Recombination between temperature-sensitive and deletion mutants of reovirus.

In standard pairwise crosses there was no detectable recombination between defective reovirus lacking the largest genomic segment and prototypes of the seven known classes of ts mutants. However, in such crosses between R2A (201) and the various prototypes frequencies of ts+ recombinants between 2.6 and 6.1% were observed, as others have found (Fields, 1971; Fields and Joklik, 1969). An infectious center assay was devised to measure recombination in this system, and it was found that all mixedly infected cells gave rise to ts+ recombinants in crosses between prototype ts mutants, but no recombination was detectable when the defective virus was crossed with three different ts mutants. The ts mutation of mutant R2A (201) was efficiently rescued when crossed with UV-inactivated wild-type virus but not when crossed with UV-inactivated defective virus. It is concluded from these various experiments that if there is any recombination between these defective reovirions and any known class of ts mutants it is too low to be measured by methods presently available. The kinetics of recombination were measured in cells mixedly infected with R2A (201) and R2B (352) mutants. At the earliest time progeny virus could be found in the cells the frequency of ts+ recombinants was 4.5%, and this frequency remained unchanged despite a subsequent 1,000-fold increase in progeny virus.     

Infectious center assay for complementation and recombination between mutant of reovirus.

An infectious center assay has been developed to measure recombination and complementation in L cells mixedly infected with ts mutants of reovirus. The mutants studied so far fall into complementation groups that correspond to the recombination groups previously defined by other laboratories (B. N. Fields, 1971; B. N. Filds and W. K. Joklik, 1969).     

Complementation of defective reovirus by ts mutants.

Defective reovirions lacking the largest (L-1) of the normal 10 genomic segments grow only in association with helper reovirus. Because of the similarity in properties of defective and infectious virions, separation of the two populations by physical methods has been unseccessful. Controlled digestion of purified virus removes the outer capsomeres of the virions. The resulting core particles containing the viral genome have a buoyant density of 1.43/ml if derived from infectious virions and of 1.415g/ml if they originate in defectives, and this difference permits ready separation of the two types of cores. With the purpose of obtaining a pure population of defective virions, L cells were co-infected with defective cores and a class E temperature-sensitive mutant which has a mutation in an early function. After three serial passages at the permissive temperature (31 C) to build up the defective population, a fourth passage was made at 39 C, the nonpermissive temperature. The virus purified from this passage was predominantly defective; it contained practically no E mutant and had a low background of wild-type virus. Complementation was thus asymmetric; the L-1 function required for growth of defective virus was supplied by the E mutant and is thus a trans-function, while defective virus did not complement the E mutation which is thus in a cis-acting function. Defective virions were indistinguishable from infectious virions except for the absence of the L-1 genomic segment in the defectives. Such defective virions could be complemented at 39 C by class A and B temperature-sensitive mutants, both of which have lesions in late functions.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type 7.

Fifty temperature-sensitive mutants, which replicate at 32 degrees C but not at 39.5 degrees C, were isolated after mutagenesis of the vaccine strain of adenovirus type 7 with hydroxylamine (mutation frequency of 9.0%) or nitrous acid (mutation frequency of 3.8%). Intratypic complementation analyses separated 46 of these mutants into seven groups. Intertypic complementation tests with temperature-sensitive mutants of adenovirus type 5 showed that the mutant in complementation group A failed to complement H5ts125 (a DNA-binding protein mutant), that mutants in group B and C did not complement adenovirus type 5 hexon mutants, and that none of the mutants was defective in fiber production. Further phenotypic characterization showed that at the nonpermissive temperature the mutant in group A failed to make immunologically reactive DNA-binding protein, mutants in groups B and C were defective in transport of trimeric hexons to the nucleus, mutants in groups D, E, and F assembled empty capsids, and mutants in group G assembled DNA-containing capsids as well as empty capsids. The mutants of the complementation groups were physically mapped by marker rescue, and the mutations were localized between the following map coordinates: groups B and C between 50.4 and 60.2 map units (m.u.), groups D and E between 29.6 and 36.7 m.u., and group G between 36.7 and 42.0 m.u. or 44.0 and 47.0 m.u. The mutant in group A proved to be a double mutant.     

