Host specific gene expression of the baculovirus, Spodoptera frugiperda nuclear polyhedrosis virus, in insect cell lines



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Texas Tech University


Baculoviruses (family: Baculoviridae) infect insects with high lethality and specificity and are experimental insecticides for suppressing pest populations in the field. Recently, recombinant DNA constructions of baculoviruses have been successfully utilized for the expression of foreign genes. In order to construct safe and stable new viral strains with desirable properties and to engineer suitable vectors for the expression of foreign genes, it is essential to understand the molecular mechanisms of virulence and specificity of baculovirus infection.

Our laboratory has developed three tissue culture model systems to study host specificity. This research focuses on System II, developed by Carpenter and Bilimoria, in which Spodoptera fingiperda nuclear polyhedrosis virus (Sf MNPV) permissively infects Si fiiigiperda IPLB-21 (SF IPLB-21) cells but semipermissively infects Trichoplusia ni 368 (TN-368) cells. Previous electron microscopy data showed that in the semipermissive infection virogenic stroma is formed, indicating that infection is initiated and some subunits are synthesized (16). However, nucleocapsid formation and subsequent steps are not observed, suggesting a block prior to virus assembly.

In this study, the kinetics of viral DNA and virus-specific polypeptide synthesis in System II have been determined. In permissively infected S^ frugiperda cells, a total of 28 infected cell-specific polypeptides (ICSP) were identified, and 11 of them are structural polypeptides. A model, in which ICSP synthesis is coordinately regulated and sequentially ordered in a cascade fashion (a—>â—>y), was confirmed by preventing polypeptide synthesis at various stages with the metabolic inhibitor, cycloheximide. Blocking of DNA synthesis demonstrated that DNA replication is a requirement for the synthesis of late (y) ICSPs. However, in semipermissively infected X. ni cells only a 97K p ICSP and a 29K y ICSP were detected. In this semipermissive infection, viral DNA replication was initiated but at a suboptimal level; no inhibition of host polypeptide synthesis was detected. In comparison with die permissive infection, viral replication in the semipermissive infection was blocked at or prior to a ICSP synthesis.

Since at least one each of the p and y ICSPs are synthesized in the semipermissive infection, and one of the a ICSPs detected in the permissive infection appears to be a component of the virus particle, I postulate that the latter induces just one of several putative cascade pathways in baculovirus infections.

The results obtained from this host-range study help determine more precisely the stage of restriction in System II and open the way for further experiments aimed at determining the exact molecular mechanism of restriction in the semipermissive infection. These developments will allow the identification of genes or regulatory sequences governing host specificity and allow the intelligent construction of ideal, hybrid "supervirus pesticides" and gene expression vectors.



Deoxyribonucleic acid (DNA) viruses, Gene expression, Baculoviruses, Fall armyworm