Temperature-sensitive herpes simplex virus type 1 mutants defective in the shutoff of cellular DNA synthesis and host polypeptide synthesis

Two temperature-sensitive herpes simplex virus type 1 mutants, ts 1-8 and ts 199, belonging to different complementation groups, were isolated. Both mutants were defective in the shutoff of host DNA synthesis at 39.5 degrees C (nonpermissive temperature). ts 1-8 exhibited intermediate levels of viral DNA synthesis at 39.5 degrees C, while ts 199 was completely deficient in viral DNA synthesis at 39.5 degrees C. Comparative polyacrylamide gel electrophoresis of the ts 1-8, ts 199 and wild-type viral-coded polypeptides and cellular proteins produced in vivo at 34 degrees C and 39.5 degrees C during various periods post infection was performed. The results indicated that ts 1-8 and ts 199 were temperature-sensitive for the secondary suppression of host polypeptide synthesis. Production of the beta (early) and gamma (late) viral polypeptides was slightly delayed in the mutant-infected cells at early times post infection at both 34 degrees C and 39.5 degrees C. This delayed protein production was not evident at later times post-infection. The ts 1-8 and ts 199 mutants were distinct from the HSV-1 viron-associated host shutoff (vhs) mutants of Read and Frenkel (J. Virol. 46 (1983) 498).     

Identification of the point mutations in two vaccinia virus nucleoside triphosphate phosphohydrolase I temperature-sensitive mutants and role of this DNA-dependent ATPase enzyme in virus gene expression

The biological function of the nucleoside triphosphate phosphohydrolase I (NTPase I) enzyme of vaccinia virus is not yet known. In this investigation we have identified the genetic lesion of two temperature-sensitive mutants of vaccinia virus, ts50 and ts36, as single point mutations contained within the 5'615 nucleotides of the NTPase I gene (ts50, G to A at position 131; ts36, C to T at position 556). The point mutations result in amino acid substitutions of Gly to Glu-44 (ts50) and Pro to Ser-186 (ts36). In monkey BSC-40 cells, ts50 and ts36 behave phenotypically like wild-type virus with respect to replication and synthesis of viral DNA but are defective in late polypeptide synthesis. However, these two ts mutants displayed a drastically different phenotype in virus-infected human HeLa cells at the restrictive temperature; viral DNA replication did not occur and late polypeptide synthesis was absent. Moreover, if the early block was overcome by a temperature shift-up, then HeLa cells infected with the ts mutants displayed a profile characteristic of defective late viral polypeptide synthesis. Our results reveal that vaccinia NTPase I enzyme functions early and late in the viral replication cycle and that the phenotype of these ts mutants is dependent upon the cell type.     

Some characteristics of an early protein (ICP 22) synthesized in cells infected with herpes simplex virus

In Vero cells incubated at 40 degrees C or treated with azetidine at 37 degrees C, synthesis of a polypeptide ('C') of apparent mol. wt. 66000 was stimulated. It was not phosphorylated and was found in the cytoplasmic fraction of cell lysates. In cells infected with herpes simplex virus type 1 (HSV-1) in the presence of azetidine, synthesis of cellular proteins, including polypeptide C, was suppressed and infected cell polypeptides ICP 4, 0, 22 and 27 (apparent mol. wt. 170000, 120000, 75000 and 60000, respectively) were made. All were phosphorylated and accumulated in the nucleus. Messenger RNA for the same four polypeptides was made in cells infected in the presence of cycloheximide. Thus, ICP 22 is distinct from cellular polypeptide C and is probably a virus-specific alpha polypeptide, although it differs from alpha ICP 4, 0 and 27 in that its rate of synthesis does not decline rapidly when later polypeptides are produced. It is modified after synthesis in at least two steps, the second of which may require a later virus-specific polypeptide. In cells infected with HSV-2 the synthesis of a polypeptide analogous to ICP 22 could not be detected.     

Regulation of herpesvirus macromolecular synthesis. V. Properties of alpha polypeptides made in HSV-1 and HSV-2 infected cells.

