Mutants Of Complementation Group Research Articles

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 deoxyribonucleo-protein 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.

A mutant of sindbis virus with a host-dependent defect in maturation associated with hyperglycosylation of E2

Following serial passage of Sindbis virus (SV) on Aedes albopictus mosquito cells a mutant (SV ap15,21) was isolated which in chick cells produced small plaques and was temperature sensitive ( ts). At 34.5° this mutant replicated normally in mosquito cells, but only poorly in chick or BHK cells. In the vertebrate cells SV ap15/21 was RNA + at both 34.5 and 40° and on the basis of complementation tests carried out at 40°, was assigned to complementation group E. The block in the replication of this mutant, like that of ts20 the prototype mutant of complementation group E, was at the level of nucleocapsid envelopment. The PE2 and E2 glycoproteins of SV ap15/21 were found to be hyperglycosylated relative to the corresponding glycoproteins of the parent virus (SV std). Analysis of revertants of SV ap15/21 suggests a causal relationship between PE2 and E2 hyperglycosylation and the host-specific defect in virus maturation. The association of a host-specific defect in virion assembly with hyperglycosylation of a viral structural protein points to the potential importance of host-specific glycosylation patterns in the determination of viral host range.

Genetic analysis of temperature-sensitive mutants of HSV-1: The combined use of complementation and physical mapping for cistron assignment

To date, mutations in mutants representing 19 of the 33 recognized HSV-1 complementation groups have been mapped. The physical map locations of mutations in 10 ts mutants of HSV-1 strain KOS representing 8 of the 19 complementation groups are reported herein. The mutations in three mutants were found to lie between coordinates 0.086 and 0.194—two of these were mapped finely to between coordinates 0.095 and 0.108—and in seven mutants, between 0.301 and 0.448. The mutation in 1 of the 10 mutants tsQ26, was mapped finely to a sequence between 500 and 1000 base pairs to the left of the 3′ end of the TK gene (0.301–0.304). The availability of physical mapping data has (1) confirmed the usefulness of the complementation test as a means of identifying viral gene functions, (2) facilitated the rapid assignment of mutants to new and recognized cistrons, and (3) prompted a reevaluation of previously ambiguous complementation for mutants in 2 complementation groups. Thus, the 10 mutants whose ts mutations were mapped in this study had been assigned previously to 8 complementation groups based on the assumption that complementation indices of 2 or greater signified that 2 mutants were in different genes. Combined with physical mapping data, however, the results of complementation tests now indicate that indices between 2 and 10 may reflect either inter- or intragenic complementation. Thus, the 10 mutants have now been assigned to 7 complementation groups. Although physical mapping data have confirmed the results of previous complementation tests for 6 of 8 groups analyzed, reevaluation of complementation data in the light of physical mapping data has resulted in a more precise genetic definition of the locus for viral DNA polymerase and of a locus (represented by mutants in complementation group 1–10) which maps in the left hand portion of U L.

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.

Genetic analysis of temperature-sensitive mutants which define the gene for the major herpes simplex virus type 1 DNA-binding protein.

We have assigned eight temperature-sensitive mutants of herpes simplex virus type 1 to complementation group 1-1. Members of this group fail to complement mutants in herpes simplex virus type 2 complementation group 2-2. The mutation of one member of group 1-1, tsHA1 of strain mP, has been shown to map in or near the sequence which encodes the major herpes simplex virus type 1 DNA-binding protein (Conley et al., J. Virol. 37:191-206, 1981). The mutations of five other members of group 1-1 map in or near the sequence in which the tsHA1 mutation maps, a sequence which lies near the center of UL between the genes for the viral DNA polymerase and viral glycoprotein gAgB. These mutants can be divided into two groups; the mutations of one group map between coordinates 0.385 and 0.398, and the mutations of the other group map between coordinates 0.398 and 0.413. At the nonpermissive temperature mutants in group 1-1 are viral DNA negative, and mutant-infected cells fail to react with monoclonal antibody to the 130,000-dalton DNA-binding protein. Taken together, these data indicate that mutants in complementation groups 1-1 and 2-2 define the gene for the major herpes simplex virus DNA-binding protein, an early gene product required for viral DNA synthesis.

Thermostabilities of virion activities of Newcastle disease virus: evidence that the temperature-sensitive mutants in complementation groups B, BC, and C have altered HN proteins.

