Isolation and preliminary characterization of temperature-sensitive mutants of Newcastle disease virus

Abstract

Temperature-sensitive (ts) mutants of Newcastle disease virus have been isolated and characterized genetically (complementation), biochemically (RNA synthesis), and biologically (fusion from within and hemadsorption). Fifteen of these mutants have been divided into five complementation groups. Groups A (five mutants) and E (one mutant) are ts for RNA synthesis (RNA-) as well as for the other functions. Group B contains four RNA+ mutants of which one is ts for fusion, one for hemadsorption and two for neither function. Group C contains one RNA+ mutant which is a poor cell fuser. Group D contains two RNA+ mutants which are ts for fusion. In addition, two noncomplementing mutants (group BC) fail to complement both group B and group C mutants while exhibiting complementation with mutants in groups A, D, and E.

Isolation and Characterization of Temperature‐Sensitive Mutants of Sendai Virus

“…After 8 days of incubation at 31 C, well-isolated plaques were picked with capillary tubes, suspended in a small amount of MEM and seeded onto CEF monolayers for further plaquing at 31 C and 39 C. Well-isolated plaques that developed at 31 C were then picked from those samples which either had a much lower PFU titer at 39 C than at 31 C or had formed "pin-hole" size plaques at 39 C (33) and ordinary ones at 31 C. The putative ts mutants selected in this manner were further purified by at least two successive single-plaque isolations in CEF cells at 31 C. Immediately before plaquing, virus samples were sonicated at 20 kc for 5 sec and then filtered through a Millipore filter of 0.45-,a porosity to eliminate virion aggregates. Final determination as a ts mutant was based on significantly reduced virus production at 39 C relative to that at 31 C or "pin-hole" plaques at 39 C (33) in contrast to plaques of ordinary size, at 31 C (Table 1). Ten ts mutants were obtained and were given numerical designations .…”

Intracellular processing of the paramyxovirus F protein: critical role of the predicted amphipathic alpha helix adjacent to the fusion domain

“…The ts mutants had been selected from individually cloned stocks of AV and grouped by complementation (50). Revertants were previously selected from individually cloned ts mutant stocks by their abilities to form plaques at the nonpermissive temperature (39).…”

“…Each nucleotide difference resulted in a deduced amino acid change ( Table 2). Mutants D2 and D3 have the same nucleotide change, despite the fact that they were independently isolated (50).…”

“…The group D temperature-sensitive (ts) mutants (50) were isolated from the Australia-Victoria (AV) (3) strain of NDV. genes are functional, and can produce hemadsorbing activity (38), indicating that its HN glycoprotein is functional.…”

“…genes are functional, and can produce hemadsorbing activity (38), indicating that its HN glycoprotein is functional. Therefore, the group D lesions are probably in one of the two remaining genes, M or F. Evidence for alteration in both proteins or their behaviors has been described: the M protein of mutant Dl migrates more rapidly upon electrophoresis (39), mutant Dl does not induce fusion from within at a nonpermissive temperature (50), and the F,+F2 glycoprotein is lacking in virions of all three group D mutants produced at a nonpermissive temperature (39).…”