Distribution and Phosphorylation of the Basic Protein P6.9 of Autographa californica Nucleopolyhedrovirus

Abstract

A protamine-like protein named P6.9 is thought to play a role in the condensation of genomes of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) during an infection. Previous studies have shown that P6.9 is phosphorylated immediately upon synthesis and dephosphorylated upon the entry of the P6.9-DNA complex into the capsid. Here, we investigate the dynamic distribution of P6.9 in AcMNPV-infected Spodoptera frugiperda cells using an influenza virus hemagglutinin (HA)-tagged P6.9. Although a portion of P6.9-HA localized to the virogenic stroma, which is the center of viral DNA replication, transcription, and nucleocapsid assembly, the majority of P6.9-HA was distributed near the inner nuclear membrane throughout the course of infection. Antiserum against P6.9 detected specific phosphorylated forms of P6.9 at the edge of, but not within, the electron-dense matte regions of the virogenic stroma. Further analysis using immunoblotting revealed that at least 11 different phosphorylated forms of P6.9, as well as dephosphorylated P6.9, were present in association with occlusionderived virions, although only dephosphorylated P6.9 was associated with budded virions.

The Autographa californica Multiple Nucleopolyhedrovirus ac54 Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

“…However, the capsid structures resulting from loss of the aforementioned genes were located not only in the RZ but also in the VS (interspace of the electron-dense mats) (16,(18)(19)(20)22), whereas in vAc54KO-transfected cells, no capsid structures were observed in the VS (Fig. 2B).…”

“…In addition, abnormal electron-dense bodies were found in the VS, and empty capsid structures were found in the RZ in cells transfected with vAc54KO, indicating that ablation of ac54 disrupted the normal assembly of nucleocapsids. In addition to ac54, some other AcMNPV genes (i.e., 38K, ac53, ac83, pk-1, and p6.9) have also been implicated in nucleocapsid assembly, and their individual deletion results in the cessation of assembly and the appearance of empty capsid structures in both the RZ and VS (16)(17)(18)(19)(20)22). However, in the present study, neither capsid structures nor VP39 was observed inside the VS in Sf9 cells transfected with vAc54KO, implying a special role of VP1054 in the baculovirus life cycle besides nucleocapsid assembly.…”

“…Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most intensively studied baculovirus. In addition to the major capsid protein VP39 (11,12), AcMNPV nucleocapsids contain several minor components (6,13), such as the following: P6.9, a protamine-like protein that is responsible for the condensation of viral DNA to form the nucleocapsid core (14)(15)(16); PK-1, a virus-encoded protein kinase that is required for the hyperphosphorylation of P6.9, which facilitates the release of viral DNA from the nucleocapsid (17); and 38K, Ac53, VP91 (encoded by ac83), and VLF-1, which are also classified as minor capsid proteins although their exact functions are poorly understood (18)(19)(20)(21). Single deletion of the genes encoding each of the six minor capsid proteins mentioned above results in the failure of nucleocapsid assembly and the distribution of abnormally long electron-lucent tubular structures at the electrondense edges of the stroma, indicating that the packaging of DNA and nucleoproteins into capsids is interrupted (16,(18)(19)(20)(21)(22).…”

Proteomic analysis of the occlusion-derived virus of Clostera anachoreta granulovirus

“…Furthermore, many different dephosphorylated forms of P6.9, as well as phosphorylated P6.9, were present in association with ODVs, although only dephosphorylated P6.9 was associated with BVs . P6.9 was also detected in the BV proteomes of HearNPV and AcMNPV, but not in the BV proteome of AgMNPV, which might be a result of P6.9 cleavage (Braconi et al, 2014;Liu et al, 2012). The biological significance of the cleavage of P6.9 remains unclear.…”

Autographa californica Multiple Nucleopolyhedrovirus orf132 Encodes a Nucleocapsid-Associated Protein Required for Budded-Virus and Multiply Enveloped Occlusion-Derived Virus Production

“…To explore the mechanism by which ac132 influences assembly and envelopment of nucleocapsids, experiments were performed to detect interaction between AC132 and other viral proteins. The candidate proteins selected for examination included the major capsid protein VP39 (39), the viral genome-binding protein P6.9 (41,42), BV and ODV envelope proteins E25 (40) and E18 (43), VS-associated proteins IE1 (44) and PP31 (45), and BV/ODV-C42, involved in virus-induced nuclear actin polymerization (46). The extracts of Sf9 cells infected by AcMNPV were immunoprecipitated with AC132-specific antibodies.…”