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Minus strand RNA synthesis by the positive strand alphaviruses, Sindbis and Semliki Forest viruses, normally occurs early in infection, is coupled to synthesis of viral nonstructural proteins and to formation of viral replication complexes, and terminates and does not occur late in infection. Previously, ts24 of the A complementation group of Sindbis virus RNA-negative mutants was found to possess, among its other temperature sensitive defects, a temperature sensitivity in the normal cessation of minus strand synthesis which enabled minus strands to be synthesized late in infection at 40 degrees in the absence of protein synthesis. Revertants of ts24 (ts24R1, ts24R2) retained the defect in the shutoff of minus strand synthesis, indicating the lesion was not conditionally lethal and could map outside the A cistron. The studies reported here used an infectious clone of Sindbis virus to identify the mutation responsible for this phenotype. Hybrid viruses were prepared from constructs containing restriction fragments of the cDNA of ts24R1 in place of the corresponding fragments in the infectious SIN HR clone and screened for their ability to synthesize minus strands at 40 degrees in the presence of cycloheximide. A unique base change of an A for a C residue at nt 6339, predicting a change from glutamine to lysine at amino acid 195 in nsP4, was found in genomes of ts24, ts24R1, and ts24R2. Other nucleotide changes present at the 5' and 3' termini did not affect minus strand synthesis. The substitution of the parental Sindbis virus sequence that encompassed nt 6339 in an infectious clone of the ts24R1 revertant eliminated the mutant phenotype. We conclude that the ability to continue minus strand synthesis at 40 degrees exhibited by ts24 and its revertants is caused by an alteration in nsP4, which is the alphavirus replicase or an essential component of the replicase. We hypothesize that this domain of nsP4 functions to fix the minus strand as the stable template of alphavirus replication complexes.

The 10 members of the A complementation group of temperature-sensitive (ts) mutants of SIN HR, the heat-resistant strain of Sindbis virus, were divided into two phenotypic subgroups. Subgroup I mutants (ts15, ts17, ts21, ts24, and ts133) demonstrated temperature-sensitive 26 S mRNA synthesis, whereas subgroup II mutants (ts4, ts14, ts16, ts19, and ts138) did not; both ts4 and ts19 demonstrated defective 26 S mRNA synthesis at 30 degrees. None of the A group mutants demonstrated temperature-sensitive 49 S plus-strand synthesis. Temperature-sensitive cleavage of ns230 was demonstrated by subgroup I mutants, except ts21, but not by subgroup II mutants. A revertant of ts133 that grew at 40 degrees retained temperature-sensitive 26 S mRNA synthesis but lost temperature-sensitive cleavage of ns230 and the RNA-negative phenotype. Only ts4, like ts11 of the B complementation group, demonstrated temperature-sensitive minus-strand RNA synthesis. In addition to ts24, cells infected with ts17 or ts133 continued to synthesize minus strands after shiftup in the absence of continued protein synthesis, and resumed synthesis of minus strands if shifted to the nonpermissive temperature after minus-strand synthesis had ceased at the permissive temperature.

We determined the effect of the overproduction of viral nonstructural proteins on alphavirus plus-strand and minus-strand RNA synthesis. Because alphavirus minus-strand synthesis ceases normally at 3 to 4 hr postinfection and requires continuous protein synthesis [D. L. Sawicki and S. G. Sawicki, J. Virol. 34, 108-118 (1980); D. L. Sawicki, S. G. Sawicki, S. Keranen, and L. Kaariainen, J. Virol. 39, 348-358 (1981a)], we determined if the cessation of minus-strand synthesis was the result of the failure to continue synthesis of viral nonstructural proteins after 3-4 hr postinfection and if the overproduction of viral nonstructural proteins would increase the rate of plus-strand synthesis. Cells infected with ts1, an RNA-positive mutant of Semliki Forest virus (SFV) which overproduced the viral nonstructural proteins and underproduced the viral structural proteins at the nonpermissive temperature, did not cause the synthesis of increased amounts of viral minus strands relative to parental SFV and did not affect the time at which minus-strand synthesis ceased. All four viral nonstructural proteins were synthesized at early and late times after infection in the same relative proportions. The overproduction and the continued synthesis of nonstructural proteins late in infection did not increase the maximal rate of plus-strand synthesis above that in wild-type SFV-infected cells.

During the replication cycle of Sindbis virus minus-strand synthesis stops normally at the time that plus-strand synthesis reaches a maximum rate. We have isolated and characterized revertants of ts24, a member of the A complementation group of Sindbis HR mutants, that we had demonstrated previously to have a temperature-sensitive defect in the regulation of minus-strand synthesis. These revertants of ts24 replicated efficiently at 40 degrees but nevertheless retained the temperature sensitive defect in the regulation of minus-strand synthesis: they continued to synthesize minus strands late in the replication cycle at 40 degrees but not at 30 degrees and in the presence or absence of protein synthesis. Although failure to regulate the synthesis of minus strands resulted in continuous minus-strand synthesis and in the accumulation of minus strands, the rate of plus-strand synthesis was not increased concertedly. Minus strands synthesized after the rate of plus-strand synthesis had become constant were demonstrated to be utilized as templates for 26 S mRNA synthesis. Thus, the change from an increasing to a constant rate of plus-strand synthesis during the alphavirus replication cycle cannot be governed solely by the number of minus strands that accumulate in infected cells. We present a model for the preferential utilization of minus strands as a mechanism for the cessation of minus-strand synthesis that occurs normally during alphavirus replication.