Previous reports from this laboratory have defined as α those viral polypeptides whose production in infected cells does not require prior protein synthesis. Two subsequent groups, β and γ, depend on prior a and β polypeptide synthesis, respectively. Comparison of the synthesis and properties of a polypeptides specified by herpes simplex viruses (HSV) 1 and 2 showed the following: (i) The three earliest virus-specific infected cell polypeptides (ICP) made in HSV-2 infected cells migrated slightly more slowly in polyacrylamide gels than the HSV-1 α polypeptides, ICP 4.0 and 27. (ii) Cells treated with canavanine from the time of infection with HSV-2 produced all α, a subset of β, and a small amount of one γ polypeptide; the synthesis of these polypeptides continued for many hours, at rates related to the multiplicity of infection. The transition from a to this subset of β polypeptide synthesis did not appear to be affected by the arginine analogue, whereas transition to the other sets of β and to γ polypeptide synthesis was blocked. A similar discrimination between different subsets of β proteins was seen in treated HSV-1 infected cells. (iii) All a and a number of β polypeptides observed in this study underwent translocation into the nucleus, posttranslational modification resulting in a reduced electrophoretic mobility, and phosphorylation. For example, the modification of HSV-1 and HSV-2 ICP 4 was in at least two steps from the translational product ICP 4a, labeled during a pulse, to slower-migrating forms ICP 4b and 4c. All three forms were phosphorylated but only 4b and 4c were found in the nucleus. In untreated infected cells, ICP 4 ultimately accumulated in form ICP 4c. ICP 4a made in the presence of canavanine was not processed efficiently into ICP 4c. In another instance, the polypeptide made in the presence of canavanine were not translocated into nuclei.     

Synthesis of virus-specific polypaptides by temperature-sensitive mutants of herpes simplex virus type 1.

The polypeptide phenotypes of 22 temperature-sensitive (ts) mutants of herpes simplex virus type 1 were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis of mutant-infected cells at permissive and nonpermissive temperatures. Following analysis of isotopically labeled polypeptides synthesized from 4–24 hr postinfection, the mutants were divided into four major phenotypic groups which include: (1) DNA^?ts mutants which share several common polypeptide defects, (2) DNA^±ts mutants which exhibit polypeptide profiles resembling the DNA^?ts mutants, (3) DNA⁺ts mutants which exhibit polypeptide phenotypes differing only slightly from that observed in wild-type virus-infected cells grown at 39°, and (4) DNA⁺ts mutants which exhibit no detectable alterations in their polypeptide profiles when compared with that of the wildtype virus. When the polypeptide phenotypes of the mutants were compared with previously determined mutant characteristics, including synthesis of viral DNA, thymidine kinase, DNA polymerase, and physical virus particles, a correlation was consistently observed between mutant polypeptide and viral DNA phenotypes.     

Studies on the mechanisms of vaccinia virus cytopathic effects. II. Early cell rounding is associated with virus polypeptide synthesis

Vaccinia virus-induced morphological lesions were studied in LLC-MK2, HeLa and L cells. In LLC-MK2 cells, cell rounding occurs within 30 to 60 min after infection with 300, 900 or 2700 particles/cell and the time of appearance of these changes is dependent on the multiplicity of infection (m.o.i.). When cycloheximide (300 microgram/ml) is added to cultures at the time of infection, early cell rounding is prevented regardless of the m.o.i. However, cell rounding does occur when cycloheximide is removed, and its time of appearance and extent depends upon the time of removal of the compound and the m.o.i. Upon removal of cycloheximide at I or 2 h after infection early cell rounding occurs, and virus polypeptide synthesis is evident in cells infected at all three multiplicities. However, when the drug is removed at 4 hr after infection, cell rounding and virus polypeptide synthesis occur only in cultures infected at 300 particles/cell. Early morphological changes are also prevented in HeLa and L cells infected at 300 particles/cell in the presence of cycloheximide. These changes occur only if the compound is removed up to 2 h after infection in HeLa cells and up to 40 min after infection in L cells. Early morphological lesions are not seen if the compound is removed at later times. The occurrence of early morphological changes in HeLa and L cells is also correlated with the synthesis of virus polypeptides. All cell types, when infected at 2700 particles/cell in the presence of cycloheximide, or inhibitors of RNA synthesis, display cell fusion. Thus, whereas early morphological changes require virus protein synthesis to become manifest, cell fusion occurs in the absence of virus RNA or protein synthesis and may be mediated by a component of the virion.     

