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

Functional Relationships within the New Jersey Serotype of Vesicular Stomatitis Virus: Genetic and Physiological Comparisons of the Hazelhurst and Concan Subtypes

“…The ts lesion in the Hazelhurst mutant A1 has been assigned to the N protein on the basis of thermolability in vitro, degradation at the restrictive temperature in vivo, and altered electrophoretic mobility of Kennedy-Morrow & Lesnaw, 1984;Lesnaw & Dickson, 1978;Belle Isle & Emerson, 1982;Lesnaw et al, 1979), the Concan A complementation group can be assigned to the N gene. On the basis of the demonstration of a thermolabile L protein in the Hazelhurst mutant B t, the L gene has been assigned to the Hazelhurst B complementation group (Lesnaw & Dickson, 1978;Belle Isle & Emerson, 1982).…”

“…Duplicate confluent monolayers of approximately 5 x 106 cells per monolayer were infected at an m.o.i, of 25 and incubated for 6 h, one at 31 °C and one at 39.5 °C, in the presence of [5,6 -3 H]uridine (30 ~Ci/monolayer, New England Nuclear) and actinomycin D (5 ~tg/ml, Calbiochem-Behring). Specific details of the following nucleocapsid isolation procedure have been published previously (KennedyMorrow & Lesnaw, 1984). The contents of each flask (the monolayer plus the overlay medium containing released virions) were frozen, thawed, and separated into supernatant (unbound) and pellet (bound) fractions by low-speed centrifugation.…”

“…Bound fractions (low-speed pellets) were extracted with buffer containing 1% NP40 and 2 M-urea, clarified, and analysed by sucrose density gradient centrifugation in the presence of 0.1~ NP40 and 2M-urea. Gradients were fractionated and assayed for radioactivity by liquid scintillation spectroscopy as described by Kennedy-Morrow & Lesnaw (1984).…”

“…We have recently shown that intracellular nucleocapsids of the Hazelhurst subtype differ from those of the Indiana serotype in that they are not released from infected cells by freezethaw, and we have developed a procedure for their isolation which is based on extraction of freeze-disrupted cells with NP40 and urea (Kennedy-Morrow & Lesnaw, 1984). In order to determine whether this property was unique to the Hazelhurst subtype or a general property of the New Jersey serotype, we examined the distribution of Concan intracellular nucleocapsids and found it to be identical to that of the Hazelhurst nucleocapsids (Fig.…”

“…The ability of the Concan prototype mutants (Table 2) to complement the prototype mutants of four Hazelhurst complementation groups was tested in a formal complementation assay (Table 3). The Hazelhurst mutants D1 and F1 were omitted from this assay because no unique genetic assignment is possible in the case of D1 (Wunner & Pringle, 1974;Pringle et al, 1981 ;Kennedy-Morrow & Lesnaw, 1984) and because F1, like B1, has a defective L protein (Belie Isle & Emerson, 1982 …”