Four virion activities of Newcastle disease virus (hemagglutinating, neuraminidase, hemolytic, and infectious activities) were examined before and after heat stress in low-salt buffer and physiological salt buffer (phosphate-buffered saline). The hemagglutinating and neuraminidase activities of the Australia-Victoria wild-type (AV-WT) strain were thermostable at both salt concentrations tested, whereas the thermostabilities of the hemolytic and infectious activities were salt dependent (thermostable in phosphate-buffered saline but not in low-salt buffer). Virions of RNA(+) temperature-sensitive (ts) mutants of AV-WT were tested for the stabilities of the four activities. Some mutants in groups B, BC, and C were as stable as AV-WT in all functions, but others were much less stable in all functions. The unstable mutants in groups B, BC, and C affirmed the assignment of the ts lesions of these mutants to the hemagglutinin/neuraminidase (HN) protein gene because HN function(s) are required for all four activities. The instability of these ts mutants was not related to their decreased virion HN protein content and was not due to physical loss of the HN protein from the virions. Three of four ts(+) plaque-forming revertants of the least stable mutant, BC2, coreverted for stability, confirming that the unstable phenotype is indeed the result of the mutation responsible for the ts phenotype. Group D mutants were approximately as stable as AV-WT in hemagglutinating, neuraminidase, and hemolytic activities; this is consistent with this group representing a lesion in a gene other than the HN protein gene. However, the infectivities of two of the three group D mutants were less stable than the infectivity of AV-WT in low-salt buffer.

Control of expression of the herpes simplex virus-induced deoxypyrimidine triphosphatase in cells infected with mutants of herpes simplex virus types 1 and 2 and intertypic recombinants.

Infection of cells with herpes simplex virus type 1 (HSV-1) induces high levels of deoxypyrimidine triphosphatase. The majority of the enzyme activity is found in infected cell nuclei. A similar activity is induced by HSV type 2 (HSV-2) which, in contrast to the HSV-1 enzyme, fractionates to more than 99% in the soluble cytoplasmic extract. Of a series of temperature-sensitive mutants of HSV-1 studied, only the immediate-early mutants in complementation group 1-2 (strain 17 mutants tsD and tsK and strain KOS mutant tsB2) induced reduced levels of triphosphatase at nonpermissive temperature. Of a series of temperature-sensitive mutants of HSV-2 strain HG52, ts9 and ts13 failed to induce wild-type levels of the enzyme at nonpermissive temperature; ts9 was the most defective mutant with regard to triphosphatase expression of both herpes simplex virus serotypes. After shift-up from permissive to nonpermissive temperature, triphosphatase activity in cells infected with ts9 decreased rapidly, whereas all other mutants continued to exhibit enzyme levels comparable with controls kept at the permissive temperature. The type 1-specific nuclear expression of the triphosphatase was mapped physically by the use of HSV-1 x HSV-2 intertypic recombinants, based on enzyme levels different by more than two orders of magnitude found in nuclei of HSV-1- and HSV-2-infected cells. The locus for the type-specific expression maps between 0.67 and 0.68 fractional length on the HSV genome.

Cross-reactive, cell-associated antigen on L929 cells infected with temperature-sensitive mutants of sindbis virus.

Temperature-sensitive (ts) mutants of Sindbis virus (SIN) were used to aid in the identification of alphavirus cross-reactive proteins on the surface of infected cells by antibody-dependent, complement-mediated cytolysis. Antisera prepared in rabbits against purified SIN or Semliki Forest viruses were highly cytotoxic for cells infected with wild-type SIN and for cells infected at the permissive temperature with maturation-defective, ts mutants of SIN belonging to several distinct complementation groups. When these SIN mutants were analyzed by antibody-dependent, complement-mediated cytolysis at the restrictive temperature only cells infected with the SIN mutant of complementation group E, ts20, participated in both homologous (with anti-SIN serum) and heterologous (with anti-Semliki Forest virus serum) antibody-dependent, complement-mediated cytolysis reactions. These data and the known defect of ts20 suggested that the cell-associated viral E1 glycoprotein was a functional target antigen for homologous and cross-immunoreactivity in alphavirus-infected cells. At the restrictive temperature there were quantitative differences in antibody-dependent, complement-mediated cytolysis reactivity of ts20- versus wild type-infected cells consistent with the suggestion that ts20-infected cells do not fully express all of the homologous or the cross-reactive antigenic determinants found in wild-type infection. Additional potential sites for antigenic determinants involved in alphavirus-immune cross-reactivity are discussed in relation to events in virus maturation.

Specific Sindbis virus-coded function for minus-strand RNA synthesis.

