Further characterization of ts1-8, a mutant of herpes simplex virus type 1.

Our initial characterization of a herpes simplex virus type 1, temperature sensitive host shutoff mutant, called ts1-8, revealed that it has a low plaquing efficiency and exhibits a defect in the shutoff of host polypeptide synthesis and host DNA replication at the nonpermissive temperature of 39.5 degrees C. Using intratypic marker rescue experiments the ts plaquing mutation was mapped to a 557 bp region. Sequence analysis and complementation studies revealed that the low plaquing efficiency phenotype is due to a mutation in the glycoprotein B gene converting Pro-357 to Ser. This novel tsgB mutation is located in a conserved region of gB and it is distinct from the delayed host shutoff mutation (dhs).     

Substitution in the poliovirus replicase gene determines actinomycin D sensitivity of viral replication at elevated temperature

A series of ts+ revertants and recombinants derived from a temperature-sensitive plurimutant of poliovirus type 1 showed identical plaquing efficiencies at 37 degrees C and at 39 degrees C and exhibited similar yields and plaque morphology to wild-type virus. However, these viruses were characterized by clear inhibition of viral RNA synthesis at 39 degrees C, as measured by uridine incorporation in the presence of actinomycin D. Similarly, virus yields were decreased by one log in the presence of actinomycin D during infection at 39 degrees C. All the ts+ recombinants formed between temperature-sensitive mutants of poliovirus that were inhibited by actinomycin D carried a glutamine----histidine modification at residue 170 of their viral replicase (polypeptide 3D), due to a G----U substitution at nucleotide 6496. Inhibition of viral growth was increased by pretreatment of cells with actinomycin D for 3 h prior to infection, suggesting that actinomycin D sensitivity could reflect an increased dependence of viral RNA replication on host factor(s).     

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 °.     

Herpesvirus proteins: DNA polymerase and pyrimidine deoxynucleoside kinase activities in temperature-sensitive mutants of herpes simplex virus type 2.

Eleven temperature-sensitive mutants of herpes simplex virus type 2 strain HG52 were examined for ability to induce DNA polymerase activity in BHK 21/C13 cells. All mutants induced DNA polymerase at a permissive temperature, (31 degrees C) and all DNA-positive mutants at a non-permissive temperature (38 degrees C). Three DNA-negative mutants induced no DNA polymerase (ts 6, ts 9) or very little DNA polymerase (ts 11), at a non-permissive temperature, while ts 1, also DNA negative, induced a little more DNA polymerase than wild-type, often at both temperatures. The DNA polymerase induced by ts 6 at 31 degrees C was temperature-sensitive in vivo, but only slightly so in vitro. These results were confirmed immunologically and suggest that HSV-2 codes for at least part of a DNA polymerase activity, necessary for infection, and that full expression of this enzyme involves at least three viral genes.     

Coordinate control of host DNA synthesis and measles virus production in persistently infected BHK-21 cells

Summary Measles virus, upon infection, caused cytopathic effects in BHK-21 cells while it readily established persistent infection in a mutant strain of BHK-21 cells which possessed a temperature sensitive defect in DNA synthesis (BN2). This finding demonstrated that host function can be involved in allowing the establishment of persistent infection. When BN2 and infected BN2 cells were incubated at the non-permissive temperature (39.5° C), DNA synthesis was inhibited in a similar manner in both cells types, suggesting that viral functions do not interfere with the expression of the host mutation. Thets mutation which blocked the DNA synthesis also caused inhibition of viral yield, viral antigen expression and viral RNA synthesis at 39.5° C. The results suggest that continued DNA synthesis is necessary for the expression of measles functions.With 3 Figures     

Stress-induced increase of hexose transport as a novel index of cytopathic effects in virus-infected cells: role of the L protein in the action of vesicular stomatitis virus