Characterization of the polypeptides synthesized in cells infected with a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Summary The intracellular synthesis of virus-specific polypeptides in cells infected with the wild-type virus of HVJ (HVJ-W) (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) and with a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture has been analysed by polyacrylamide gel electrophoresis. At the permissive temperature (32° C), all of the known virus structural polypeptides were identified in cells infected with each strain of virus and in addition to the non-structural polypeptides B and C, another polypeptide at the region with a molecular weight of 26,000 to 27,000 (26 to 27K) could be detected in infected cells. At the non-permissive temperature (38° C), the synthesis of the polypeptide M was markedly restrained in cells infected with HVJ-pB, while other major virus polypeptides were present in approximately comparable amounts to cells infected with the wild-type virus. A non-structural polypeptide with a molecular weight of 105K was dominant ints mutant infected cells at higher temperatures and disappeared after temperature-shift from 38° to 32° C. The production of the non-structural polypeptides B and 27K was also temperature-sensitive. The molecular weights of the polypeptides B, M and 27K in HVJ-pB infected cells were larger than those of the corresponding polypeptides in HVJ-W infected cells. The synthesis of the M protein in HVJ-pB infected cells started just after lowering the incubation temperature and the newly made M protein was successfully incorporated into virus particles.With 4 Figures     

Role of gene 8 product in morphogenesis of bacteriophage T3

The product of gene 8 (gp8) of T3 phage is one of the minor head proteins located at the phage head-tail junction. To determine the role of gp8, an amber (8-) and four temperature-sensitive mutants (ts8) were characterized by sedimentation analysis, polyacrylamide gel electrophoresis, and extract complementation. Neither DNA-containing particles nor empty particles were formed in cells infected with 8-. In addition, prohead assembly was greatly reduced. Prohead assembly was also blocked in cells infected with all ts8 mutants at 42 degrees and with some ts8 even at 37 degrees. Proheads containing gpts8 were converted to empty heads when cell lysates were treated with chloroform. The protein compositions of proheads showed that the minor head proteins, gp8, gp15, and gp16, were lost from proheads formed in cells infected with ts8, but these minor proteins were present in proheads formed in cells infected with double mutants of ts8 and 5- or 19-, which are defective in DNA synthesis or DNA maturation, respectively. In vitro complementation experiments suggested that a ts mutation in gene 8 affected not only DNA packaging but also subsequent assembly steps. From these results, it is concluded that gp8 plays multiple roles in T3 phage morphogenesis, including prohead assembly, prohead stabilization, DNA packaging, and subsequent events.     

Induction of cellular DNA synthesis by adenovirus type 12 in a set of temperature-sensitive mutants of rat 3Y1 fibroblasts blocked in G1 phase

Four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, which represent separate complementation groups, cease to proliferate predominantly with a 2C DNA content, either at 39.8 degrees (temperature arrest), or at 33.8 degrees at a confluent cell density (density arrest). When infected at 39.8 degrees with adenovirus type 12 (Ad12), cells of all four ts mutants in both arrest states entered the S phase, thereby suggesting that Ad12 overcomes the four independent functional blocks to cellular entry into S phase. Results of experiments using Ad12 E1-region mutants suggest that the E1A gene product(s) is indispensable to overcoming the ts block, whereas the E1B product(s) may be dispensable. The cell killing observed in 3Y1 cells infected with wild-type Ad12 did not occur in infection with one of the E1-region mutants with a 6-bp insertion in the E1A 13 S mRNA unique region. When infected with this mutant at 39.8 degrees, two ts mutants of 3Y1 (3Y1tsF121 and 3Y1tsG125) in both arrested states proliferated through at least one generation. Another mutant (3Y1tsD123) was accelerated to die following entry into the S phase. In the other mutant (3Y1tsH203), the cell number was either unchanged (temperature arrest) or was increased less than twofold and then decreased (density arrest). The findings with the latter two mutant lines suggest that induction of cellular DNA synthesis is not sufficient for the subsequent proliferation of the infected cells, and that the Ad12 gene function(s) does not directly rescue the primary lesions in these ts mutants but does overcome some of the blocks to concomitantly occurring events. In the former two mutant lines, however, Ad12 gene function(s) may directly rescue the ts lesions. We propose that the Ad12 gene product(s) can overcome blocks to the initiation of cellular DNA synthesis but cannot overcome blocks to events related to cell survival.     

