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The polydnavirus associated with the parasitic wasp Campoletis sonorensis is injected into the lepidopteran insect, Heliothis virescens, during parasitization, after which viral gene products suppress the cellular immune system of the hosts. Four related cysteine‐rich polydnavirus genes have been identified in parasitized H. virescens larvae and grouped into a family. In this study, we investigated the expression and hemocyte targeting of the cysteine‐rich Vhv1.4 protein. Full‐ length and truncated Vhv1.4 proteins were produced in a bacterial expression system, and the purified proteins were used to raise polyclonal antisera. In immunoblots the Vhv1.4 protein was detected in parasitized insects as early as 6 h and throughout the entire course of parasitism. The Vhv1.4 protein appeared predominantly in the plasma fraction of hemolymph from parasitized larvae, suggesting that this protein is secreted. The Vhv1.4 protein expressed from a recombinant baculovirus was secreted in two lepidopteran cell lines and in larvae injected with the recombinant virus. Digestion with endoglycosidases suggests that the Vhv1.4 protein is glycosylated at multiple N‐glycosylation sites. Immunofluorescence assays showed that the Vhv1.4 protein binds to the hemocytes, most notably the granulocytes, in H. virescens larvae. After binding, the Vhv1.4 protein was internalized, probably by endocytosis. Specific binding of the Vhv1.4 to granulocytes implies an important function in the suppression of host cellular encapsulation response. Arch. Insect Biochem. Physiol. 36:251–271, 1997. © 1997 Wiley‐Liss, Inc.

We have cloned and sequenced a novel alcohol oxidase (Hv-p68) from the filamentous fungus Helminthosporium (Cochliobolus) victoriae that copurifies with mycoviral double-stranded RNAs. Sequence analysis revealed that Hv-p68 belongs to the large family of FADdependent glucose methanol choline oxidoreductases and that it shares significant sequence identity (>67%) with the alcohol oxidases of the methylotrophic yeasts. Unlike the intronless alcohol oxidases from methylotrophic yeasts, a genomic fragment of the Hv-p68 gene was found to contain four introns. Hv-p68, purified from fungal extracts, showed only limited methanol oxidizing activity, and its expression was not induced in cultures supplemented with methanol as the sole carbon source. Northern hybridization analysis indicated that overexpression of Hv-p68 is associated with virus infection, because significantly higher Hv-p68 mRNA levels (10-to 20-fold) were detected in virus-infected isolates compared with virus-free ones. We confirmed by Northwestern blot analysis that Hv-p68 exhibits RNA binding activity and demonstrated that the RNA-binding domain is localized within the N-terminal region that contains a typical ADP-binding ␤-␣-␤ fold motif. The Hv-p68 gene, or closely similar genes, was present in all species of the genus Cochliobolus but absent in the filamentous fungus, Penicillium chrysogenum, as well as in two nonmethylotrophic yeasts examined. This study represents the first reported case that a member of the FAD-dependent glucose methanol choline oxidoreductase family, Hv-p68, may function as an RNA-binding protein. victoriae 190S virus (Hv190SV) has been extensively studied (2-9), and is classified as a definitive member of the genus Totivirus in the family Totiviridae (10, 11). The Hv145SV, on the other hand, has only been subjected to limited biochemical characterization (2). Because of similarity in size and number of dsRNA segments between the Hv145SV and viruses in the genus Chrysovirus in the family Partitiviridae, the Hv145SV was tentatively classified as a member of the genus Chrysovirus (12). We have recently (13) isolated a cellular protein, Hv-p68, that copurifies with viral dsRNA from H. victoriae isolates infected with both Hv190SV and Hv145SV. Additionally, Hv-p68 was demonstrated to be present as a minor component in the viral capsids (13). Our initial biochemical characterization studies of Hv-p68 indicated that it occurs in vivo as an octamer and that it is consistently present in higher levels in virus-infected H. victoriae isolates than in virus-free ones. We have also demonstrated by a gel retardation assay that Hv-p68 has RNA binding activity (13).In the present study, we report the isolation and complete nucleotide sequence of a cDNA clone of Hv-p68. We present evidence that Hv-p68 belongs to the FAD-dependent GMC family of oxidoreductases and that it has high sequence similarity to the alcohol oxidases of methylotrophic yeasts. Furthermore, we show that overexpression of Hv-p68 is associated with virus infection and that...

A cellular protein that co-purifies with mycoviral dsRNA was isolated from the plant pathogenic fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae) infected with two viruses, the totivirus Helminthosporium victoriae 190S virus and the chrysovirus-like Helminthosporium victoriae 145S virus (Hv145SV). The cellular protein, which was, designated Hv-p68, accumulated to higher levels in virus-infected isolates compared to virus-free ones. The majority of the Hv145S dsRNAs were found in association with Hv-p68 and not packaged in virions. Hv-p68 could also be detected as a minor component of the virus capsid. Evidence is presented that Hv-p68 occurs in vivo as an octamer and that it possesses RNA-binding activities. Based on partial amino acid sequence analysis, Hv-p68 was shown to share significant sequence identity with alcohol oxidases from methylotrophic yeasts. Hv-p68 is proposed to play a role in viral RNA packaging/replication and in regulating viral pathogenesis.

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