Transactivator IE1 Is Required for Baculovirus Early Replication Events That Trigger Apoptosis in Permissive and Nonpermissive Cells

Abstract

Immediate early viral protein IE1 is a potent transcriptional activator encoded by baculoviruses. Although the requirement of IE1 for multiplication of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is well established, the functional roles of IE1 during infection are unclear. Here, we used RNA interference to ablate IE1, plus its splice variant IE0, and thereby define in vivo activities of these early proteins, including gene-specific regulation and induction of host cell apoptosis. Confirming an essential replicative role, simultaneous ablation of IE1 and IE0 by gene-specific double-stranded RNAs inhibited AcMNPV late gene expression, reduced yields of budded virus by more than 1,000-fold, and blocked production of occluded virus particles. Depletion of IE1 and IE0 had no effect on early expression of the envelope fusion protein gene gp64 but abolished early expression of the caspase inhibitor gene p35, which is required for prevention of virus-induced apoptosis. Thus, IE1 is a positive, gene-specific transactivator. Whereas an AcMNPV p35 deletion mutant caused widespread apoptosis in permissive Spodoptera frugiperda cells, ablation of IE1 and IE0 prevented this apoptosis. Silencing of ie-1 also prevented AcMNPV-induced apoptosis in nonpermissive Drosophila melanogaster cells. Thus, de novo synthesis of IE1 is required for virus-induced apoptosis. We concluded that IE1 causes apoptosis directly or contributes indirectly by promoting virus replication events that subsequently trigger cell death. This study reveals that IE1 is a gene-selective transcriptional activator which is required not only for expedition of virus multiplication but also for blocking of its own proapoptotic activity by upregulation of baculovirus apoptotic suppressors.

Baculoviruses Modulate a Proapoptotic DNA Damage Response To Promote Virus Multiplication

“…(i) dsRNA production. pBluescript K/S ϩ (Invitrogen) plasmids containing portions of the enhanced green fluorescence protein (GFP) gene (egfp) or the AcMNPV genes ie-1, p143, lef-1, or p47 were described previously (54,55). Portions of Drosophila atm (GenBank accession number NM_001043247) and atr (GenBank accession number NM_078645) open reading frames (ORFs) were PCR amplified using DL-1 genomic DNA and the following primer sets: ATM #1, 5=-CGAGC TCCGGCAATGCGAAAAATCTCCG-3= and 5=-GGGTACCCGATCTC AAAGTTAAGGCCTCGC-3= (ORF nucleotides 1305 to 2005); ATM #2, 5=-GGAGCTCAGCGTTCTGCTGGAAGATGC-3= and 5=-AGGTACCG GTCAATTGGTTGGCGGC-3= (ORF nucleotides 6097 to 6834); and ATR, 5=-CGAGCTCGAGAGCTGCCTAAGTGAACCG-3= and 5=-AGGT ACCGATGGCTCCCAGTTCTCCG-3= (ORF nucleotides 2911 to 3639).…”

“…Transfection with dsRNA and plasmid DNA. RNA silencing (RNAi) was conducted by using gene-specific double-stranded RNA (dsRNA) as described previously (55). After transfection with dsRNA mixed with cationic liposomes consisting of N- [1-(2, 3 dioleoyloxy)propyl]-N,N, N-trimethylammonium methyl sulfate-L-␣-phophatidylethanolamine, dioleoyl (C 18:1 , [cis]-9) (DOTAP-DOPE), DL-1 cells were maintained as monolayers for 2 days at 27°C, after which they were harvested, counted, and treated as indicated.…”

Baculovirus DNA Replication-Specific Expression Factors Trigger Apoptosis and Shutoff of Host Protein Synthesis during Infection

“…RNA silencing is an effective means by which to selectively reduce viral or cellular gene expression during infection of invertebrates (13,35,46,51). An important advantage to our approach was the capacity to evaluate the function of individual genes during replication of fully infectious virus after normal receptor-mediated entry.…”

“…However, it was unclear whether DNA replication is the direct trigger or if virus DNA synthesis is indirectly involved because it promotes late multiplicative events, which could trigger apoptosis. The AcMNPV immediate-early protein IE1 is also necessary for virus-induced apoptosis (46). Because this transcriptional activator is required for viral DNA replication and replication-dependent late gene expression (43,46,48), it may trigger apoptosis by initiating DNA synthesis or promoting downstream multiplicative events.…”

“…The AcMNPV immediate-early protein IE1 is also necessary for virus-induced apoptosis (46). Because this transcriptional activator is required for viral DNA replication and replication-dependent late gene expression (43,46,48), it may trigger apoptosis by initiating DNA synthesis or promoting downstream multiplicative events. Alternatively, IE1 may directly activate host pro-death genes, perturb the cell cycle, or inhibit host biosynthetic pathways, all of which are sufficient to cause apoptosis (reviewed in references 18, 24, 29, 30, and 57).…”