Viruses and renal carcinoma of Rana pipiens. XIV. Temperature-sensitive mutants of frog virus 3 with defective encapsidation

The temperature-sensitive defects of six mutants of frog virus 3 were identified. Although each of the mutants synthesized comparable amounts of virus-specific DNA at permissive (25 C) and nonpermissive (30 C) temperatures, viral DNA was not encapsidated at 30 C. When infected cells were shifted to permissive temperature, the viral DNA made at 30 C was encapsidated. Encapsidation required RNA and protein synthesis at 25 C, and the 30 C DNA could act as template for the required RNA. The assembled virions containing 30 C DNA were infectious. Thus, the DNA synthesized at nonpermissive temperature is fully functional and the temperature-sensitive defects involve only encapsidation.     

Electron microscopic studies of temperature-sensitive mutants of herpes simplex virus type 1

Fifteen temperature-sensitive (ts) mutants of herpes simplex virus type 1, each representing a single complementation group, were found to fall into three classes with regard to the degree of defectiveness in virion synthesis which they exhibited at the nonpermissive temperature (39°) when examined by electron microscopy. Mutants in class A, one DNA and one DNA⁺, failed to synthesize detectable particles. Mutants in class B, three DNA^? and four DNA⁺, synthesized small to moderate numbers of empty nucleocapsids. An unique cylindrical core form was observed in particles synthesized after infection with a class B mutant at 39°. Six DNA⁺ mutants in class C synthesized large numbers of virus particles most of which contained apparently empty nucleocapsids. In addition, five of the six mutants synthesized small to moderate numbers of dense-cored nucleocapsids and of these, two formed enveloped, dense-cored particles. The ultrastructural appearance of cells infected with one class C mutant at 39° resembled wild-type virus-infected cells at this temperature, yet it produced 10,000-fold less infectious virus than cells infected with the wild-type virus.     

Deoxyribonucleoprotein complexes and DNA synthesis of herpes simplex virus type 1

Two temperature-sensitive mutants of herpes simplex virus type 1 in complementation group 1-1 were analyzed to determine if the major DNA-binding protein they produced was thermolabile. Cells infected with these mutants were analyzed for deoxyribonucleoprotein complexes containing the DNA-binding protein. These complexes were found in cells infected at the permissive temperature but not at the nonpermissive temperature. In temperature shift-up experiments with mutant virus infected cells, the levels of the deoxyribonucleoprotein complexes decreased with time of incubation at the nonpermissive temperature. Viral DNA synthesis terminated in cells infected with these mutants after temperature shift-up. The kinetics of termination of viral DNA synthesis were similar to the kinetics of dissociation of the deoxyribonucleoprotein complexes. These results indicate that two mutants in complementation group 1-1 produce a thermolabile DNA-binding protein and that this protein is required for viral DNA synthesis. Furthermore, they suggest that the major DNA-binding protein of herpes simplex virus type 1 functions in viral DNA synthesis as a component of deoxyribonucleoprotein complexes.     

Studies on temperature-sensitive events in synthesis of poliovirus ts mutants

Summary Studies on temperature-sensitive events of four poliovirus ts mutants indicated that the temperature-sensitive stage of ts2 and ts5 mutants occurs late in the reproduction cycle, but for ts 11 and tsM/23 it occurs both at early and at late stages of the cycle. RNA production by ts2, ts11 and tsM/23 mutants (RNA^–) under nonpermissive conditions (40° C) is 13–14 per cent as compared with RNA production at 36° C. All the mutants under study form both 35S and 20S RNA under nonpermissive conditions, but the relative amount of 35S RNA synthesized at 40° C is considerably lower than at 36° C. A degradation of viral RNA under nonpermissive conditions is detected. Obtained data suggest that ts5 mutant (RNA⁺) under nonpermissive conditions is incapable of assembly of virus particles from synthesized components, but the multiplication of ts2, ts11 and tsM/23 mutants does not occur because of strong inhibition of RNA synthesis. Protein subvirion structures induced by ts5 mutant under nonpermissive conditions after subsequent transfer of the infected cells to permissive conditions, are incorporated into mature virus particles. Subvirion structures induced by ts M/23 mutant do not participate in further stages of morphogenesis after the infected cells are shifted to permissive conditions.     