Isolation, complementation and partial characterization of mutants of the methanol autotroph Xanthobacter H4-14 defective in methanol dissimilation

Seven mutants of Xanthobacter H4-14, unable to grow on methanol but capable of growth on formate, were isolated and complemented with a chromosomal clone bank constructed in the broad-host-range cosmid pVK100. One mutant could not be complemented but the others fell into four distinct complementation groups that involved three different recombinant clones. All of the complementing regions were separated by at least 10 kbp. The five complementation classes had different phenotypic characteristics and were defective in different aspects of methanol and formaldehyde oxidation. Class I mutants were defective in methanol oxidation, class II mutants were impaired in formaldehyde oxidation, class III mutants appeared to be defective in a regulatory element involving the methanol oxidation system, and class IV mutants appeared to be defective in a regulatory element involving formaldehyde oxidation. Class V mutants exhibited a methanol-sensitive phenotype, which was correlated with an imbalance between methanol and formaldehyde dehydrogenase activities. Analysis of this class suggested it was defective in a repressor that regulated methanol dissimilation functions.     

Defective Particles in BHK Cells Infected with Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Defective particles were the major product after undiluted passage of certain temperature-sensitive (ts) mutants of the Indiana C strain of vesicular stomatitis virus in BHK-21 cells at the permissive temperature (31 C). Essentially homogeneous preparations of defective particles were obtained with the wild-type and individual ts mutants. The defective particles associated with some of the ts mutants, however, were morphologically and physically distinguishable from wild type and from each other. All varieties of defective particle interfered with the multiplication of mutant and wild-type virus at the permissive temperature at early times of infection but failed to complement virions of different complementation groups at the restrictive temperature (39 C) at any time during infection.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type2.

Fourteen temperature-sensitive mutants of human adenovirus type2, which differed in their plaquing efficiencies at at the permissive and nonpermissive temperatures by 4 to 5 orders of magnitude, were isolated. These mutants, which could be assigned to seven complementation groups, were tested for their capacity to synthesize adenovirus DNA at the nonpermissive temperature. Three mutants in three different complementation groups proved deficient in viral DNA synthesis. The DNA-negative mutant H2ts206 complemented the DNA-negative mutants H5ts36 and H5ts125, whereas mutant H2ts201 complemented H5ts36 only. Among the DNA-negative mutants, H2ts206 synthesized the smallest amount of viral DNA at the nonpermissive temperature (39.5 C). Data obtained in temperature shift experiments indicated that a very early function was involved in temperature sensitivity. In keeping with this observation, early virus-specific mRNA was not detected in cells infected with H2ts206 and maintained at 39.5 C. Prolonged (52 h) incubation of cells infected with H2ts206 at the nonpermissive temperature led to the synthesis of a high-molecular-weight form of viral DNA.     

Generation of defective virus after infection of newborn rats with reovirus.

When 2-day-old rats were inoculated subcutaneously with the R2 strain of reovirus type 3 or with a class B (352) or class C (447) temperature-sensitive (ts) mutant, 5 to 10% of the animals died from acute encephalitis within 12 days. Approximately half of the survivors recovered rapidly and grew normally, but the remainder became runted. Two phases of infection are distinguished in the animals: an acute phase during which infectious virus reaches a maximum titer in brain and other tissues by 10 days p.i. and thes runting of the rats and the slow disappearance of virus from their brains over a period of 2 months or so. Virus isolated from chronically infected brains generally retained the genetic character (ts or wild type) of the inoculated virus, but two exceptions to this are described. Defective virions lacking the L1 segment of the viral genome (L1 defectives) were generated in rat brains during the acute phase of infection. Defective virus was also generated during the chronic phase, but during this period defectives were found with multiple segments deleted from the genome in addition to L1 defectives. In another type of experiment defective virus exerted a marked protective effect when inoculated intracerebrally with R2 virus. In the absence of defectives all animals died, but in their presence 17 of 23 animals survived and 15 of 23 became runted and chronically infected. The formation and evolution of defective particles in the brains of these rats were similar to those found in rats chronically infected after subcutaneous inoculation of reovirus. We conclude that the formation of defective virus particles may play a role in the initiation and maintenance of chronic neutropic infections with reovirus.     

