Assignment of the temperature-sensitive lesion in the replication mutant A1 of vesicular stomatitis virus to the N gene.

The replication defect in the temperature-sensitive mutant A1 of the New Jersey serotype (Hazelhurst subtype) of vesicular stomatitis virus was confirmed by the absence of intracellular nucleocapsids in infected cells incubated at the restrictive temperature. After preamplification, the relative yield of the A1 N protein accumulated intracellularly after 1 h of incubation at the restrictive temperature was decreased by 50% that of the wild-type or revertant A1 N protein. This difference was not as apparent in pulse-chase experiments. The functional lesion in A1 was correlated with a structural alteration in the N protein on the basis of the thermolability of the template activity of the A1 N protein-RNA complex in in vitro transcription reactions and the covariance of this phenotype with the temperature-sensitive phenotype in a spontaneous A1 revertant. This correlation was consistent with a direct role of the N protein in replication and allowed the assignment of the N gene to complementation group A.

Functional relationships within the New Jersey serotype of vesicular stomatitis virus: genetic and physiological comparisons of the Hazelhurst and Concan subtypes.

Seventeen temperature-sensitive mutants of the Concan subtype of the New Jersey serotype of vesicular stomatitis virus have been isolated following mutagenesis and assigned to two complementation groups: CC/A, containing three mutants, and CC/B, containing 14 mutants. Prototype mutants of these two Concan groups efficiently complemented prototype mutants of the Hazelhurst complementation groups (with the exception of the corresponding group) which correspond to genes specifying the L, N, M and NS proteins. The pattern of intersubtypic complementation allowed the correlation of the Concan CC/B group with the Hazelhurst B group (L gene) and of the Concan CC/A group with the Hazelhurst A group (N gene). In contrast, the Concan prototype mutants failed to complement the prototype mutant of each of the five Indiana complementation groups for which genetic assignments have been made. The partitioning of intracellular nucleocapsids of the Concan and Hazelhurst subtypes during isolation was identical, and distinct from that of Indiana serotype intracellular nucleocapsids. The M protein of the Concan, but not of the Hazelhurst, subtype was observed to migrate as a doublet on SDS-polyacrylamide gels electrophoresed in a phosphate buffer.

Structural and functional characterization of the RNA-positive complementation groups, C and D, of the New Jersey serotype of vesicular stomatitis virus: assignment of the M gene to the C complementation group.

The structural and functional lesions in the RNA-positive complementation groups, C and D, of the New Jersey serotype (Hazelhurst subtype) of vesicular stomatitis virus have been characterized. The M protein of the temperature-sensitive mutant C1, the prototype of the C complementation group, was degraded at the restrictive temperature in vivo, and was resolved from the wild-type M protein by SDS-polyacrylamide gel electrophoresis and nonequilibrium pH gradient electrophoresis. Coreversion of these properties and the temperature-sensitive phenotype was observed in a spontaneous revertant. On the basis of these results, the M gene was assigned to the C complementation group. Intracellular nucleocapsids could not be isolated from New Jersey serotype infections by procedures developed for Indiana serotype infections. Therefore, in order to assess the ability of New Jersey ts mutants to accumulate nucleocapsids at the restrictive temperature, a procedure for their isolation was developed. Hypertranscription was observed in C1-infected cells incubated at the restrictive temperature, but was not accompanied by proportionate increases in intracellular viral nucleocapsids or protein synthesis. The G and N proteins of the temperature-sensitive mutant D1, the sole representative of the D complementation group, were electrophoretic variants. The relative yield of intracellular D1 N protein was lower at the restrictive than at the permissive temperature, and the D1 L protein was thermolabile. No intracellular viral nucleocapsids were detected in D1 infected cells incubated at the restrictive temperature; however, more 40 S and less message-sized RNA were synthesized at the restrictive than at the permissive temperature. These results suggested functional defects in both the N protein and polymerase of D1.