Functions of the major nonstructural DNA binding protein of a herpesvirus (pseudorabies)

Eight mutants of pseudorabies virus belonging to complementation group 3 and situated between 0.14 and 0.18 units on the physical map of the genome were analyzed. All the mutants tested in this respect (seven) recombined with one another, indicating that the mutations were located in different regions of the gene. All mutants were DNA-; the first round, as well as subsequent rounds, of DNA replication was completely blocked at the nonpermissive temperature in the mutant-infected cells. After shift-up from the permissive to the nonpermissive temperature, viral DNA synthesis continued for a short period of time only and viral DNA which had accumulated at the permissive temperature became degraded. Parental viral DNA, however, retained its integrity at the nonpermissive temperature and viral DNA synthesis started immediately after shift-down of the mutant-infected cells from the nonpermissive to the permissive temperature (even in the absence of protein synthesis). All mutants belonging to complementation group 3 tested (5 out of 8) produced a thermolabile nonstructural DNA binding protein (136K). In some of the mutant virus-infected cells this protein failed to migrate to the nucleus. We conclude that the pseudorabies virus mutants in complementation group 3 code for a defective 136K protein and that this protein is not only essential to the process of viral DNA synthesis but also plays a role in the stabilization of progeny DNA (but not of nonreplicating parental DNA) within the infected cells.     

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.     

Characterization of a temperature-sensitive mutant of frog virus 3 defective in DNA replication

We have characterized the defect of a temperature-sensitive (ts) DNA^? mutant (ts 6642) of frog virus 3 (FV 3). At the nonpermissive temperature (30°) ts 6642 synthesized <3% of the viral DNA that was synthesized at the permissive temperature (23°). When ts 6642-infected cells were incubated at 30° for 4.0 hr and then shifted to permissive temperature, viral DNA synthesis started immediately even when protein synthesis was inhibited at the time of shiftdown. This result implies that at 30°, ts 6642 synthesized all the proteins required for viral DNA replication but that one of these was nonfunctional at the nonpermissive temperature. Further characterization revealed that ts 6642 was probably defective in the initiation of DNA replication. This conclusion was based on the following data: When ts 6642-infected cells incubated at 23° for 4.0 hr were shifted to 30°, there was a gradual decrease in viral DNA synthesis. By 1 to 1.5 hr after the shiftup, viral DNA synthesis was completely inhibited. Analysis of the density of the DNA synthesized after a shiftup in the presence of BUdR and FUdR suggested that residual viral DNA synthesis represented chain elongation, and not initiation of new rounds of DNA replication. The defective protein was therefore involved in the initiation process. Both wild-type FV 3 (FV 3⁺) and ts 6642 induced the synthesis of thymidine kinase and DNA polymerase at 30°. Therefore, neither of these enzymes was involved in the DNA replication defect of ts 6642.At the nonpermissive temperature, ts 6642 synthesized all the viral proteins that were detectable at the permissive temperature. However, synthesis of late proteins was delayed, and never reached wild-type levels. Furthermore, the rate of synthesis of late proteins at 30° became dependent upon the multiplicity of infection. These results reinforce our previous conclusion (R. Goorha and A. Granoff, 1974, Virology60, 237–250) that in FV3⁺-infected cells late proteins (and by implication late mRNAs) were synthesized in the absence of viral DNA replication.     

