The Baculovirus Uses a Captured Host Phosphatase to Induce Enhanced Locomotory Activity in Host Caterpillars

The baculovirus is a classic example of a parasite that alters the behavior or physiology of its host so that progeny transmission is maximized. Baculoviruses do this by inducing enhanced locomotory activity (ELA) that causes the host caterpillars to climb to the upper foliage of plants. We previously reported that this behavior is not induced in silkworms that are infected with a mutant baculovirus lacking its protein tyrosine phosphatase (ptp) gene, a gene likely captured from an ancestral host. Here we show that the product of the ptp gene, PTP, associates with baculovirus ORF1629 as a virion structural protein, but surprisingly phosphatase activity associated with PTP was not required for the induction of ELA. Interestingly, the ptp knockout baculovirus showed significantly reduced infectivity of larval brain tissues. Collectively, we show that the modern baculovirus uses the host-derived phosphatase to establish adequate infection for ELA as a virion-associated structural protein rather than as an enzyme

High-Frequency Homologous Recombination between Baculoviruses Involves DNA Replication

We determined the frequency of DNA recombination between Bombyx mori nucleopolyhedroviruses (BmNPVs) and between BmNPV and the closely related Autographa californica NPV (AcMNPV) in BmN cells, Sf-21 cells, and larvae of Heliothis virescens. The BmN cells were coinfected with two BmNPVs, one with a mutation at the polyhedrin gene (polh) locus and a second carrying a lacZ gene marker cassette. Eleven different BmNPV mutants carrying the lacZ gene marker at various distances (1.4 to 61.7 kb) from polh were used for the coinfections. The Sf-21 cells and larvae of H. virescens were coinfected with wild-type AcMNPV and 1 of the 11 lacZ-marked BmNPV mutants. In BmN cells, high-frequency recombination was detected as early as 15 h postcoinfection but not at 12 h postcoinfection. At 18 h postcoinfection, the mean frequency of recombination ranged between 20.0 and 35.4% when the polh and lacZ marker genes were separated by at least 9.7 kb. When these marker genes were separated by only 1.4 kb, the mean frequency of recombination was 2.7%. In BmN cells, the mean recombination frequency between two BmNPVs increased only marginally when the multiplicity of infection of each virus was increased 10-fold. In Sf-21 cells and the larvae of H. virescens, the recombination frequency between BmNPV and AcMNPV was ≤1.0%. AcMNPV DNA replication occurred normally after the coinfection of Sf-21 cells. BmNPV DNA replication, however, was not detected, indicating that normal DNA replication by both viruses is required for high-frequency recombination

BmPTPD produces fewer progeny in 5^th instar <i>B. mori</i> and shows a delay in late gene expression in BmN cells.

<p>Production of OBs (A) and BVs (B) in the hemolymph of larvae infected with BmNPV, BmPTPD, BmPTPDR or BmPTP-C119S at 4 d p.i. Data shown are means ± standard deviation (SD) (N = 4). *<i>p</i><0.05, one-way ANOVA with Tukey's post test in comparison to BmPTPD. (C) Western blot analysis of the expression of viral gene products in BmN cells infected with BmNPV, BmPTPD or BmPTPDR. The proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with antibodies that recognize BmNPV early-expressed (DBP, BRO, and LEF3) or late-expressed (V-CHIA) proteins or actin. Similar results were obtained in two independent experiments. Abbreviations: WT, BmNPV; PTPD, BmPTPD; DR, BmPTPDR; and CS, BmPTPD-C119S.</p

Reduced expression of viral genes in tissues of larvae infected with a <i>ptp</i>-disrupted virus.

<p>(A) Heatmaps of viral gene expression in 16 tissues of 5^th instar <i>B. mori</i> infected with BmNPV (WT) or BmPTPD (PTPD). The tissues were dissected from virus-infected larvae at 1, 2, 3, and 4 d p.i., and the expression of the early/late and very late genes <i>gp64</i> and <i>polh</i>, respectively, were quantified by qRT-PCR. Tissues from 5 to 30 larvae were mixed and used for the preparation of cDNAs. Abbreviations: FB, fat body; TR, trachea; BR, brain; CN, central nerve; PG, prothoracic gland; CA, corpora allata; HE, hemocyte; ASG, anterior silk gland; MSG, middle silk gland; PSG, posterior silk gland; MI, midgut; MT, Malpighian tubule; MU, muscle; IN, integument; OV, ovary; and TE, testis. (B) Expression of <i>polh</i> in fat body, trachea, central nerve, and brain. Tissues were dissected from four individual larvae at 4 d p.i. First strand cDNAs were generated from individual larvae and qRT-PCR was performed using primers that targeted the <i>polh</i> gene. Data shown are means ± SD (N = 4). *<i>p</i><0.05, Student's t-test.</p

PTP interacts with ORF1629 in BmNPV-infected cells.

