Temperature-sensitive Mutants of Vesicular Stomatitis Virus; Homology and Nomenclature

In vitro transcriptase activity of three group I temperature-sensitive (ts) mutants of vesicular stomatitis virus restricted at 39 C was restored by Lprotein fractions derived from wild-type (wt) vesicular stomatitis virion nucleocapsids. Soluble NS protein from wt nucleocapsids did not reconstitute restricted transcription of the group I RNAts mutants. NS protein activity, but not L protein activity, was purified from the group I ts mutants; this NS fraction always displayed the wt phenotype in reconstitution assays. Neither the L nor the NS protein was capable of restoring the defective transcriptive activity of the group IV vesicular stomatitis virus mutant tsW16B.

Section: Methodssupporting

confidence: 80%

mentioning

confidence: 99%

RNA- temperature-sensitive mutants of vesicular stomatitis virus: L-protein thermosensitivity accounts for transcriptase restriction of group I mutants

Hunt¹,

Emerson²,

Wagner³

1976

“…Over this base layer was poured a suspension of 107 L cells in the same agar medium which was allowed to solidify. Some plates were also seeded with 4 x 106 L cells freshly infected with tsGll at an MOI of 5 in the top layer in addition to the uninfected cells. The plaques to be tested were stabbed with a sterile toothpick, and the toothpick was then stabbed into identical positions on three plates of L cells in agar suspension, the last plate containing tsGll-infected cells.…”

Section: Pringlementioning

confidence: 99%

“…Complementation in high-MOI-infected monolayer cultures. The complementation groups of ts mutants were determined by a modification of the method of Cormack et al (4). Monolayer cultures of 2.5 x 10' L cells were mixedly infected with pairs of ts mutants, each at an input MOI of 5.…”

Section: Pringlementioning

confidence: 99%

Screening procedure for complementation-dependent mutants of vesicular stomatitis virus

Rettenmier¹,

Dumont²,

Baltimore³

1975

To isolate new types of vesicular stomatitis virus (VSV) mutants, a four-stage screen was developed which identifies and characterizes mutants capable of complementing the defect in the VSV temperature-sensitive mutant tsGll. Two types of mutants of VSV, Indiana serotype, have been found by using the screen; they are new temperature-sensitive mutants which are, of necessity, not in complementation group I and mutants which do not produce plaques under conditions of single infection at 31 C (the normal permissive temperature) and are, therefore, called complementation-dependent mutants. The newly isolated, temperature-sensitive mutants fall into three complementation groups, two of which are congruent with known complementation groups; the newly identified group extends to six the number of complementation groups of VSV Indiana. The nature of the complementation-dependent mutants has not been established, but one was shown to not contain a significant deletion in its nucleic acid.

“…Temperature-sensitive (ts) mutants (11,13,19) of VS virus have been isolated in Glasgow (G group), Orsay (O group), and Winnipeg (W group) and provide a sensitive probe for deline-ating the physiological parameters of viral transcription. Mutants of complementation groups I and IV (7,12,19) are classified as RNA-owing to marked reduction of' RNA synthesis in vivo at restrictive temperatures (17,20,26). Definitive evidence has also been provided for defective transcription at the restrictive temperature for certain group I mutants (6,8,21,22).…”

mentioning

confidence: 99%

Location of the Transcription Defect in Group I Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Hunt

Wagner

1974

The ribonucleoprotein-dependent RNA transcriptase in vesicular stomatitis B virions of four temperature-sensitive (ts) mutants belonging to complementation group I was analyzed in vitro at permissive (31 C) and restrictive (39 C) temperatures. The RNA-synthesizing activity of all four ts mutants was more labile at 39 C than was the transcriptive activity of wild-type (wt) virions. In order to locate the temperature-sensitive transcription defect in the mutants, wt and ts mutant virions were fractionated by Triton X-100-high salt solubilizer into a sedimentable ribonucleoprotein template and a nonsedimentable enzyme fraction, each of which alone had little or no transcriptive activity. The templateand enzyme-containing fractions of wt virions were then tested for their capacity to restore transcriptive activity at 39 C to corresponding template and enzyme preparations of ts mutant virions. Recombination of' wt template and ts enzymes resulted in no significant restoration of capacity to synthesize RNA at restrictive temperature. In contrast, transcriptive function at 39 C was reconstituted by recombining the wt enzyme with the template component of ts mutants. It appears, therefore, that the enzyme, rather than the template, is the temperature-sensitive component of the transcription complex of group I vesicular stomatitis virus mutants.