The synthesis of minus-strand RNA was studied in cell cultures infected with the heat-resistant strain of Sindbis virus and with temperature-sensitive (ts) belonging to complementation groups A, B, F, and G, all of which exhibited an RNA-negative (RNA-) phenotype when infection was initiated and maintained at 39 degrees C, the nonpermissive temperature. When infected cultures were shifted from 28 degrees C (the permissive temperature) to 39 degrees C at 3 h postinfection, the synthesis of viral minus-strand RNA ceased in cultures infected with ts mutants of complementation groups B and F, but continued in cultures infected with the parental virus and mutans of complementation groups A and G. In cultures infected with ts11 of complementation group B, the synthesis of viral minus-strand RNA ceased, whereas the synthesis of 42S and 26S plus-strand RNAs continued for at least 5 h after the shift to 39 degrees C. However, when ts11-infected cultures were returned to 28 degrees C 1 h after the shift to 39 degrees C, the synthesis of viral minus-strand RNA resumed, and the rate of viral RNA synthesis increased. The recovery of minus-strand synthesis translation of new proteins. We conclude that at least one viral function is required for alphavirus minus-strand synthesis that is not required for plus-strand synthesis. In cultures infected with ts6 of complementation group F, the syntheses of both viral plus-strand and minus-strand RNAs were drastically reduced after the shift to 39 degrees C. Since ts6 failed to synthesize both plus-strand and minus-strand RNAs after the shift to 39 degrees C, at least one common viral component appears to be required for the synthesis of both minus-strand and plus-strand RNAs.

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.

Characterization of vesicular stomatitis virus mutants by partial proteolysis.

Structural proteins of temperature-sensitive (ts) mutants of vesicular stomatitis virus, Indiana serotype, were compared with those of wild-type and revertant virions by electrophoresis on polyacrylamide gels of partial digests with Staphylococcus aureus V8 protease. Mutants of complementation groups III (tsG31 and tsG33), II (tsG22), and IV (tsG41) differed from the wild-type virion in peptide profiles of their M, NS, and N proteins, respectively. The differences were only detectable over a narrow range of enzyme-substrate ratios and were due to peptides transiently generated during incomplete digestion. Proteins of revertants to tsG31, tsG22, and tsG41 exhibited the wild-type virion peptide pattern, indicating that reversion had restored their original conformation. However, in the case of tsG22, the NS peptide profile reverted to the wild-type phenotype only partially, suggesting that a silent mutation might have taken place during either the original chemical mutagenesis or the following repeated laboratory passages. The apparent alteration in protein conformation and its restoration upon reversion of the mutants indicated that the lesions of groups III and IV were located in the M and N proteins, respectively. Moreover, for the first time, the site of mutation of group II could be positively identified as the NS protein cistron.

Mutant analysis of herpes simplex virus-induced cell surface antigens: resistance to complement-mediated immune cytolysis.

BHK-21 cells infected with temperature-sensitive mutants of herpes simplex virus type 1 strain KOS representing 16 complementation groups were tested for susceptibility to complement-mediated immune cytolysis at permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures. Only cells infected by mutants in complementation group E were resistant to immune cytolysis in a temperature-sensitive manner compared with wild-type infections. The expression of group E mutant cell surface antigens during infections at 34 and 39 degrees C was characterized by a combination of cell surface radioiodination, specific immunoprecipitation, and gel electrophoretic analysis of immunoprecipitates. Resistance to immune lysis at 39 degrees C correlated with the absence of viral antigens exposed at the cell surface. Intrinsic radiolabeling of group E mutant infections with [14C]glucosamine revealed that normal glycoproteins were produced at 34 degrees C but none were synthesized at 39 degrees C. The effect of 2-deoxy-D-glucose on glycosylation of group E mutants at 39 degrees C suggested that the viral glycoprotein precursors were not synthesized. The complementation group E mutants failed to complement herpes simplex virus type 1 mutants isolated by other workers. These included the group B mutants of strain KOS, the temperature-sensitive group D mutants of strain 17, and the LB2 mutant of strain HFEM. These mutants should be considered members of herpes simplex virus type 1 complementation group 1.2, in keeping with the new herpes simplex virus type 1 nomenclature.

Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses.

Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.