The VSV-specific increase in hexose transport by BHK cells has been measured by assay of the [3H]dGlc/[14C]AIB uptake ratio. The effect was abolished by uv-irradiation of the virus, indicating that viral gene expression is required. Cells infected with the T1026 R1 mutant of VSV, which causes only slight cytopathic changes, exhibited only a slight increase in hexose uptake. Cells infected with temperature-sensitive (ts) mutants of VSV that are defective in the function of the viral N, NS, G, or M proteins at the restrictive temperature (39.5 degrees) exhibited increased [3H]dGLC/[14C]AIB uptake ratios typical of wild-type virus at either restrictive (39.5 degrees) or permissive temperature (34 degrees). Cells infected with a mutant defective in the function of the viral L protein exhibited an increased [3H]dGlc/[14C]AIB uptake ratio at permissive temperature (34 degrees) only; at restrictive temperature (39.5 degrees) the uptake ratio was essentially the same as that of mock-infected cells. Temperature-shift experiments indicated that the effect on hexose transport persisted for at least 6 hr in cells which no longer expressed function L protein, and that when expression of L was restricted to the first 2 hr of infection, an almost complete stimulation of hexose transport was observed 4 hr later. These results indicate that expression of the L gene is a necessary factor for inducing an increased hexose uptake in VSV-infected BHK cells. They also suggest that the action of the L protein on hexose transport is indirect, and is presumably mediated by other cellular constituents. The studies support the concept that an increased dGlc uptake may be a useful index of the cytopathic consequences of virus infection.     

Protein synthesis following infection of the blue-green alga Plectonema boryanum with the temperate virus SPI and its ts mutants

The structural proteins of the purified phage and proteins synthesized in virus-infected cells were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and subsequent autoradiography. A total of 24 viral-induced proteins were identified, 12 of which are present in the mature virus. The kinetics of synthesis of the various viral proteins demonstrates the presence of “early” and “late” genes. Specific suppression of host protein synthesis by the infecting phage takes place late in the infection cycle. Analysis of the proteins synthesized at the nonpermissive temperature by phage temperature-sensitive (ts) mutants demonstrates that some of the viral genes have complex pleiotropic effects on viral protein synthesis. The results suggest the presence of positive regulatory genes, and of specific genes involved in the shutoff of host protein synthesis.     

Integration of herpes simplex virus type 1 DNA into the DNA of growth-arrested BHK-21 cells.

The ability of HSV-1 DNA to become associated with host cell DNA in an alkaline-stable form has been demonstrated following infection of a ts baby hamster kidney growth mutant (ts BTN-1), at the non-permissive temperature (39.5 depgrees C). After 8 h pre-incubation at 39.5 degrees C, ts BTN-1 cells infected at this temperature using m.o.i. ranging from 0.5 to 200 p.f.u./cell fail to replicate virus DNA even though transport of input virus genomes to the nucleus is the same at both permissive and non-permissive temperatures. Virions containing 3H-labelled DNA were used to infect ts BTN-1 cells at 39.5 degrees C, and the total cellular DNA isolated from these cells was resolved into host and virus material by repeated CsCl equilibrium gradient centrifugation. A significant amount of the input radioactivity was found as a distinct band in the host region in both neutral and alkaline CsCl gradients, strongly suggesting a covalent association between host and virus DNAs. Evidence for this association was strengthened by demonstrating that radioactive material (virus DNA) banding in the host region of CsCl gradients could be driven towards the density expected for virus DNA following degradation of the putative hybrid molecules by shearing.     

Autographa californica M nuclear polyhedrosis virus and cytochalasin D: antagonists in the regulation of protein synthesis

During infection by the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), synthesis of host proteins in IPLB-Sf-21 cells is inhibited. We report here that cytochalasin D (CD), a drug that specifically interacts with actin, behaved as an antagonist in the virus-mediated shutdown of host proteins actin and tubulin. In uninfected cells, CD caused an increase in actin synthesis but had no apparent effect on tubulin synthesis. In infected cells, CD similarly enhanced actin synthesis early in infection and delayed the virus shutoff of actin synthesis by 14 hr. The shutoff of tubulin synthesis was delayed by 8 hr. Addition of CD to infected cells after host protein synthesis ceased resulted in an induction of actin synthesis reversing viral inhibitory effects. Similarly, the removal of CD resulted in virus-induced inhibition of actin synthesis. Treatment of infected cells with CD caused a delay in the onset and/or shutoff of at least five viral proteins and inhibited the amplification of polyhedrin synthesis by at least 8 hr.     