Preliminary characterization of coxsackievirus B3 temperature-sensitive mutants.

Prototype temperature-sensitive (ts) mutants of a coxsackievirus B3 parent virus capable of replication to similar levels at 34 or 39.5 degrees C were examined for the nature of the temperature-sensitive event restricting replication in HeLa cells at 39.5 degrees C. The ts mutant prototypes represented three different non-overlapping complementation groups. The ts1 mutant (complementation group III) synthesized less than 1% of the infectious genomic RNA synthesized by the coxsackievirus B3 parent virus at 39.5 degrees C and was designated an RNA- mutant. Agarose gel analysis of glyoxal-treated RNA from cells inoculated with ts1 virus revealed that cell RNA synthesis continued in the presence of synthesis of the small amount of viral RNA. This mutant was comparatively ineffective in inducing cell cytopathology and in directing synthesis of viral polypeptides, likely due to the paucity of nascent genomes for translation. The ts5 mutant (complementation group II) directed synthesis of appreciable quantities of both viral genomes (RNA+) and capsid polypeptides; however, assembly of these products into virions occurred at a low frequency, and virions assembled at 39.5 degrees C were highly unstable at that temperature. Shift-down experiments with ts5-inoculated cells showed that capsid precursor materials synthesized at 39.5 degrees C can, after shift to 34 degrees C, be incorporated into ts5 virions. We suggest that the temperature-sensitive defect in this prototype is in the synthesis of one of the capsid polypeptides that cannot renature into the correct configuration required for stability in the capsid at 39.5 degrees C. The ts11 mutant (complementation group I) also synthesized appreciable amounts of viral genomes (RNA+) and viral polypeptides at 39.5 degrees C. Assembly of ts11 virions at 39.5 degrees C occurred at a low frequency, and the stability of these virions at 39.5 degrees C was similar to that of the parent coxsackievirus B3 virions. The temperature-sensitive defect in the ts11 prototype is apparently in assembly. The differences in biochemical properties of the three prototype ts mutants at temperatures above 34 degrees C may ultimately offer insight into the differences in pathogenicity observed in neonatal mice for the three prototype ts mutants.     

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.     

Genetic studies of the ploidy of Moloney murine leukemia virus.

An assay for Moloney murine leukemia virus was developed that made use of the production of morphologically altered foci in nonproducer mouse cells (15F) carrying murine sarcoma virus. Wild-type (wt) virus gave a ratio of titers at 39 degrees C/34degrees C = 1.05 +/- 0.45 (standard deviation;n = 20). A spontaneous, thermosensitive (ts) mutant of Moloney murine leukemia virus, ts3, defective in a late viral function, gave 39 degrees C/34degrees C = 0. A murine cell line (TB) was mixedly infected with ts3 and wt (multiplicities of infection, 7.8:4.3), cloned after infection, and shown to be infected by both viruses. At 34 degrees C it produced wt, ts, and particles of mixed parentage. The heterozygotes (hz) had ratios of assays 39 degrees C/34 degrees C = 0.06 to 0.84 (mean, 0.36). To eliminate possible interference by multiploid particles with determination of the proportions of the three types of particles, the virus produced by the mixedly infected, cloned cell line at 34 degrees C was distributed by velocity sedimentation in a sucrose gradient, and virus was picked from the lightest part of the gradient. The proportions of ts, wt, and hz were 0.27, 0.26, and 0.47. Those particles identified as hz segreated ts, wt, and hz in the proportions 0.24, 0.27, and 0.49, respectively. These values were not significantly different from those predicted from a diploid model of the genome.     

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.     

Temperature-sensitive mutants of vesicular stomatitis virus are conditionally defective particles that interfere with and are rescued by wild-type virus.