Temperature-Sensitive Mutants of Complementation Group E of Vesicular Stomatitis Virus New Jersey Serotype Possess Altered NS Polypeptides

In vesicular stomatitis virus New Jersey serotype polyacrylamide gel electrophoresis was unable to distinguish the polypeptides of the temperature-sensitive (ts) mutants of complementation groups A, B, C, and F from those of the wild-type virus. However, the NS polypeptide of the representative mutant of group E, ts E1, had a significantly greater electrophoretic mobility than that of the wild-type virus NS polypeptide. The electrophoretic mobilities of the NS polypeptides of the three mutants of complementation group E varied, being greatest in the case of ts E1, slightly less for ts E2, and only a little greater than that of wild-type virus NS polypeptide in the case of ts E3. Since the NS polypeptides of the revertant clones ts E1/R1 and ts E3/R1 have mobilities identical to that of wild-type NS polypeptide, the observed altered mobilities of the group E mutants are almost certainly the direct result of the ts mutations in the E locus. The electrophoretic mobilities of the intracellular NS polypeptides of the group E mutants were indistinguishable from those of their virion NS polypeptides. The electrophoretic mobilities of the NS polypeptides of the group E mutants synthesized in vitro using mRNA synthesized in vitro by TNP were identical to those of the NS polypeptides of their purified virions. The NS polypeptides of all three mutants were labeled with (32)P(i) to approximately the same extent as wild-type virus NS polypeptide, indicating that gross differences in phosphorylation of this polypeptide are unlikely to account for the altered mobilities. We propose a model in which the NS polypeptide consists of at least three loops held in this configuration by hydrophobic or ionic forces or both and stabilized by phosphodiester bridges. If a mutation affects one of the amino acids to which the phosphate is covalently linked, the phosphodiester bridge cannot be formed, and, as a result, in the presence of sodium dodecyl sulfate the affected loop opens and thus the NS polypeptide migrates further into the gel. Such a configuration may also explain the multifunctional nature of the NS polypeptide.

Effect of Temperature-Sensitive Mutation on Activity of the RNA Transcriptase of Vesicular Stomatitis Virus New Jersey

The virion-associated RNA transcriptase activity of vesicular stomatitis virus New Jersey temperature-sensitive (ts) mutants was assayed in vitro at the permissive (31 degrees C) and restrictive (39 degrees C) temperatures. RNA synthesis at 39 degrees C by the RNA-negative ts A1 and the RNA-positive ts C1 and ts D1 mutants was similar to that of wild-type virus. The RNA-negative ts B1 synthesized only small amounts of RNA in vitro at 39 degrees C. The three mutants of complementation group E were dissimilar in the amounts of RNA they synthesized at 39 degrees C: ts E1 synthesized very little RNA, ts E2 synthesized moderate amounts, and RNA synthesis by ts E3 was not inhibited. The two mutants of group F were also dissimilar, since ts F1 synthesized very little RNA at 39 degrees C, whereas ts F2 synthesized as much RNA as wild-type virus. The revertant clones ts B1/R1, ts E1/R1, and ts F1/R1 synthesized RNA at 39 degrees C in amounts comparable to wild-type virus, indicating that the heat sensitivity of the transcriptase activity of the mutants ts B1, ts E1, and ts F1 was associated with temperature sensitivity. Similar heat sensitivities were observed when transcribing nucleoprotein complexes were used in the assays, showing that the mutated polypeptides were part of the viral core. The heat stability of the mutant ts B1 was similar to that of wild-type virus, and in vitro RNA synthesis was fully restored when the temperature was lowered to 31 degrees C after 30 min of preincubation at 39 degrees C, showing that the inhibition was due to reversible configurational change of the mutated polypeptide. When virions of the mutant ts E1 were heated for 5 h at 39 degrees C, their infectivity and transcriptase activity were as stable as those of the wild-type virus, whereas transcriptase activity became very heat labile after disruption of the viral coat with a neutral detergent. This suggests an interaction between the mutated polypeptide and a coat polypeptide which stabilizes the activity of the transcriptase. The RNA transcriptase activity of the mutant ts F1 was also heat labile, although to a lesser extent than that of ts E1. Thus, the defects in transcriptase activity of groups B, E, and F suggest that all three polypeptides of the virus core, polypeptides L, N, and NS, are involved in the transcription. In addition, we postulate that the mutated gene products of groups E and F are multifunctional, being required both in transcription and replication, and that the gene product of group E may also be involved in some late stage of virus development.

Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein

We describe a procedure that enriches for temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV), Indiana serotype, which are conditionally defective in the biosynthesis of the viral glycoprotein. The selection procedure depends on the rescue of pseudotypes of known ts VSV mutants in complementation group V (corresponding to the viral G protein) by growth at 39.5 degrees C in cells preinfected with the avian retrovirus Rous-associated virus 1 (RAV-1). Seventeen nonleaky ts mutants were isolated from mutagenized stocks of VSV. Eight induced no synthesis of VSV proteins at the nonpermissive temperature and hence were not studied further. Four mutants belonged to complementation group V and resembled other ts (V) mutations in their thermolability, production at 39.5 degrees C of noninfectious particles specifically deficient in VSV G protein, synthesis at 39.5 degrees C of normal levels of viral RNA and protein, and ability to be rescued at 39.5 degrees C by preinfection of cells by avian retroviruses. Five new ts mutants were, unexpectedly, in complementation group IV, the putative structural gene for the viral nucleocapsid (N) protein. At 39.5 degrees C these mutants also induced formation of noninfectious particles relatively deficient in G protein, and production of infectious virus at 39.5 degrees C was also enhanced by preinfection with RAV-1, although not to the same extent as in the case of the group V mutants. We believe that the primary effect of the ts mutation is a reduced synthesis of the nucleocapsid and thus an inhibition of synthesis of all viral proteins; apparently, the accumulation of G protein at the surface is not sufficient to envelope all the viral nucleocapsids, or the mutation in the nucleocapsid prevents proper assembly of G into virions. The selection procedure, based on pseudotype formation with glycoproteins encoded by an unrelated virus, has potential use for the isolation of new glycoprotein mutants of diverse groups of enveloped viruses.

Defective transforming capacity of adenovirus type 5 host-range mutants

Host-range (hr) mutants of human adenovirus type 5 (Ad 5), selected for their ability to grow preferentially on transformed human embryo kidney cells (293 cells) and shown to fall into two complementation groups (I and II), have been tested for ability to transform rat embryo, rat embryo brain, and baby rat kidney cells. When embryo or embryo brain cells were used, mutants of both complementation groups either failed to transform or transformed at a frequency much lower than that observed with wild-type ( wt) virus. Moreover, the small number of transformed colonies which did arise in hr-infected cultures failed to grow when isolated and subcultured, unlike wt transformants from which lines could be established with a high success rate. When assays were carried out with baby rat kidney cells, hr mutants of complementation group II were again transformation negative, while mutants of group I induced transformation with several times the efficiency of wt Ad 5. Again, attempts to establish transformed lines from single hr-transformed foci were consistently unsuccessful, in contrast to an almost 100% success rate with wild-type transformants; hr mutant-transformed lines could only be established with difficulty by passaging whole cultures. Our results are consistent with the idea that group I mutants are able to initiate an abortive or abnormal transformation but are defective in some aspects of its maintenance; group II mutants, on the other hand, appear to be defective in initiation of transformation.

DNA-negative temperature-sensitive mutants of herpes simplex virus type 1: patterns of viral DNA synthesis after temperature shift-up

Temperature-sensitive mutants of herpes simplex virus type 1 belonging to four DNA- complementation groups exhibited two distinct patterns of viral DNA synthesis after shift-up to the nonpermissive temperature. In cultures infected with mutants belonging to complementation groups A, C, and D, little or no viral DNA was synthesized after shift-up. In cultures infected with a mutant in complementation group B, nearly normal amounts of viral DNA were synthesized after shift-up.

Parental G protein reincorporation by a vesicular stomatitis virus temperature-sensitive mutant of complementation group V at nonpermissive temperature

Rescue virions obtained after superinfection of ts O45(V) Indiana serotype-infected cells by uv-irradiated vesicular stomatitis virus (VSV) at a nonpermissive temperature do not incorporate G protein synthesized at this temperature. They apparently contain G protein in their envelope since they are neutralized by homotypic antivirion and antispike sera. Rescue of ts O45(V) mutant Indiana serotype by uv-irradiated VSV New Jersey serotype is demonstrated. Ultraviolet-inactivated VSV of New Jersey serotype has therefore been used to determine the origin of the incorporated G protein molecules. The progeny-rescue virions belong genetically to VSV Indiana complementation group V. They are however neutralized by anti-New Jersey serum and not by anti-Indiana serum. Rescue ts O45(V) virions have thus probably reincorporated G protein molecules supplied by the uv-irradiated New Jersey virus. This provides further evidence suggesting that protein G is encoded for by gene V.

Temperature-sensitive mutants of type 12 adenovirus defective in a late function: protein synthesis and evidence for recombination between mutants in complementation group D.

Four temperature sensitive mutants of adenovirus type 12, classified into complementation group D, synthesize most or all of the late virus-specific polypeptides at the restrictive temperature, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Infections by these mutants under restrictive conditions apparently result in some over production of the virion hexon polypeptide compared to wild type infection. Genetic recombination between the four mutants has been demonstrated. A partial linear genetic map of the D cistron is presented.