<p>(A) A yeast two-hybrid screen was performed to identify interactions between BmNPV PTP and proteins in BmNPV-infected (12 h p.i.) BmN cells or in epidermal tissues from BmNPV-infected (2 d p.i.) 5^th instar <i>B. mori</i>. This screening identified 5 PTP-interacting clones (12h-3, 12h-11, 12h-4, 12h-16, and 2d-2) by X-gal and 3-AT assays. Clones 12h-3, 12h-11, 12h-4, and 12h-16 were derived from BmNPV-infected BmN cells whereas clone 2d-2 was derived from BmNPV-infected larval <i>B. mori</i>. A legend showing the location of positive standards and PTP-interacting clones (streaked in triplicate) is shown to the right. (B) Interaction of PTP and ORF1629 in BmNPV-infected BmN cells. BmN cells were inoculated with BmNPV (WT) or BmPTPD-wt (D-wt) at an MOI of 5 or mock-infected (mock). BmPTPD-wt expresses FLAG-tagged PTP under an authentic <i>ptp</i> promoter. At 72 h p.i., the cells were harvested and immunoprecipitated with anti-FLAG antibody, and then subjected to western blot analysis (left panels) with anti-ORF1629 antibody or anti-FLAG antibody. The right “Input” panels show western blot analysis using whole cell extracts.</p

Effect of mutation of the BmNPV <i>ptp</i> gene on virus-induced ELA in 5^th instar <i>B. mori</i>.

<p>(A) Schematic representation of the <i>ptp</i> gene locus of wild-type (WT) and mutant BmNPVs. The locations of PCR primers (ptpF1 and ptp_B) used in the genotyping experiments are represented by the arrows. In BmPTPD (PTPD), nucleotides −8 to 377 of the <i>ptp</i> gene are replaced by a <i>hsp</i>70-<i>lac</i>Z gene cassette (3.7 kbp). BmPTPDR (DR) is a repair mutant in which the <i>hsp</i>70-<i>lac</i>Z gene cassette of BmPTPD was replaced with the original BmNPV sequence. BmPTP-C119S (CS) contains a point mutation within the <i>ptp</i> gene which results in an amino acid residue substitution (C119S) within the predicted P-loop motif that is required for phosphatase activity. BmPTP-Y9stop (Y9) and BmPTP-E93stop (E93) contain point mutations within the <i>ptp</i> gene which generate stop codons at Tyr-9 and Glu-93, respectively, but the RNA structures of the respective transcripts are likely to be unchanged. (B) Induction of ELA in 5^th instar <i>B. mori</i> injected with wild-type or mutant BmNPVs. Distances traveled at 90 h p.i. are shown by <i>box-and-whisker diagrams</i>. The boxes represent the median and 25–75 percentile ranges of the distances traveled. The w<i>hiskers</i> indicate the most extreme data points, which were no more than 1.5 times the interquartile range from the <i>boxes</i>. The dots indicate outliers predicted by Prism software. BmPTPD did not induce any ELA during the assay period. *<i>p</i><0.05, Kruskal-Wallis analysis with Dunn's post test in comparison to the value obtained for BmPTPD. The abbreviations of the viruses are the same as in A.</p

PTP is an envelope-associated protein required for the production of normal virions.

<p>(A) Localization of PTP in the envelope and capsid fractions of budded virus. Western blot analysis of envelope (E) and capsid (C) fractions of budded virus (BV) of BmNPV or BmPTPD-wt were performed with anti-FLAG, anti-GP64 or anti-ORF1629 antibodies. (B) Localization of GP64 and ORF1629 in PTP-deficient BV. Western blot analysis of envelope (E) and capsid (C) fractions of BV of BmNPV, BmPTPD, and BmPTPDR were performed with anti-GP64 or anti-ORF1629 antibody.</p