Activation of herpes simplex virus (HSV) type 1 genome by temperature-sensitive mutants of HSV type 2

Previous studies have demonstrated that herpes simplex viruses (HSV) type 1 (HSV-1) and type 2 (HSV-2) can be maintained in a repressed form in human embryo lung cells. Reducing the incubation temperature or superinfecting with a heterologous herpesvirus, human cytomegalovirus (HCMV), results in activation of virus replication. We now report that superinfection with a partially homologous herpesvirus, HSV-2, also resulted in activation of HSV-1. To minimize excessive synthesis of infectious HSV-2 while allowing virus gene expression, repressed HSV-l-infected cultures were superinfected with HSV-2 temperature-sensitive (ts) mutants (tsF3, tsB5, or tsH9). The predominant virus replicated after HSV-2 ts mutant superinfection at a nonpermissive temperature was identified as activated parental-like HSV-1 by (i) plaquing efficiency at permissive (34 degrees) and nonpermissive (40.5 degrees) temperatures, (ii) sensitivity to inhibition by the HSV-l-specific antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine, and (iii) restriction endonuclease cleavage analysis. In addition, the fact that superinfection with HSV-2 tsB5 or tsH9, which are unable to synthesize virus DNA and express only early virus genes at nonpermissive temperature, resulted in synthesis of virus demonstrated that HSV-2 DNA synthesis is not required for activation. This system has provided the basis for further studies concerning the regulation of HSV gene expression in human cells.     

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.     

Viral interference phenomena induced by foot-and-mouth disease temperature-sensitive mutants in bovine kidney cells

Summary Cultures of bovine kidney (BK) cells infected with temperature-sensitive(ts) mutants of foot-and-mouth disease virus (FMDV) were incubated at 38.5°C, a temperature nonpermissive for mutant virus growth and RNA synthesis. The cells were subsequently resistant to viral growth and RNA synthesis when super-infected with wild-type FMDV and with heterologous fowl plague virus. The extent of interference was proportional to the multiplicity of infection of thets mutant. It increased with time elapsed between infection with mutant and challenge infection, becoming greater than 99 percent after 24 hours. Interference was not proportional to decreased levels of cellular protein synthesis. The interference could be produced in the presence of actinomycin D, and thus was apparently mostly caused by thets mutant itself rather than by interferon. The interference could not be produced in other less susceptible cell lines. Supernatant fluids from the BK cells infected withts mutant virus interfered with wild-type FMD viral growth and RNA synthesis in fresh BK cells, and also showed low levels of activity in a vesicular stomatitis virus-plaque reduction assay. The properties of the supernatant fluid-interfering agent resembled to some extent those of an interferon. Thets mutant-mediated interference factor was apparently not able to diffuse into the supernatant fluid.Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Activation of cellular stress protein genes by herpes simplex virus temperature-sensitive mutants which overproduce immediate early polypeptides

This paper describes the activation of cellular genes after infection of chick embryo fibroblasts (CEF) with temperature-sensitive (ts) mutants of herpes simplex virus type 1. One mutant, tsK, has a mutation in the gene sequences which encode the immediate-early (IE) polypeptide Vmw175. After infection of CEF with tsK at the nonpermissive temperature (38.5°), IE polypeptides are overproduced but early and late viral gene products cannot be detected. In addition, the synthesis of three cellular polypeptides is stimulated in a manner which closely resembles the “stress” or “heat-shock” response. Peptide mapping confirmed that the tsK-induced polypeptides are stress proteins. No induction was observed when viral gene expression was prevented, leading to the conclusion that one or more IE polypeptides is responsible for the stress response. Two other mutants, tsD and tsT, which have mutations in different regions of the Vmw175 coding sequences, also induce stress proteins at 38.5°.     

Alfalfa mosaic virus temperature-sensitive mutants. I. Mutants defective in viral RNA and protein synthesis

The replication in cowpea protoplasts of temperature-sensitive (ts) mutants of alfalfa mosaic virus (AIMV) was studied at the permissive (25 degrees) and the restrictive (30 degrees) temperature. Using the Northern blot hybridization technique, it was shown that at the restrictive temperature two RNA 1 mutants, Bts 03 and Bts 04, and two RNA 2 mutants, Mts 03 and Mts 04, were all defective in the synthesis of viral minus-strand RNA, whereas the synthesis of the plus-strand genomic RNAs 1, 2, and 3 and the subgenomic coat protein messenger, RNA 4, was relatively unimpaired. In Bts 04 inoculated protoplasts the RNA 4 produced at 30 degrees was translated into coat protein and viral RNA was encapsidated to give infectious virus. RNA 4 in Bts 03 and Mts 04 infected protoplasts was not translated into coat protein at 30 and consequently there was no assembly of infectious virus. Protein synthesis by Mts 03 was not investigated. A1MV RNAs 1 and 2 encoded proteins are both involved in the synthesis of viral minus-strand RNA and the translation of RNA 4 and possibly other viral messengers. The results with Bts 03 and Bts 04 show that the two functions of the RNA 1 encoded protein can be mutated separately.     

