Genetic analysis of murine hepatitis virus strain JHM.

We performed a genetic analysis of 37 temperature-sensitive mutants of murine hepatitis virus strain JHM. Of our mutants, 32 did not induce murine hepatitis virus-specific RNA synthesis in infected cells at the restrictive temperature, 39 degrees C. By complementation testing we have identified at least seven nonoverlapping complementation groups. Six of the genes identified in this way are required for murine hepatitis virus-specific RNA synthesis. The seventh complementation group is made up of five mutants which induced virus-specific RNA synthesis at 39 degrees C.     

Isolation and characterization of temperature-sensitive mutants of Sendai virus

Sixteen temperature-sensitive mutants of Sendai virus were isolated from mutagenized stocks (10 mutants, designated numerically) and persistently infected cultures (6 mutants, designated alphabetically). Based on complementation tests, virion-associated activities, thermal inactivation, and viral RNA and hemadsorbing antigen synthesis as well as virion production in chick lung embryo cells at nonpermissive temperature, these mutants were divided into seven groups as follows. i) HANA group mutants (ts-5, -9, -10, -201), defective in hemagglutinin-neuraminidase protein, complementation group I. ii) F group mutants (ts-18, -108), defective in hemolytic and cell-fusing activity, complementation group II. iii) Ts-43, defective in RNA polymerase activity, complementation group III. iv) Ts-23, defective in RNA polymerase activity, interfered with the other mutants in complementation tests. v) Ts-25, defective in the incorporation of hemagglutinin-neuraminidase protein into the virion at the stage of virus assembly. vi) Ts-110, belongs to F group mutants on one hand, but is considered to carry another undetermined defect. vii) C group (carrier culture-borne group) mutants (ts-a, -b, -c, -d, -e, -f), defective lesion not yet determined and belong to neither complementation group I nor II. Assignment of mutants in groups iv), v), vi), and vii) to complementation groups could not be achieved.     

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.     

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.     

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.     

Temperature-sensitive mutants of frog virus 3: biochemical and genetic characterization

Nineteen frog virus 3 temperature-sensitive mutants were isolated after mutagenesis with nitrosoguanidine and assayed for viral DNA, RNA, and protein synthesis, as well as assembly site formation at permissive (25 degrees C) and nonpermissive (30 degrees C) temperatures. In addition, mutants were characterized for complementation by both quantitative and qualitative assays. Based on the genetic and biochemical data, the 19 mutants, along with 9 mutants isolated earlier, were ordered into four phenotypic classes which define defects in virion morphogenesis (class I), late mRNA synthesis (class II), viral assembly site formation (class III), and viral DNA synthesis (class IV). In addition, we used two-factor crosses to order 11 mutants, comprising 7 complementation groups, onto a linkage map spanning 77 recombination units.     

Isolation and characterization of temperature-sensitive mutants of measles virus.

Nine temperature-sensitive (ts) mutants of nonattenuated Edmonston strain measles virus were isolated from wild-type virus which was grown in the presence of 5-fluorouracil. Adsorption, temperature shift, and complementation experiments indicated that all these mutants were restricted at an intracellular stage of infection. However, all the mutants were more rapidly inactivated at 41 C than was wild-type virus, suggesting that the ts product of each mutant either influences or is a structural component of the virus. Three complementation groups were found to be represented among the mutants. Group A contained one mutant and it did not induce synthesis of detectable amounts of viral antigen at the nonpermissive temperature (39 C). Group B consisted of six mutants which did not induce viral antigen synthesis at 39 C and one mutant which did. Group C was represented by one mutant and it induced viral antigen synthesis at 39 C. The two mutants which induced sythesis of viral antigen also induced synthesis of relatively small amounts of virus-specific RNA at 39 C. These mutants, while producing cytoplasmic and nuclear accumulations of viral antigen at 39 C, were restricted in production of syncytia and hemadsorption. All the mutants were less neurovirulent than wild-type virus, as indicated by their inability to produce acute disease in newborn hamsters.     

Isolation and Characterization of Sendai Virus Temperature-Sensitive Mutants

Ten temperature-sensitive mutants of Sendai virus, a paramyxovirus, were isolated and partially characterized. The mutants replicated in chicken embryo lung cells at 30 C, but not at 38 C; wild-type virus grew equally well at both temperatures. Complementation tests divided the mutants into seven groups. Six groups synthesized neither infectious virus nor RNA when incubated at 38 C from the beginning of infection. Temperature shift-up experiments demonstrated that three of these complementation groups were blocked in early steps required for RNA synthesis, but these gene functions were not needed throughout the replicative cycle. In contrast, the other three RNA-negative complementation groups were defective throughout the replicative cycle in functions required for virus-specific RNA synthesis. Only one mutant, which complemented all of the above, synthesized RNA but not infectious virus when placed at 38 C; the hemagglutinin of this mutant functioned only at the permissive temperature.     

