Comparative replication of Autographa californica nuclear polyhedrosis virus in abortive and productive infections of insect cell lines



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Texas Tech University


Nuclear polyhedrosis viruses (NPVs) are enveloped, rod-shaped insect viruses containing a circular double-strand DNA genome (Family: Baculoviridae). NPV genes are expressed according to a temporally regulated program consisting of four phases: immediate early, delayed-early, late and very late. With the exception of the immediate-early genes, which do not require viral factors for expression, each phase is triggered by genes of a previous class.

The NPVs have become important gene expression vectors and are being developed as biopesticides. The ideal NPV would have high virulence for a broad range of pests and yet be harmless for beneficial insects and nontarget organisms. However, before such strains can be developed by genetic engineering approaches, it is necessary to understand the molecular mechanisms of NPV specificity and virulence.

Our laboratory has developed a tissue culture model system for the study of NPV host specificity. This model is based on earlier observations that Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) does not produce polyhedral inclusion bodies (PIBs) in Bombyx mori (BM) cells, but 90% of the cells contain viral antigen. This constitutes the nonpermissive or abortive component of our model. The permissive or productive component is AcMNPV-infected Spodopterafrugiperda (SF) cells. Previous studies in our laboratory have shown that AcMNPV initiates infection in BM cells and produces unique cytopathic effect (CPE). The CPE was characterized as large "sac-like bodies" at the cell periphery and elongated structures referred to as "protrusions." PIBs were not observed. SDS-PAGE analyses showed that some viral proteins are synthesized in infected BM cells. In addition, novel proteins, not present in the permissive (SF) cells, are also observed.

In the present study, I examined additional aspects of die differential replication of AcMNPV in SF and BM cell lines. I designed this study to see if viral particles of any type are formed in BM cells and whether restriction of virus replication is due to a block at the transcription level. I also tested for replication of viral DNA. Electron microscopy data showed that both cell lines displayed nuclear hypertrophy and virogenic stroma upon infection; however, BM cells did not contain polyhedral inclusion bodies or virions. Nucleocapsids were detected in approximately one of every five cells and were defective. Viral DNA replicated in BM cells but more slowly and at a much lower level than in SF cells. Northem blot analysis showed that: (a) AcMNPV immediate-early genes (IE-1 and pe-38) were transcribed at normal levels at early times post infection in both cell lines; however, in BM cells they were transcribed at higher than expected levels at late times post infection; (b) a delayed-early gene (dnapol) and a late gene which codes for the major component of the nucleocapsid (ccp) were transcribed at relatively low levels in the abortive infection; and (c) a very late gene (polh), responsible for the matrix of PIBs, was transcribed in BM cells at barely detectable levels.

The results strongly suggest that delayed-early, but not immediate-early, genes require host factor(s) for expression, I propose that virus replication in BM cells is primarily restricted between the immediate-early and delayed-early stages and that the block occurs at the transcriptional level; other aspects of sub-optimal gene expression and particle assembly in this abortive infection appear to be consequences of the primary block.



Baculoviruses -- Genetics, Host-virus relationships, Autographa californica, Fall armyworm -- Viruses, Silkworms -- Viruses