Temperature-sensitive defect of influenza A/Ann Arbor/6/60 cold-adapted variant leads to a blockage of matrix polypeptide incorporation into the plasma membrane of the infected cells

A temperature-sensitive (ts) defect in growth of the A/Ann Arbor/6/60 (A/AA/60) cold-adapted (ca) and ts variant strain has been studied. At the restrictive temperature of 38.5 degrees C, the variant synthesized all the viral polypeptides in normal amounts within the infected cells, but the virions released into the culture fluid contained greatly reduced amounts of the matrix (M1) polypeptide and showed significantly low infectivity per unit hemagglutinin activity. Cell fractionation experiments revealed that incorporation of the M1 polypeptide into plasma membranes of the variant-infected cells was selectively reduced at 38.5 degrees C, whilst it occurred normally at 34 degrees C. The ts reassortants between the A/AA/60 variant and the A/AA/1/80 wild type (wt) strain (non-ts), which had the M gene derived from the wt parent, also showed similar patterns. These results suggest that the ts defect of the variant and its ts reassortants involves the process of incorporation of the M1 polypeptide into the plasma membranes of the infected cells and that this defect is not attributable to the M gene of the variant.     

Establishment of persistent infection in BHK-21 cells by temperature-sensitive mutants of Sindbis virus

Twelve temperature-sensitive (ts) mutants of Sindbis were examined for their ability to establish persistent infection in BHK-21 cells at 39 degrees C. Five of these mutants were able to initiate colony formation in infected cultures, which followed an extensive c.p.e. Two of the mutants were able to establish persistent infections which survived beyond the fifth cell passage p.i. The ability to initiate colony formation was correlated with low reversion of the ts mutation, or with ability to interfer with the multiplication of the wild-type virus. Virus released from persistently infected cultures was not temperature-sensitive. The restriction of virus multiplication in persistently infected cells operated prior to virus-specified RNA synthesis. It is concluded that in this system establishment of persistent infection depends on an inhibition of virus multiplication early in infection and occurs in only a small proportion of infected cells.     

Polypeptides synthesized in rabbit cells infected with murine herpesvirus (MHV): a comparison of proteins specified by various MHV strains.

Polypeptides synthesized in rabbit embryo fibroblasts (REF) infected with six isolates of murine herpesvirus (MHV) coming from different localities in CSFR were compared by electrophoresis (SDS-PAGE). Altogether 28 virus-coded polypeptides with apparent molecular weights ranging from 240 kD to 16 kD were identified. While isolates MHV-60, MHV-72, and MHV-76 had identical polypeptide profiles, the isolates MHV-68 and MHV-78 lacked a polypeptide with apparent molecular weight of 46 kD, the rest of polypeptides being identical as well. The polypeptide profile of MHV Sumava A.f. was most different from that of other MHV isolates. This result is in agreement with some different biological properties of this isolate. At a high multiplicity of infection (40 TCID50 per cell) first polypeptides with apparent molecular weights 125 kD and 84 kD were detected by 5-9 hr post-infection (p.i.). All other proteins appeared 14-18 p.i.     