Genetic Characteristics of Conditional Lethal Mutants of Vesicular Stomatitis Virus Induced by 5-Fluorouracil, 5-Azacytidine, and Ethyl Methane Sulfonate

One hundred and seventy-five temperature-sensitive (ts) mutants of vesicular stomatitis virus (type Indiana-C) induced by 5-fluorouracil (FU), 5-azacytidine (ACR), and ethyl methane sulfonate (EMS) have been assigned to four complementation groups by a qualitative test. Group I contains 151 mutants; group II, 2 mutants; group III, 1 mutant; and group IV, 15 mutants; 6 are unclassified. FU was much more effective as a mutagen than either ACR or EMS. The proportion of the mutants belonging to groups I and IV, however, was similar in the case of all three mutagens. Fifteen mutants from groups I and IV have been used to obtain quantitative complementation data. Both groups appear to be homogeneous. Complementation yields increase with increasing multiplicity, but the number of particles per cell required to elicit maximal complementation is small. The pattern of genetic recombination parallels that of complementation. No recombination could be detected in crosses within group I (<0.001%) or group IV (<0.07%), whereas recombination (0.31 to 3.4%) was observed in crosses between groups I and IV. Recombination frequency did not increase with multiplicity above an input of 0.6 plaque-forming units per cell. Many group I mutants have very low reversion rates, and BHK 21 clone 13 cells infected with one of these mutants have been "cured" of infection by prolonged exposure at the restrictive temperature.     

Selection and Preliminary Characterization of Temperature-Sensitive Mutants of Type 5 Adenovirus

Eight temperature-sensitive (ts) mutants that replicate normally at 32 C but poorly, if at all, at 39.5 C have been isolated from mutagenized stocks of a wild-type strain of type 5 adenovirus. Three mutagens were employed: nitrous acid, hydroxylamine, and nitrosoguanidine. Ts mutants were isolated from mutagenized viral stocks with frequencies between 0.01 and 0.1%. All eight mutants had reversion frequencies of 10(-5) or less. Complementation experiments in doubly infected cultures at the nonpermissive temperature separated the mutants into three nonoverlapping complementation groups. Complementation yields ranged from a 2.3- to a 3,000-fold increase over the sums of the yields from the two singly infected controls. Genetic recombination was also demonstrated; approximate recombination frequencies ranged from 0.1 to 15%. Preliminary biochemical and immunological characterization of the mutants indicated that: (i) the single mutant in complementation group I did not replicate its deoxyribonucleic acid (DNA) or synthesize late proteins at the nonpermissive temperature but did inhibit host DNA synthesis to 25% of an uninfected control; (ii) the four group II mutants replicated viral DNA, shut off host DNA synthesis, synthesized penton base and fiber, but did not synthesize immunologically detectable hexon; the three mutants in complementation group III synthesized viral DNA, shut off host DNA synthesis, and made immunologically reactive capsid proteins (hexon, penton base, and fiber).     

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.     

Isolation and preliminary characterization of temperature-sensitive mutants of measles virus.

Twenty-four genetically stable temperature-sensitive mutants of measles virus were isolated after mutangenesis by 5-azacytidine, 5 fluorouracil, or proflavine. The restricted replication of all mutants at 39 C was blocked subsequent to cell penetration and could not be attributed to heat inactivation of virus infectivity. Complementation analysis was made possible through the use of poly-L-ornithine. The members of one complementation group exhibited wild-type RNA synthesis at the nonpermissive temperature and induced the synthesis of virus antigens. These mutants were found defective in both hemolysin antigen synthesis and cell fusion "from within," supporting the unitary hypothesis for these functions. The members of the other two complementation groups synthesized neither virion RNA nor detectable virus antigens at the nonpermissive temperature.     

Temperature-sensitive defect of vesicular stomatitis virus in complementation group II.

The prototype member of the complementation group II temperature-sensitive (ts) mutants of vesicular stomatitis virus, ts II 052, has been investigated. In ts II 052-infected HeLa cells at the restrictive temperature (39.5 degrees C), reduced viral RNA synthesis was observed by comparison with infections conducted at the permissive temperature (30 degrees C). It was found that for an infection conducted at 39.5 degrees C, no 38S RNA or intracytoplasmic nucleocapsids were present. For nucleocapsids isolated from ts II 052 purified virions or from ts II 052-infected cells at 30 degrees C, the RNA was sensitive to pancreatic RNase after an exposure at 39.5 degrees C in contrast to the resistance observed for wild-type virus. The nucleocapsid stability of wild-type virus when heated to 63 degrees C or submitted to varying pH was not found in nucleocapsids extracted from ts II 052 purified virions. The data suggest that for ts II 052 there is an altered relationship between the viral 38S RNA and the nucleocapsid protein(s) by comparison with wild-type virus. Such results argue for the complementation group II gene product being N protein, so that the ts defect in ts II 052 represents an altered N protein.