Characterisation of temperature-sensitive mutants of herpes simplex virus type 2. Growth and DNA synthesis

The growth of 10 temperature-sensitive (ts) mutants of type 2 herpes simplex virus has been studied at permissive (31 °) and restrictive (38 °) temperatures. At 31 °, maximum virus yields were attained by all mutants 18–24 hours after infection—some 8–12 hr later than for wild-type virus. At 38 °, the mutants failed to replicate, although they all adsorbed to cells as efficiently as at 31 °. Five mutants were slightly, but not markedly, more thermolabile than wild-type virus. Three mutants synthesised viral DNA at 38 °, the others did not. One DNA-negative mutant, ts9, did not shut off host cell DNA or histone synthesis at 38 °.     

Study of fowl plague virus RNA synthesis in temperature-sensitive mutants.

A temperature-sensitive clear mutant of the temperate cyanophage SPI was isolated. This mutant, SPIcts1, enters a stable lysogenic state following infection of its blue-green algal host Plectonema boryanum. Induction takes place upon transfer of the culture to 40°.Prophage induction requires light energy during its early stage. Induction is prevented by the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) but can take place in the absence of CO₂. Infection by SPIcts1, on the other hand, can proceed in the dark. Shortly after infection there is extensive leakage of macromolecules from the host cell accompanied by cessation of CO₂ fixation. In contrast, the induced prophage does not cause early cellular leakage or early shutoff of CO₂ fixation of the host.     

Studies on temperature-sensitive mutants of Chinese hamster ovary cells affected in DNA synthesis

DNA synthesis in two mutants of Chinese hamster overy cells,ts 13A andts 15C, which were temperature sensitive for growth, was found to be shut off rapidly at the nonpermissive temperature. The mutants did not complement each other and thets lesion was not located on the X chromosome. Both isolates were found to be considerably more sensitive to the alkylating agents, ethylmethanesulfonate (EMS) and methylmethanesulfonate (MMS), as compared to the parental cells, but showed normal sensitivity to UV irradiation. The mutants also showed interesting differences in their response to EMS-induced mutation frequencies at the ouabain-resistant and thioguanine-resistant loci. At high survival (50%) the frequencies of mutations at these genetic loci were markedly low in thets mutants as compared to the parental cells. Ints ^* revertants isolated from the mutants, thets phenotype and the increased sensitivity to EMS and MMS were affected simultaneously, indicating that both these characteristics resulted from a single genetic lesion.     

Multiple transformation phenotypes among revertants of temperature-sensitive mutants in the type 5 adenovirus DNA-binding protein

Seven independently isolated revertants of temperature-sensitive mutants in the 72K protein of Ad5 were tested for the ability to transform rat cells under a variety of conditions. Using the continuous cell line designated CREF, at 36° the range of transformation phenotypes of the different revertants included a frequency characteristic of WT and also the elevated frequency associated with the parental temperature-sensitive alleles. Transformation frequency did not correlate with other phenotypes, such as plaque size or plaquing efficiency on HeLa cells at 38.5°–39°. Therefore, although it is likely that the 72K protein modulates transformation, it is possible to dissociate genetically this regulatory function of the protein from its replicative function in permissive infection.     

Phenotypic heterogeneity among temperature-sensitive mutants of rous sarcoma virus. Studies with inhibitors of protein synthesis

Fourteen temperature-sensitive (ts), transformation-defective mutants have been isolated from mutagenized Schmidt-Ruppin Rous sarcoma virus. We report that, while in cells infected with most of the mutants all parameters of cell transformation were coordinately suppressed, certain ts mutants induced the ability of infected cells to multiply in soft agarose and to express the tumor-specific surface antigen (TSSA), in the absence of morphological conversion. Studies with inhibitors of protein synthesis have shown that cycloheximide (Ch) and pactamycin (Pac) act differently on focus formation from Pu and that the heat-labile src gene product of these mutants may be spontaneously reactivated following a downshift to permissive temperature. Our data also indicate the existence of two classes of mutants regarding the response of infected cells to Pu. In most cases, focus formation is inhibited by Pu when infected cells are shifted to 37°. However, cells infected with three ts mutants resume morphological transformation at permissive temperature in the presence of this inhibitor. In addition, the fact that the same mutants induce high levels of TSSA expression at restrictive temperatures suggests that the two properties may be linked.     