Recombination between temperature-sensitive mutants of simian virus 40

Seven temperature-sensitive (ts) SV40 mutants have been isolated and characterized. All of the mutants were defective in a late function. Four of the mutants were assigned to complementation group B, one to a new group designated C and two could not be assigned to a complementation group. Recombination occurred between mutants in the B and C complementation groups and between mutants in the B group. The recombination frequency (RF) between tsB302 and tsB306 was about 2.0 × 10^?4 when virus was used for infection, but was 10-fold higher when infectious ts SV40 DNAs were used. Treatment of doubly infected cells with 1-β-d-arabinofuranosylcytosine (ara-C) to interrupt DNA synthesis increased the RF 3- to 14-fold. Ultraviolet irradiation of viral inocula to 1–5% survival resulted in a 25- to 40-fold increase in RF. However, only a 2-fold increase in RF was obtained when uv-irradiated ts SV40 DNAs were used for infection. Ultraviolet irradiation of host CV-1 cells or pretreatment of host CV-1 cells with nonirradiated or uv-irradiated CV-1 DNA prior to infection failed to increase the RF between tsB302 and tsB306.     

Different host-cell shutoff strategies related to the matrix protein lead to persistence of vesicular stomatitis virus mutants on fibroblast cells

Acute infection of fibroblastic cell lines by the Indiana strain of vesicular stomatitis virus (VSV) usually induces dramatic cytopathic effects and shutoff of cellular gene expression. We have compared a series of independent mutants with differences in shutoff induction and found that M was mutated either in the N-terminus (M(51)R) or C-terminus (V(221)F and S(226)R). Furthermore, only double mutants (M mutation and a ts mutation related or not to M) were able to persist on fibroblast cell lines at 39 degrees C. A more detailed investigation of the infection was performed for the mutants T1026, TP3 and G31, differing in their host shutoff effects related to M protein. Viral activity in persistently infected mouse L-929 and monkey Vero cell lines was followed by viral proteins detection, RNA synthesis throughout infection and finally detection of infectious particles. All three mutants cause extensive CPE followed by emergence of persistently infected cells on Vero cells. The same thing is seen on L-929 cells except for T1026 which causes little CPE. Taken together, the results form a basis of further studies to clarify how various viral and cellular factors interact in the establishment of a persistent infection by VSV mutants.     

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.     

Control of protein synthesis in herpesvirus-infected cells: analysis of the polypeptides induced by wild type and sixteen temperature-sensitive mutants of HSV strain 17.

The polypeptides induced in cells infected with a Glasgow isolate of HSV-I (17 syn+) have been characterized by SDS polyacrylamide gel electrophoresis. Study of the kinetics of synthesis in three cell lines has detected a total of 52 polypeptides, 33 of which can be identified in polypeptide profiles of purified virions. These include six low mol. wt. polypeptides that have not been previously reported. Several polypeptides were labelled with glucosamine in infected BHK cells. The different polypeptide patterns obtained at permissive (31 degrees C) and nonpermissive (38 degrees C) temperature in cells infected with 16 temperature-sensitive (ts) mutants are reported. The effect of multiplicity of infection (m.o.i.) on the polypeptide profile has been examined for two of the DNA -ve mutants: below ten, the profile varied with the m.o.i. whereas above ten it was constant. All mutants were therefore examined at an m.o.i. of approx. 20. Mutants from the same complementation group showed very similar profiles. A number of general conclusions concerning control of protein synthesis in HSV infected cells can be made: (I) As most of the 16 ts mutants affected the synthesis of several or many polypeptides it follows that a large proportion of genome specifies controlling functions. (2) The high frequency with which some polypeptides were affected suggests they are at or near the terminus of biosynthetic pathways which are under multiple control. (3) Conversely, some polypeptides were affected with a low frequency suggesting that their synthesis is not dependent on the expression of many virus functions. (4) Several individual ts mutations lead to the synthesis of increased amounts of different large polypeptides. (5) Analysis of every band detectably affected by at least one ts mutation has disclosed nine classes of dependence relationship between polypeptide synthesis and the DNA phenotype of the mutants, illustrating that this relationship is complex and different for different polypeptides. (6) The inhibition of host protein synthesis by the virus may not be a simple single step process.     

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.