrast, anti-p30 serum-specific gag (group specific antigen) gene products were found to be distributed in similar amounts in both the microsomal and postmicrosomal supernatant fractions of pulse-labeled cells.     

Measles virus polypeptides in infected cells studied by immune precipitation and one-dimensional peptide mapping.

Measles virus does not turn off host cell polypeptide synthesis, making it difficult to precisely identify the polypeptides specified by the virus during the infectious cycle. By using the technique of immune precipitation with measles-specific antisera, the host cell background has been eliminated, and new observations have been made concerning measles virus polypeptides H, P, NP, F, and M. The H polypeptide is first synthesized as a monomer which is processed by further glycosylation and by the formation of disulfide-bonded dimers. Polypeptide P (70,000 daltons) has been found to occur also as a 65,000-dalton molecule, P2, and both forms of the molecule are equally phosphorylated. Polypeptide NP is processed from a cleavage-sensitive form (which undergoes cleavage during the process of isolation to form polypeptide 6 [41,000 daltons]) to a form which is resistant to this cleavage. The fusion and hemolysin polypeptide is first found in the cells as a 55,000-dalton precursor, F0, which is clearly resolved from the NP polypeptide on gel electrophoresis. The measles virus F0 protein identified in previous reports had not been resolved from the 60,000-dalton NP polypeptide. The M protein occurs in the infected cells as two distinct bands, and, as in the case of Sendai virus, one of these two M protein bands represents a phosphorylated form of the other.     

腮腺炎病毒的多肽及其在感染细胞中的合成

以差异离心和蔗糖密度梯度离心祛提纯了在鸡胚尿囊腔中繁殖的腮腺炎病毒粒子。并用SDS—PAGE分析病毒粒子的结构多肽,发现其结构多肽为11种,分子量在35K到72K之间。同时还检测到HN蛋白的多聚体和F蛋白的大亚基F1。将腮腺炎病毒分别感染Hela,Vero和CE细胞,比较这三种细胞对ME株腮腺炎病毒的敏感性,发现CE细胞是ME株的敏感宿主。用[31S]蛋氨酸标记病毒感染的CE细胞,以SDS-PAGE及放射自显影法检测到腮腺炎病毒在宿主细胞中合成了至少8种多肽,分子量在26．5K到94K之间。对这些多肽在细胞中不同时期合成情况进行了研究。还用脉冲追踪(pulsechase)技术在感染细胞中发现了FO到F这一转译后加工(Postttanslational procession)现象。此外也研究了放线菌素D和高沈度氯化钠对细胞蛋白质合成的抑制作用。     

Identification of herpesvirus sylvilagus-induced polypeptides in productively infected cells.

Polypeptides synthesized in cell cultures infected with high multiplicities of herpesvirus sylvilagus were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts. Initiation of polypeptide synthesis was detected by 6 h after infection. The maximum intensity of many [35S]methionine-labeled viral bands was observed at 45 h after infection. Production of detectable infectious virus began between 18 and 24 h and reached a plateau at 48 h after infection. Immunoprecipitation of cell extracts identified a minimum of 45 virus-induced polypeptides ranging in molecular weight from 230,000 to 27,000. The major polypeptide appeared to have a molecular weight of 150,000. The pattern of these extracts suggested that the synthesis of host polypeptides is stimulated during the first 12 h and thereafter reduced, but not completely inhibited, during the remaining course of infection.     

Cycl AMP in Gl-arrested BHK21 cells infected with adenovirus type 12

Changes in intracellular levels of cyclic AMP and activity of adenyl cyclase and cyclic AMP phosphodiesterase were studied in G1-arrested BHK21 cells infected with adenovirus type 12 (Ad12). The intracellular concentration of cyclic AMP is reduced at 8 hours after infection (h.p.i.) and reaches a minimum at 14 h.p.i. The decrease in activity of adenyl cyclase is detected at 7.5 h.p.i.; at 9 h.p.i. the activity is about 50% of that in mock-infected cells. No significant changes were observed in the activity of cyclic AMP phosphodiesterase until 13 h.p.i., thereafter the activity of cyclic AMP phosphodiesterase decreased. It is suggested that the observed changes in cyclic AMP metabolism are related to expression of the viral genome, as the first changes are detected only after appearance of Ad12 mRNA in the infected cells.     

Defective interfering particles of Sindbis virus do not interfere with the homologous virus obtained from persistently infected BHK cells but do interfere with Semliki Forest virus.

Defective interfering particles derived from wild-type Sindbis virus no longer interfere with the infectious virus cloned from BHK cells persistently infected with Sindbis virus for 16 months. These particles do interfere with the replication of Semliki Forest virus.     

Identification of acyl donors and acceptor proteins for fatty acid acylation in BHK cells infected with Semliki Forest virus

The modification of viral glycoproteins through the covalent attachment of fatty acids was studied in baby hamster kidney (BHK) cells infected with Semliki Forest virus (SFV). Comparative pulse-chase experiments with [3H]palmitic acid and [35S]methionine revealed that a precursor polypeptide, designated p62, of the structural SFV glycoprotein and E1 serve as the primary acceptors of acyl chains. Acylation of p62 occurs immediately prior to its proteolytical cleavage to E2 and E3 emphasizing the post-translational and specific nature of this hydrophobic modification. To trace the acyl donor(s) for protein acylation the covalent attachment of fatty acids to p62 was studied after extremely short labeling periods with [3H]palmitic acid and correlated to the metabolism of the exogenous tritiated fatty acid. The shortest possible labeling time, a 10 s pulse with [3H]palmitic acid, was sufficient to acylate SFV p62. Analysis of the labeled lipids extracted from the same cells revealed that palmitoyl-CoA and phosphatidic acid showed the highest specific radioactivity among the tritiated lipid species. Out of these lipid species palmitoyl-CoA was identified as the functional acyl donor lipid in a cell-free system for the acylation of polypeptides.     

A fluorescence photobleaching study of vesicular stomatitis virus infected BHK cells. Modulation of G protein mobility by M protein

The mobility of vesicular stomatitis virus (VSV) G protein on the surface of infected BHK cells was studied by using the technique of fluorescence photobleaching recovery. The fraction of surface G protein that was mobile in that time scale of the measurement (minutes) was at least 75%, a relatively high value among cell surface proteins so far observed. For studies of the effect of an internal viral protein (M protein) on G protein mobility, cells infected with wild-type VSV were compared with those infected with temperature-sensitive VSV mutants of complementation group III, which contains lesions in the M protein. At the permissive temperature, a pronounced decrease in the mobile fraction of surface G was observed for each of three mutants studied, while mobility of surface G at the nonpermissive temperature was indistinguishable in mutant and wild-type infected cells. A significantly lower mobile fraction of G protein was also observed in SV40 transformed 3T3 cells infected with wild-type VSV, but not in 3T3 or chick embryo fibroblast cells similarly infected. None of the variables tested had a measurable effect on the lateral diffusion coefficient of the mobile G protein. These results are interpreted as modulation of the mobility of a specific cell surface protein by a specific intracellular protein.