Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication

Abstract

The gene encoding the 35-kDa protein (35K gene) located within the EcoRI-S genome fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) is transcribed early in infection. To examine its function(s) with respect to virus multiplication, we introduced specific mutations of this early gene into the AcMNPV genome. In Spodoptera frugiperda (SF21) culture, deletion of the 35K gene reduced yields of extracellular, budded virus from 200to 15,000-fold, depending on input multiplicity. Mutant replication was characterized by dramatically diminished levels of late and very late (occlusion-specific) virus gene expression and premature cell lysis. In contrast, 35K gene inactivation had no effect on virus growth in cultured Trichoplusia ni (TN368) cells. Insertion of the 35K gene and its promoter at an alternate site (polyhedrin locus) restored virus replication to wild-type levels in SF21 culture. Subsequent insertion of 4 bp after codon 81 generated a frameshift mutant that exhibited a virus phenotype indistinguishable from that of 35K deletion mutants and demonstrated that the 35K gene product (p35) was required for wild-type replication in SF21 cells. Mutagenesis also indicated that the C terminus of p35, including the last 12 residues, was required for function. In complementation assays, wild-type virus bearing a functional 35K gene allele stimulated all aspects of 35K null mutant replication and suppressed early cell lysis. These findings indicated that p35 is a trans-dominant factor that facilitates AcMNPV growth in a cell line-specific manner.

Neurovirulence in mice of soluble receptor-resistant (srr) mutants of mouse hepatitis virus: intensive apoptosis caused by less virulent srr mutant

“…Apoptotic cell death could play an important role in host defense mechanism for eliminating infected cells. In support of this hypothesis, some viruses have acquired genes that block apoptosis, such as p35 and iap genes of baculovirus [3,12]. Apoptosis may be an important antiviral mechanism in cells that can regenerate from surviving cells, however, it may result in functional disorders in the non-regenerative cells, such as neuronal cells.…”

Expression of the Baculovirus p35 Gene Inhibits Mammalian Neural Cell Death

“…The mechanism by which p35 mediates such an effect is unknown. p35 has been shown to inhibit baculovirus-induced apoptosis in a transdominant fashion (Hershberger et al, 1992). During infection of apoptosis-sensitive insect cells, p35 is targeted primarily to the cytoplasmic compartment, but -10% is associated with the nuclear fraction; p35 does not copurify with mitochondria, heavy membranes, or light membranes (Hershberger et al, 1993).…”

“…The p35 gene encoded by Aulographa californicu nuclear polyhedrosis virus (AcMNPV) is required to block premature death of cultured Spodoptera frugiperda cells (IPLB-SF21) caused by virus-induced apoptosis (Clem et al, 1991). Suppression of apoptosis is cell line specific and is correlated with increased virus replication and dramatically higher yields (200-1 5,000-fold) of progeny virus (Hershberger et al, 1992). Although p35 and Bcl-2 proteins both function to suppress apoptosis, no sequence similarities be-tween the two genes has yet been detected.…”

A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors.

“…Insect baculoviruses contain at least two independent genes that promote the survival of the infected host cell, p35 (Friesen and Miller, 1987;Clem et al, 1991;Hershberger et al, 1992Hershberger et al, , 1994Clem and Miller, 1993;Kamita et al, 1993) and iap (Crook et al, 1993;Birnbaum et al, 1994). Recombinant baculoviruses lacking both genes induce accelerated host cell death leading to severely impaired virus production (Clem and Miller, 1994b).…”