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

Abstract

Apoptosis is an important antivirus defense. To define the poorly understood pathways by which invertebrates respond to viruses by inducing apoptosis, we have identified replication events that trigger apoptosis in baculovirusinfected cells. We used RNA silencing to ablate factors required for multiplication of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Transfection with double-stranded RNA (dsRNA) complementary to the AcMNPV late expression factors (lefs) that are designated as replicative lefs (lef-1, lef-2, lef-3, lef-11, p143, dnapol, and ie-1/ie-0) blocked virus DNA synthesis and late gene expression in permissive Spodoptera frugiperda cells. dsRNAs specific to designated nonreplicative lefs (lef-8, lef-9, p47, and pp31) blocked late gene expression without affecting virus DNA replication. Thus, both classes of lefs functioned during infection as defined. Silencing the replicative lefs prevented AcMNPV-induced apoptosis of Spodoptera cells, whereas silencing the nonreplicative lefs did not. Thus, the activity of replicative lefs or virus DNA replication is sufficient to trigger apoptosis. Confirming this conclusion, AcMNPV-induced apoptosis was suppressed by silencing the replicative lefs in cells from a divergent species, Drosophila melanogaster. Silencing replicative but not nonreplicative lefs also abrogated AcMNPV-induced shutdown of host protein synthesis, suggesting that virus DNA replication triggers inhibition of host biosynthetic processes and that apoptosis and translational arrest are linked. Our findings suggest that baculovirus DNA replication triggers a host cell response similar to the DNA damage response in vertebrates, which causes translational arrest and apoptosis. Pathways for detecting virus invasion and triggering apoptosis may therefore be conserved between insects and mammals.

Pathogenesis of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar and mechanisms of developmental resistance

“…Potentially, haemocytes of 4 48 larvae were virus-infected and undergoing apoptosis before LacZ signalling was detectable. In studies using AcNPV, apoptosis was triggered by replication of viral DNA, as evidenced by DNA polymerase expression (Schultz & Friesen, 2009). In LdMNPV, viral DNA polymerase is first expressed at 7 h p.i.…”

Baculoviruses Modulate a Proapoptotic DNA Damage Response To Promote Virus Multiplication

“…At 12 h after infection, infected cells were collected and total cellular DNA was extracted. AcMNPV DNA was quantified by using quantitative real-time PCR as described previously (54). Samples were normalized for cell equivalents by using the Spodoptera frugiperda sfiap gene.…”

“…(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).…”

“…Through unknown events associated with AcMNPV DNA replication, the principal IAP of Spodoptera is rapidly depleted as a result of instability motifs embedded within its N-terminal leader domain (9,64). Likewise, AcMNPV DNA replication causes depletion of the Drosophila melanogaster IAP (DIAP1), which leads to widespread apoptosis in cultured cells derived from this distantly related insect species (54,64). Thus, baculoviruses provide a unique opportunity to define conserved pathways by which vDNA replication engages the host apoptotic program.…”

“…A critical role for the host DDR in baculovirus-induced apoptosis was suggested by the finding that the prototype baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) triggers apoptosis through events associated with the replication of its 134-kb DNA genome ( Fig. 1) (54,64). The replication of mammalian virus DNA (vDNA) is a well-established initiator of the DDR, which is also a trigger for apoptosis in both vertebrates and invertebrates (reviewed in references 61 and 66).…”