Temperature-sensitive (ts) mutants of vesicular stomatitis virus belonging to complementation groups I, II and IV inhibited the replication of wild-type vesicular stomatitis virus when mixed infections were carried out in BHK21 cells at 32, 37, and 39.5 C. The group IV mutant (ts G 41) was most effective in this regard; wild-type virus yields were inhibited almost 1,000-fold in mixed infections with this mutant at 32 C. In the case of group I and II mutants, inhibition of wild-type virus replication at 37 and 39.5 C was accompanied by an enhancement (up to 15,000-fold) of the yields of the coinfecting ts mutant. The yields of the group IV mutant (ts G 41) were not enhanced by mixed infections with wild-type virus at any temperature, although this mutant inhibited wild-type virus replication at all temperatures. The dominance of the replication of ts mutants at 37 C provides a rationale for the selection and maintenance of ts virus in persistently infected cells.     

Physiological characterization of temperature-sensitive mutants of mengovirus.

Twenty-four temperature-sensitive mutants of mengovirus were characterized physiologically with respect to phenotype. The mutants were separated into four classes on the basis of viral RNA synthesis. L-67-S cells infected with five of the mutants synthesized little viral RNA at 39.5 C. These mutants are designated RNA-. One mutant is designated RNA* since its RNA synthesis is altered at both 39.5 and 31.5 C. The other mutants were divided into two groups, RNA plus or minus (25 TO 49% of wild-type RNA synthesis) and RNA plus (50 to 100% of wild-type RNA synthesis). The time of expression of the mutation in the RNA- mutants was estimated from the results of reciprocal temperature-shift experiments. The mutatation in ts12 appears to be expressed at the time RNA synthesis normally begins. The defect in three of the mutants was expressed 1 to 2 h before RNA synthesis is normally detectable. Protein synthesis is required before RNA synthesis begins when the cells are shifted from 39.5 to 31.5 C. The RNA polymerase synthesized by cells infected with these RNA- mutants at 31.5 C was stable and fully active when assayed at 39.5 C in vitro. The sedimentation profiles of the viral RNA synthesized by cells infected with RNA plus and RNA plus or minus mutants are similar to wild-type profiles with the exception of ts148. Cells infected with this RNA plus or minus mutant synthesize RNA that sediments in a sucrose gradient like replicative-intermediate RNA, but little mature viral RNA is evident. The results of step-up experiments indicate that the temperature-sensitive period for the majority of the RNA plus and RNA plus and minus mutants extends through most of the replicative cycle. The temperature-sensitive defect of four of the mutants, however, was expressed in the first hour, suggesting that some undefined early function is required for the eventual maturation of mengovirus. The virions of three of the RNA- mutants were more thermolabile than wild-type virions. Five of the RNA plus and RNA plus or minus mutants were also thermolabile. Genetic complementation at a significant level was not detectable in mixed infections of the mutants described.     

Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus.

The synthesis and processing of virus-specific precursor polypeptides in NIH/3T3 cells infected at the permissive temperature (31 degrees C) with temperature-sensitive (ts) mutants of Rauscher murine leukemia virus was studied in pulse-chase experiments at the permissive and nonpermissive (39 degrees C) temperatures. The newly synthesized virus-specific polypeptides were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera against Rauscher murine leukemia virus proteins. In cells infected with ts mutants defective in early replication steps (the early mutants ts17 and ts29), and ts mutants defective in postintegration steps (the late mutants ts25 and ts26), the processing of the primary gag gene product was impaired at the nonpermissive temperature. gag-pr75 of all four mutants was converted into gag-pr65; however, gag-pr65 accumulated at the nonpermissive temperature, and the main internal virion polypeptide p30 was not formed. Therefore, the proteolytic cleavage is blocked beyond gag-pr65. Concomitantly, the formation of the env gene-related polypeptide p12(E) of all four mutants was blocked at the restrictive temperature. In contrast, cells infected with the late mutant ts28, which produced noninfectious virions at 39 degrees C, showed a normal turnover of the gag and env precursor polypeptides.