Phenotypic characterization and physical mapping of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus defective in DNA synthesis

A ts mutant of Autographa californica nuclear polyhedrosis virus (AcMNPV), ts8, was shown to be defective in viral DNA synthesis at the nonpermissive temperature. Ts8-infected cells synthesized only early viral polypeptides at the nonpermissive temperature, and in contrast to wild-type (WT)-infected cells, showed no inhibition of host cell protein synthesis. The effect of the mutation on viral DNA synthesis was not immediately reversed after shifting infected cells down from the nonpermissive temperature to the permissive temperature; rather, a delay of several hours occurred before viral DNA synthesis was detected. The rate of accumulation of viral DNA in ts8-infected cells failed to increase after a shift from the permissive temperature to the nonpermissive temperature. This indicated that the ts8 mutation was involved in the synthesis of proteins required for viral DNA synthesis. The mutation was mapped by marker rescue to the region lying between 60.1 and 62.0% of the AcMNPV physical map.     

Isolation, complementation and preliminary phenotypic characterization of temperature-sensitive mutants of herpes simplex virus type 2

Eight temperature-sensitive (ts) mutants of herpes simplex virus type 2, isolated following mutagenesis with 5-bromodeoxyuridine, were assigned to seven nonover-lapping complementation groups (A through G). Partial phenotypic characterization of the mutants at the nonpermissive temperature (38 °) revealed the following: (1) Mutants of 3 complementation groups had a DNA^? phenotype, the remaining being DNA⁺. (2) The DNA^? mutants were deficient in DNA polymerase activity, whereas the DNA⁺ mutants were not. (3) Two mutants, one DNA⁺ and one DNA^?, failed to induce thymidine kinase. (4) Except for one DNA^? mutant, all mutants synthesized physical particles; however, only DNA⁺ mutants synthesized enveloped particles. (5) Mutants belonging to 4 complementation groups were markedly more thermolabile than the wild-type virus, suggesting that they possessed alterations in viral structural proteins.     

A study of TMV ts mutant Ni2519. I. Complementation experiments

Two distinct virus-specific functions, i.e., virus assembly and spreading of infection from cell to cell (transport function), are temperature-sensitive (ts) in TMV mutant Ni2519. Assembly of Ni2519 cannot be complemented by the temperature-resistant TMV strains used: A14 (a wild type strain from which Ni2519 was derived) and dolichos enation mosaic virus (DEMV, or cowpea strain of TMV), a thermophilic strain. On the other hand, Ni2519 can serve as a donor of the coat protein to complement is strain Ni118, which has a mutation in the coat protein gene. The genomic RNA can be produced by Ni2519 at a nonpermissive temperature; functionally active Ni2519 coat protein (capable of coating Ni118 RNA upon mixed infection) is produced at a nonpermissive temperature as well. The is phenotype of Ni2519 upon virus assembly probably results not from the ts behavior of any virus-coded protein(s) but is due to the ts properties of the genomic RNA molecule itself, so the possibility of the complementation of assembly of Ni2519 is ruled out. Thus, Ni2519 appears to represent a novel class of virus mutants with is virion RNA. The second is function of Ni2519 (transport of infection) can be complemented by a helper virus. The experimental system used for complementation of the transport function allowed Ni2519 to spread from cell to cell at a nonpermissive temperature. Obviously, Ni2519 infection spreads under these conditions in a form different from that in the mature virions, since its assembly cannot be complemented by the helper virus. Some aspects of the transport function are discussed.     

Biogenesis of poxviruses: Preliminary characterization of conditional lethal mutants of vaccinia virus defective in DNA synthesis

Characterization of six is mutants of the DNA^? phenotype of vaccinia 1HD-W is described. Complementation analysis revealed that four of these map into different complementation groups while one was able to complement, albeit inefficiently, with only one among the other ts mutants tested. Judging by the rate of viral DNA synthesis it became evident that all DNA^? mutants except ts 6389 became more “leaky” for DNA synthesis at the restrictive temperature in single-cycle infection if adsorption was carried out at 4° when compared with values obtained after initiation of infection at 39.5°. This suggested that these mutants are defective in some function required very early in the virus life cycle. This notion was further supported by the observation that multiple rounds of viral DNA synthesis could occur at 39.5° provided that the temperature was raised after initiating the infection at a lower temperature. Employing density-shift experiments coupled with analysis of the size of replicative intermediates by means of alkaline sucrose gradients, it was found that progeny DNA synthesized by all but ts 6389 was indistinguishable from wild-type DNA. By contrast, DNA synthesis in ts 6389-infected cytoplasm was is throughout the period of viral DNA replication, regardless of the time when temperature was shifted up, relegating this mutant to the “fast-stop” category. This idea is supported by evidence that all of the DNA^? isolates except ts 6389 are deficient at 39.5° in some of the virus-specified DNA-binding proteins. Therefore, ts 6389 is the only isolate at hand with the characteristics expected of a mutant with a defective protein related to the growing fork of the vaccinia DNA replication apparatus.     

