VP39 of Spodoptera litura multicapsid nucleopolyhedrovirus cannot efficiently rescue the nucleocapsid assembly of vp39-null Autographa californica multiple nucleopolyhedrovirus

Background Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vp39 is conserved in all sequenced baculovirus genomes. In previous studies, VP39 has been identified as the major capsid structure protein of baculoviruses and found to be essential for nucleocapsid assembly. The nucleocapsid composition and structure of Group I and II NPVs of the Alphabaculovirus genus are very similar. It is not clear whether the major capsid structure protein VP39 of Group I NPVs is functionally identical to or substitutable with the Group II NPV VP39. In this study, the function of Group II Spodoptera litura MNPV (SpltMNPV) VP39 in Group I AcMNPV was characterized. Methods Sequence alignment of AcMNPV VP39 and SpltMNPV VP39 was performed using Clustal X and edited with GeneDoc. To determine whether VP39 of Group I NPVs can be functionally substituted by Group II NPV VP39, a vp39-null AcMNPV (vAcvp39KO) and a vp39-pseudotyped AcMNPV (vAcSpltvp39:FLAG), in which the Group I AcMNPV vp39 coding sequence was replaced with that of SpltMNPV from Group II NPVs, were constructed via homologous recombination in Escherichia coli. Using an anti-FLAG monoclonal antibody, immunoblot analysis was performed to examine SpltMNPV VP39 expression. Fluorescence and light microscopy were used to monitor viral replication and infection. Viral growth curve analysis was performed using a fifty percent tissue culture infective dose (TCID50) endpoint dilution assay. Viral morphogenesis was detected using an electron microscope. Results Sequence alignment indicated that the N-termini of AcMNPV VP39 and SpltMNPV VP39 are relatively conserved, whereas the C-terminus of SpltMNPV VP39 lacks the domain of amino acid residues 306–334 homologous to AcMNPV VP39. Immunoblot analysis showed that SpltMNPV VP39 was expressed in vAcSpltvp39:FLAG. Fluorescence and light microscopy showed that vAcSpltvp39:FLAG did not spread by infection. Viral growth curve analysis confirmed a defect in infectious budded virion production. Electron microscopy revealed that although masses of abnormally elongated empty capsid structures existed inside the nuclei of Sf9 cells transfected with vAcSpltvp39:FLAG, no nucleocapsids were observed. Conclusion Altogether, our results demonstrated that VP39 from SpltMNPV cannot efficiently substitute AcMNPV VP39 during nucleocapsid assembly in AcMNPV.

Bombyx mori nucleopolyhedrovirus BmP95 plays an essential role in budded virus production and nucleocapsid assembly

Bombyx mori nucleopolyhedrovirus (BmNPV) BmP95 is a highly conserved gene that is found in all of the baculovirus genomes sequenced to date and is also found in nudiviruses. To investigate the role of BmP95 in virus infection in vitro, a BmP95 deletion virus (vBmP95-De) was generated by homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis indicated that the BmP95 deletion bacmid led to a defect in production of infectious budded virus (BV). However, deletion of BmP95 did not affect viral DNA replication. Electron microscopy showed that masses of aberrant tubular structures were present in cells transfected with the BmP95 deletion bacmid, indicating that deletion of BmP95 affected assembly of the nucleocapsid. This defect could be rescued by insertion of full-length BmP95 into the polyhedrin locus of the BmP95-knockout bacmid but not the N-terminal domain of BmP95. Together, these results showed that full-length BmP95 is essential for BV production and is required for nucleocapsid assembly.