Functional Analysis Of Autographa Californica Multiple Nucleopolyhedrovirus Ac130 (gp16) And Ac132

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf130 (ac130, gp16) and orf132(ac132) are located within a cluster of five ORFs(orf129 to133), which are oriented in the same direction on the genome. ac130 encodes a 16 kDa glycosylated protein, named GP16. gp16 gene has homologs in the genomes of all Group I and most Group II alphabaculoviruses sequenced to date. ac132 has homologs in all genome-sequenced group I alphabaculoviruses. Its role in the baculovirus replication cycle is unknown. In this article, we investigated molecular biology characteristics and functions in viral replication of gp16 and ac132 and the proteins they encode.In the study of AcMNPV gpl6, gp16-knockout and repair mutants were constructed by using a λ-Red homologous recombination system and the Bac-to-Bac system. The mutants were used to transfect or infect Sf9 cells. Effects of gp16 deletion on BV reproduction, morphogenesis, DNA replication and late gene expression in infected cells and infectivity to host larvae were evaluated. In addition, the subcellular location of the GP16 in the cells infected by wild type AcMNPV was characterized.It was shown that no significant difference in virus growth curves and BV production was observed among the wild type (wt), gp16 knockout and gp16 repair viruses. Quantitative PCR analysis of viral DNA replication in Sf9 cells infected by gp16 knockout, gp16 repair or wt virus showed that viral DNA replication was not affected by deletion of gp16. SDS-PAGE and western blot assays performed to examine expression of AcMNPV VP39 and polyhedrin in Sf9 cells infected by gp16 knockout or gp16 repair virus showed that the levels of both VP39 and polyhedrin detected in extratracts of infected cells were for gp16 knockout and gp16 repair virus, at all time points post infection. It suggested that deletion of gp16 did not have significant effect on late gene expression. Transmission electron microscopy (TEM) of the thin sections of the infected cells showed that virus morphogenesis in the cells infected with gp16 knockout virus was similar to that in the cells infected with the gp16 repair virus. The results demonstrate that, gp16 is nonessential for virus replication.The infectivity of gp16 knockout, gp16 repair and wt viruses was tested for newly molted third-instar Spodoptera exigua larvae by feeding the larvae with viral OBs. The results found no significant difference in Median lethal dose between the gp16 knockout and the wt or gp16 repair virus, but the Median Survival Time of gp16 knockout mutant was 6 h longer than that of the wt and the gp16 repair virus, at least.In an earlier study, GP16 of Orgyia Pseudotsugata multiple nucleopolyhedrovirus was found to be associated with aggregates of lamellar membrane-like structures located in the cytoplasm near the nuclear membrane. In this study, to determine the subcellular localization of GP16, a recombinant virus vAcgp16ko-rep-gfp was constructed in which EGFP was fused to the GP16 and was expressed under the control of the gpl6 native promoter. Confocal microscopy showed that the GP16-EGFP fusion protein located close around the nuclear membrane in the cells infected by vAcgp16ko-rep-gfp. In subcellular fractionation of the infected Sf9 cells, AcMNPV GP16 was detected mainly in the light membrane fraction. TEM of the ultrathin sections of infected cells showed that many newly assembled nucleocapsids were wrapped into vesicles across the nuclear envelope. These results suggest that GP16 protein may locate on golgi apparatus in the infected cells, and may be involved in releasing of the nucleocapsids from the nucleus and associated with trafficking of nucleocapsids through cytoplasm.Similarly, AcMNPV mutants with orfl29-132 or ac132 deleted from the viral genome were constructed by using the λ-Red homologous recombination system and Bac-to-Bac system. Since orf129, orf131 and gp16 had be known to be nonessential for virus replication, we constructed ac132 repair mutants basing on the orfl29-132 and ac132 knockout mutants. The mutants were used in transfection/infection of Sf9 cells, respectively, to investigate the role ac132 played in virus replication.In the Sf9 cell cultures transfected or infected by either the orfl29-132 knockout or ac132 knockout virus, infection was restricted to single cells, but the infectious BVs were produced in the cells transfected with the ac132 repair mutants. The growth curves of the two ac132 repair mutants were similar to the one of the wt virus. The results imply that ac132 is essential for BV production.SDS-PAGE and western blot assays of the proteins in extracts of the cells transfected by ac132 knockout or ac132 repair mutants, with antisera separately against AcMNPV VP39 and E25, showed that the levels of accumulation of VP39 and E25 were similar for ac132 knockout and ac132 repair viruses at all time points post transfection, suggesting that deletion of ac132 did not have significant effect on late gene expression. TEM showed that the formation of the virogenic stroma and occlusion bodies was delayed, the numbers of enveloped nucleocapsid were reduced, and the occlusion bodies contained mainly singly enveloped nucleocapsids, in the cells transfected with the ac132 knockout bacmid, compared to the cells transfected with the ac132 repair bacmid.To determine if AC132 was a structural protein, BV and ODV purified from AcMNPV infected Sf9 culture were fractionated into envelope and nucleocapsid fractions, separately, and analyzed by SDS-PAGE and western blot, with AcMNPV AC132-specific antiserum. As a result, AC 132 was detected in the fraction of nucleocapsid of both BV and ODV, not in the envelope fractions, showing that AC132 was nucleocapsid associated protein in both ODV and BV. The size of AC132 was around 28kDa.In a time course analysis of AC132 in AcMNPV-infected Sf9 cells, AC132 was first detected in the extracts of infected cells at 12 h post infection (hpi), and the peak level of accumulation occurred at 24 hpi.Confocal immunofluorescence microscopy showed that AC132 protein was first observed at 12 hpi, predominantly in the cytoplasm of the cells infected by AcMNPV. A few small dots of AC132 were also viewed at the center of the nucleus at this time. By 24 hpi, AC 132 was mainly observed in the nucleus, forming a condensed ring zone at the periphery of the nucleus. By 72 hpi, it was only present in the nucleus.To explore the mechanism by which ac132 influences replication of virus, we tried to detect interaction between AC132 and other viral proteins. The candidate proteins selected for screening included the major capsid protein VP39, virogenic stroma-associated protein 39K, BV and ODV envelope protein El 8, and the viral genome-binding protein P6.9. The lysates of Sf9 cells infected by AcMNPV were immunoprecipitated with AC132-specific antibodies. AC132-specific antibodies detected a band of 28 kD, and ODV-E18-specific antibodies detected a band of 18 kD on western blots of proteins coprecipitated with AC132-specific polyclonal antibodies, P6.9-specific antibodies detected a weak band of P6.9, while polyclonal antibodies specific to other proteins did not detect bands of the corresponding proteins. These data imply that AC 132 may be involved in nucelocapsid assembly through interaction with P6.9, it may also affect envelopment of nucelocapsid through interaction with E18.