Fine structure mapping and phenotypic analysis of five temperature-sensitive mutations in the second largest subunit of vaccinia virus DNA-dependent RNA polymerase

We have used plasmid clones spanning the region encoding the 132-kDa subunit of the cowpox virus RNA polymerase (CPV rpo 132) to marker rescue each of five vaccinia virus (VV) temperature sensitive (ts) mutants, ts 27, ts 29, ts 32, ts 47, and ts 62, which together constitute a single complementation group. The experiments fine-map the vaccinia mutations to a 1.3-kb region containing the 3' end of the CPV rpo 132 gene. Phenotypic characterization shows that all five mutants are affected to varying extents in their ability to synthesize late viral proteins at the nonpermissive temperature, similar to other ts mutants with lesions in the 22- and the 147-kDa subunits of the VV RNA polymerase. Two mutants, ts 27 and ts 32, exhibit a delay in the synthesis of late viral proteins at both the permissive and the nonpermissive temperatures. We conclude that the five VV mutants affect the 132-kDa subunit of the VV RNA polymerase. Additional genetic experiments demonstrate intragenic complementation between ts 62 and three other members of this complementation group, ts 27, ts 29, and ts 32.     

Macromolecular synthesis in cells infected by frog virus 3. I. Virus-specific protein synthesis and its regulation

Heat-inactivated frog virus 3 inhibited protein synthesis in fathead minnow and baby hamster kidney cells but did not affect the replication of superinfecting infectious frog virus 3 in these cells. This method of controlling host-cell protein synthesis enabled us to identify 20 proteins induced by frog virus 3 infection. All detectable virus-specific proteins were synthesized within 2 hr after infection. Three viral structural proteins (VSP)—7, 9, and 13—were synthesized at maximum rates early in infection, and two others, VSP 5 and 11, reached their peak rate of synthesis late in infection. All structural and nonstructural proteins were made in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. In the absence of viral DNA replication, the kinetics of synthesis of VSP 5 and 11 were similar to those during normal infection, but there was no reduction in the synthesis of VSP 7, 9, and 13 late in the infection. These results suggest that synthesis of all viral structural proteins is an early event in the FV 3 replication cycle, with progeny viral DNA required to regulate the synthesis of certain viral proteins.     

Early proteins are required for the formation of frog virus 3 assembly sites

The formation of frog virus 3 virions takes place within morphologically distinct regions of the cytoplasm termed assembly sites. These sites are formed within infected BHK cells by 6-7 hr after infection, a time when viral DNA and both early and late proteins are present. To identify macromolecules involved in assembly site formation, a temperature-sensitive mutant ( ts9467 ) was used which is not only defective in the synthesis of late RNA and proteins (D.B. Willis, R. Goorha , and A. Granoff , 1979, Virology 98, 328-335), but, as reported here, also does not form assembly sites at nonpermissive temperatures. When ts9467 -infected cells were shifted from the nonpermissive to permissive temperature, assembly sites were observed within 1 hr even when late protein synthesis was inhibited by cycloheximide. Monoclonal antibodies specific for early and late viral proteins were used to show that assembly sites formed under these conditions contained at least one early protein, but lacked four representative late proteins. These results indicate that assembly site formation involves interaction between one or more early proteins and viral DNA, and that late proteins do not play a role in this process.