Genetic analysis of subunit assembly of the tail fiber of bacteriophage T3

Bacteriophage T3 virions have six tail fibers composed of the product of gene 17 (gp17). Each tail fiber is a trimer of gp17 polypeptide. To characterize the assembly process of the tail fiber, temperature-sensitive (ts) mutants of gene 17 (ts17) were analyzed by SDS-polyacrylamide gel electrophoresis and by extract complementation. Newly synthesized gp17 polypeptide chains matured to SDS-resistant native trimers with a half time of about 7.5 min at 30 degrees. Although all ts17 mutants had similar plating efficiencies at restrictive temperature (41.5 degrees or 42 degrees), they showed different phenotypes. tsNG75, whose mutation was located in the carboxyl-terminal region of gene 17, was defective in trimer assembly at 41.5 degrees. The ts tail fibers formed at 30 degrees lost the ability to attach to the tail upon treatment at 41.5 degrees. There was a change in temperature sensitivity of tsNG75 tail fibers upon attachment to the tail, suggesting that the tail fiber may change conformation after attachment to the tail. tsNG215 and tsNG169, whose mutation sites were located in the amino-terminal region of gene 17, were not defective in the trimer assembly and attachment to the tail at the restrictive temperature. tsNG215 tail fibers formed at 41.5 degrees appear to be aberrant because they were not active in extract complementation and their attachment to fiberless particles resulted in production of noninfectious phage. Tail fibers produced by cells infected with tsNG169 at the restrictive temperature were active in extract complementation. Phage particles were formed in tsNG169-infected cells at the restrictive temperature. These particles were infectious at the permissive temperature and the mutant was non-infectious only if infection was continued at the restrictive temperature. These phenotypic differences exhibited by different gene 17 mutants may indicate the regions within the gene 17 polypeptide that play a role(s) in the folding and assembly of gp17 and in the biological activity of the mature tail fiber.     

Functional analysis of the A complementation group mutants of Sindbis HR virus

The 10 members of the A complementation group of temperature-sensitive (ts) mutants of SIN HR, the heat-resistant strain of Sindbis virus, were divided into two phenotypic subgroups. Subgroup I mutants (ts15, ts17, ts21, ts24, and ts133) demonstrated temperature-sensitive 26 S mRNA synthesis, whereas subgroup II mutants (ts4, ts14, ts16, ts19, and ts138) did not; both ts4 and ts19 demonstrated defective 26 S mRNA synthesis at 30 degrees. None of the A group mutants demonstrated temperature-sensitive 49 S plus-strand synthesis. Temperature-sensitive cleavage of ns230 was demonstrated by subgroup I mutants, except ts21, but not by subgroup II mutants. A revertant of ts133 that grew at 40 degrees retained temperature-sensitive 26 S mRNA synthesis but lost temperature-sensitive cleavage of ns230 and the RNA-negative phenotype. Only ts4, like ts11 of the B complementation group, demonstrated temperature-sensitive minus-strand RNA synthesis. In addition to ts24, cells infected with ts17 or ts133 continued to synthesize minus strands after shiftup in the absence of continued protein synthesis, and resumed synthesis of minus strands if shifted to the nonpermissive temperature after minus-strand synthesis had ceased at the permissive temperature.     

Polypeptide synthesis in alphavirus-infectedAedes albopictus cells during the establishment of persistent infection

Summary Polypeptide synthesis was examined in mosquito cells during the establishment of a persistent infection with two alphaviruses, Ross River virus (RRV) and Semliki Forest virus (SFV), and in vertebrate cells cytopathically-infected with the same viruses.InAedes albopictus cells, RRV reached peak titres at 34–48 hours p.i. At 12 hours 85 per cent of cells assayed as infected by infective centre assay; by 48 hours when persistence was established, virus production was reduced and <5 per cent of cells assayed as infected. There was no shut-down of host polypeptide synthesis during infection. Viral polypeptide synthesis was maximal between 10 and 24 hours p.i. The major viral polypeptides labelled were nucleocapsid protein and envelope protein(s). The precursor polypeptide p95 which was prominent in infected BHK cells was not detected in mosquito cells. Similar results were obtained on SFV infection.During the establishment of persistence there was a coordinate decline in the synthesis of RRV polypeptides, reaching undetectable levels by 72 hours p.i. Subculturing persistently-infected cells led to a small increase in viral polypeptide synthesis and virus titre.In contrast, during RRV growth in BHK cells host protein synthesis was severely inhibited and by 9–11 hours p.i. virus-specific polypeptide synthesis represented more than 90 per cent of total protein synthetic activity.With 6 Figures     