The polypeptides induced in Drosophila cells by Drosophila C virus (strain Ouarzazate)

The Ouarzazate strain of Drosophila virus (DCV0) was grown in Drosophila melanogaster tissue culture cells, and [35S]methionine-labeled virions were found to contain a group of major structural proteins with a molecular weight of approximately 30,000 as well as several minor proteins of higher molecular weight and a protein of approximately 10,000 daltons. Using a range of pulses, chases and gel systems, examination of the intracellular proteins induced by DCV0 showed the presence of 17 polypeptides not found in uninfected cells. The synthesis of virus-induced polypeptides was extremely asymmetric with a rapid appearance of the major virus structural proteins and a much slower appearance of the lowest molecular weight structural protein (VP4). Processing of virus-induced proteins including the appearance of VP4 was demonstrated using pulse-chase after pulsing with [35S]methionine. While the highest molecular weight induced protein found in infected cells was 146,000, pretreatment of cells with iodoacetamide resulted in the appearance of a protein with a molecular weight of approximately 200,000. The evidence presented in this paper supports the inclusion of DCV0 in the Picornaviridae group.     

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.     

Differential 2-D protein gel electrophoresis analysis of Legionella pneumophila wild type and Tat secretion mutants

The twin-arginine translocation (Tat) pathway is a secretory pathway for translocation of folded proteins with two arginines in their signal peptide across the cytoplasmic membrane. Recently, we showed the presence of the Tat secretion pathway in Legionella pneumophila Philadelphia-1 and its role in intracellular replication and biofilm formation. To analyse the importance of the Tat pathway in protein export and its role in L. pneumophila virulence, a comparative 2-D protein gel electrophoresis analysis was performed on supernatants of the wild type and two Tat secretion mutants in order to identify possible Tat substrates. Twenty proteins were identified as differential proteins, eight of which were present in a lower quantity in the supernatant of the tat mutants. Among these, one protein with a typical twin-arginine motif in its signal peptide was identified as the 3′,5′-cyclic nucleotide phosphodiesterase. Two other proteins that resulted as differential proteins from this study were flagellin and LvrE, which were studied in more detail and their Tat-dependence was further confirmed with specific antibodies. LvrE was shown to play a role in intracellular growth in differentiated U937 cells.     

RNA synthesis by temperature-sensitive mutants of vesicular stomatitis virus, New Jersey serotype.

We report the results of studies using temperature-sensitive mutants of Rous sarcoma virus (ts RSV) to study the alterations in surface proteins occurring on transformation. A large external transformation-sensitive (LETS) protein is detected by lactoperoxidase-catalyzed iodination on normal chicken embryo fibroblasts (CEF) but is reduced on RSV-transformed cells. The LETS protein is temperature sensitive in ts RSV-infected CEF and the kinetics of the alterations occurring on shifts between permissive and restrictive temperature are reported. During reversion to normality on shift-up, the LETS protein appears at the surface quite rapidly and in parallel with the morphological changes, although the change is less rapid than that in glucose transport. On transformation during shift-down, the disappearance of the LETS protein lags behind the morphological change. Cycloheximide does not inhibit reappearance of iodinatable LETS protein on shift-up, suggesting that the LETS protein is synthesized in transformed cells, although it is not present at the surface. Hypotheses to explain the absence of the LETS protein at the surface are discussed and evidence is presented for an increased rate of turnover after transformation.     

The fine structure of cells infected with temperature-sensitive mutants of herpes simplex virus type 2.

The fine structure of cells infected with the HG 52 strain of herpes simplex virus type 2 and 13 temperature-sensitive mutants derived from it was investigated. In cells infected with the wild-type virus, development of virions appeared to be similar to that described in previous reports. However there were two exceptions to this: (1) capsid envelopment apparently occurred de novo in the nucleus; (2) densely staining vacuolar accumulations were seen, frequently surrounding virus capsids. The 13 temperature-sensitive mutants of the virus were divided into three classes according to the type of capsid, if any, produced in cells infected and maintained at the non-permissive temperature. Class I mutants produced no capsids, Class II mutants produced empty and partial-cored capsids and Class III mutants produced empty, partial- and dense-cored capsids. Cellular alterations were also determined. Membranous tubular structures, previously unreported for herpes simplex virus, were observed in cells infected with Class III mutants and very occasionally with wild-type virus at the non-permissive temperature. Cytoplasmic particles were also found, but could not be correlated with any particular class of mutant.