Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115

Abstract

Per os infectivity factors PIF1 (Ac119) and PIF2 (Ac022), like P74, are essential for oral infection of lepidopteran larval hosts of Autographa californica M nucleopolyhedrovirus (AcMNPV). Here we show that Ac115 also is a PIF (PIF3) and that, unlike PIF1 and PIF2, it does not mediate specific binding of AcMNPV occlusion-derived virus (ODV) to midgut target cells. We used an improved in vivo fluorescence dequenching assay to compare binding, fusion, and competition among control AcMNPV ODV and the ODVs of AcMNPV PIF1, PIF2, and PIF3 deletion mutants. Our results showed that binding and fusion of PIF1 and PIF2 mutants, but not the PIF3 mutant, were both qualitatively and quantitatively different from those of control ODV. Unlike control and PIF3-deficient ODV, an excess of PIF1- or PIF2-deficient ODV failed to compete effectively with control ODV's binding to specific receptors on midgut epithelial cells. Moreover, the levels of PIF1- and PIF2-deficient ODV binding were depressed threefold compared to control levels. Binding, fusion, and competition by PIF3-deficient ODV, however, were all indistinguishable from those of control ODV. These results implicated PIF1 and PIF2 as ODV envelope attachment proteins that mediate specific binding to primary target cells within the midgut. In contrast, PIF3 mediates another unidentified, but critical, early event during primary infection.

FIG. 1.

Construction of plasmids used for making AcMNPV pif gene mutants. A cassette containing the β-galactosidase reporter gene driven by the Drosophila hsp70 promoter was inserted into ORFs Ac022 encoding PIF2 (A), Ac115 encoding PIF3 (B), and Ac119 encoding PIF1 (C) at the locations indicated. Subfragments containing the relevant pif gene were cloned and exposed to pairs of restriction enzymes as follows: EcoRV and NruI for Ac022, BstEII and StuI for Ac115, and BsaBI and NruI for Ac119.

FIG. 2.

(A) Correlation between specific fluorescence of AcMNPV E2 ODV_R and concentration of R18 added to ODV preparations prior to gradient centrifugation. The points represent the average of two samples taken from each treatment. The regression line was fitted by the method of least squares (y = 0.05x + 0.247; r² = 0.98). (B) Mortalities of H. virescens larvae following oral inoculation as newly molted, fourth instars with various doses of AcMNPV E2 ODV and ODV_R. The specific fluorescence of ODV_R was 1.1 × 10⁶ fluorescence units/μg for this experiment. Each point represents the final mortality from a cohort of between 30 and 32 insects. Regression lines were fitted by the method of least squares (ODV, y = 6.78x^0.384, r² = 0.99; ODV_R, y = 9.52x^0.329, r² = 0.92).

FIG. 3.

Percentages of AcMNPV ODV_R that bound and fused with midgut epithelia of newly molted, fourth instar H. virescens orally inoculated with 1.25 μg Achsp70/lacZ ODV_R, 2.1 μg AcΔPIF1, 2.4 μg AcΔPIF2 ODV_R, and 2.1 μg AcΔPIF3 ODV_R. Each histogram bar represents the mean (+1 standard error [SE]) of 10 to 15 larvae.

FIG. 4.

(A) Percentages of Achsp70/lacZ ODV_R that bound and fused with midgut epithelia of newly molted, fourth instar H. virescens orally inoculated with 0.5 μg ODV_R alone and in the presence of 100× unlabeled homologous ODV competitor. Each histogram bar represents the mean (+1 standard error [SE]) of 15 larvae. (B) Percentages of Achsp70/lacZ ODV_R that bound and fused when larvae were inoculated with 0.4 μg Achsp70/lacZ ODV_R alone and in the presence of various amounts of unlabeled AcΔPIF3 ODV competitor. Each histogram bar represents the mean (+1 standard error) of 13 to 16 larvae. (C) Percentages of AcΔPIF3 ODV_R that bound and fused with midgut epithelia of newly molted, fourth instar H. virescens orally inoculated with 0.4 μg AcPIF3 ODV_R alone and in the presence of various amounts of unlabeled Achsp70/lacZ ODV competitor. Each histogram bar represents the mean (+1 standard error) of 13 larvae.

FIG. 5.

Percentages of Achsp70/lacZ ODV_R that bound and fused when newly molted, fourth instar H. virescens were inoculated with 0.4 μg Achsp70/lacZ ODV_R alone and in the presence of various amounts of unlabeled AcΔPIF1 ODV competitor (A) or unlabeled AcΔPIF2 ODV competitor (B). Each histogram bar represents the mean (+1 standard error [SE]) of 13 to 16 larvae.