Temperature-sensitive mutants of reovirus : V. Studies on the nature of the temperature-sensitive lesion of the group C mutant ts447

Studies were carried out to determine which of the polypeptides coded by the group C reovirus temperature-sensitive mutant ts447, a double-stranded RNA-negative mutant, is mutated. Evidence was obtained from two lines of experimentation:

1. (i) In cells infected with this mutant at nonpermissive temperatures the structures within which viral double-stranded RNA is normally synthesized (DSRSS) are not formed; further, when shifted from permissive to nonpermissive temperatures, the rate of DS RNA synthesis decays at approximately the same rate at which plus-stranded template is used up during double-stranded RNA synthesis in vitro. ts447 DSRSS that are formed at permissive temperatures can synthesize double-stranded RNA at nonpermissive temperatures in vitro, but they are more heat-labile than wt DSRSS. These results suggest that the ts447 polypeptide that is mutated is a structural component of DSRSS.

2. (ii) In contrast to wt reovirus, ts447 forms two types of empty, or top component, particles: normal ones that are indistinguishable from those of wt virus, and aberrant ones which possess a slightly lower buoyant density and a capsid that (1) is only about one-half as thick as that of wt top component particles, (2) appears to consist of only one rather than two shells, and (3) lacks the two innermost core polypeptides λ1 and σ2. Independent evidence suggests that core polypeptides are among the structural components of DSRSS; the two lines of evidence therefore converge to suggest that the mutation of ts447 is either in polypeptide λ1 or in polypeptide σ2.

     

Patterns of polypeptide synthesis in human rotavirus infected cells

Summary Polypeptide analysis of three strains of human rotavirus (KUN, Wa and MO) were conducted using a hypertonic culture which suppressed host protein synthesis and unmasked rotavirus specific protein synthesis. As a result, eleven human rotavirus specific polypeptides (Vp 1–Vp 11) were detected by pulselabeling infected cells with [¹⁴C]-leucine. Among the 11 polypeptides, three polypeptides (Vp 7, Vp 10 and Vp 11) underwent post-translational processing, and two (Vp 7 and Vp 10) were glycosylated. Six polypeptides (Vp 1, 2, 3, 4, 6 and 7) were identified as viral structural proteins. Comparisons of three strains of different serotypes revealed that their polypeptide profiles differed from each other in electrophoretic mobility; in particular, profiles of the glycosylated polypeptide, Vp 7, were distinct among the three strains.With 6 Figures     

Intracellular RNA synthesis directed by temperature-sensitive mutants of simian rotavirus SA11

The kinetics of intracellular synthesis of single-stranded (ss) RNA and double-stranded (ds) RNA directed by prototype temperature-sensitive (ts) mutants representing the 10 mutant groups of rotavirus SA11 were examined. Cells were infected with individual mutants or wild type under one-step growth conditions and maintained at permissive temperature (31 degrees) or nonpermissive temperature (39 degrees). At various times postinfection, infected cells were pulse-labeled, ssRNA and dsRNA were purified, RNA species were resolved by electrophoresis and autoradiography, and RNA synthesis was quantitated by computer-assisted densitometry. The mutants representing all groups synthesized significantly less ssRNA and dsRNA at both 31 degrees and 39 degrees, when compared to wild type. When the ratio of synthesis at 39 degrees/31 degrees was determined for ssRNA and dsRNA of each mutant, three RNA synthesis phenotypes were evident. The tsB(339), tsC(606), and tsE(1400) mutants synthesized both ssRNA and dsRNA in a temperature-dependent manner. The group G mutant, tsG(2130), synthesized ssRNA in temperature-independent fashion but was temperature-dependent for the synthesis of dsRNA. The remaining mutants, tsA(778), tsD(975), tsF(2124), tsH(2384), tsI(2403), and tsJ(2131), synthesized both ssRNA and dsRNA in a temperature-independent fashion. The RNA synthesis phenotypes of the ts mutants are discussed in terms of what is known of the function(s) of the protein species to which ts lesions have been assigned.