Association of a Baculovirus-Encoded Protein with the Capsid Basal Region

It has been shown that the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) 38K (ac98) is required for nucleocapsid assembly. However, the exact role of 38K in nucleocapsid assembly remains unknown. In the present study, we investigated the relationship between 38K and the nucleocapsid. Western blotting using polyclonal antibodies raised against 38K revealed that 38K was expressed in the late phase of infection in AcMNPV-infected Spodoptera frugiperda cells and copurified with budded virus (BV) and occlusionderived virus (ODV). Biochemical fractionation of BV and ODV into the nucleocapsid and envelope components followed by Western blotting showed that 38K was associated with the nucleocapsids. Immunoelectron microscopic analysis revealed that 38K was specifically localized to the nucleocapsids in infected cells and appeared to be distributed over the cylindrical capsid sheath of nucleocapsid. Yeast two-hybrid assays were performed to examine potential interactions between 38K and nine known nucleocapsid shell-associated proteins (PP78/83, PCNA, VP1054, FP25, VLF-1, VP39, BV/ODV-C42, VP80, and P24), three non-nucleocapsid shell-associated proteins (P6.9, PP31, and BV/ODV-E26), and itself. The results revealed that 38K interacted with the nucleocapsid proteins VP1054, VP39, VP80, and 38K itself. These interactions were confirmed by coimmunoprecipitation assays in vivo. These data demonstrate that 38K is a novel nucleocapsid protein and provide a rationale for why 38K is essential for nucleocapsid assembly.

Section: Discussionmentioning

confidence: 69%

mentioning

confidence: 99%

Autographa californica Multiple Nucleopolyhedrovirus 38K Is a Novel Nucleocapsid Protein That Interacts with VP1054, VP39, VP80, and Itself

Liang

Kan

et al. 2008

“…To confirm that the interactions predicted by yeast two-hybrid analysis reflected interactions which occurred in vivo, blue native gel and Western blot analysis was performed using fractions prepared from infected cells (28 h p.i.). This analysis showed that C42 and p78/83 (p78/83 is also a structural protein of baculovirus [18,21]) comigrate, consistent with complex formation, and that they are present in two complexes, a complex at 180 kDa and a larger complex (approximately 500 to 600 kDa) that also contains EC27.…”

Section: Discussionmentioning

confidence: 79%

Identification of BV/ODV-C42, an Autographa californica Nucleopolyhedrovirus orf101 -Encoded Structural Protein Detected in Infected-Cell Complexes with ODV-EC27 and p78/83

Braunagel

Guidry

Rosas-Acosta

et al. 2001

orf101 is a late gene of Autographa californica nucleopolyhedrovirus (AcMNPV). It encodes a protein of 42 kDa which is a component of the nucleocapsid of budded virus (BV) and occlusion-derived virus (ODV). To reflect this viral localization, the product of orf101 was named BV/ODV-C42 (C42). C42 is predominantly detected within the infected-cell nucleus: at 24 h postinfection (p.i.), it is coincident with the virogenic stroma, but by 72 h p.i., the stroma is minimally labeled while C42 is more uniformly located throughout the nucleus. Yeast two-hybrid screens indicate that C42 is capable of directly interacting with the viral proteins p78/83 (1629K) and ODV-EC27 (orf144). These interactions were confirmed using blue native gels and Western blot analyses. At 28 h p.i., C42 and p78/83 are detected in two complexes: one at approximately 180 kDa and a high-molecular-mass complex (500 to 600 kDa) which also contains EC27.Occlusion-derived virus (ODV) of Autographa californica nucleopolyhedrovirus (AcMNPV) initiates primary infection through gut cells of the susceptible insect, where the columnar cells are the primary sites of infection (13, 31). Columnar cells, however, are in a differentiated state (7). Thus, AcMNPV must have the ability to progress the infected columnar cell from G 0 to G 1 and further progress the cell to a phase conducive to viral DNA replication (S phase). These events occur rapidly: in virus-challenged Trichoplusia ni larvae, midgut infection is detectable by 4 h postinoculation and systemic infection is detected by 12 h (31).Viral regulation of the host cell cycle could be accomplished through several mechanisms. One mechanism is the early transcription-translation of viral genes which interact with cellular proteins or protein complexes. Some evidence suggests that AcMNPV may be utilizing this mechanism. The ie-2 gene is sufficient to arrest transfected Sf9 cells in S phase: the arrested cells do not undergo mitosis, have abnormally large nuclei, and contain greater than 4N DNA content (19). Infected Sf9 cells, however, show only a transient increase of cells in S phase, and the cells quickly progress to G 2 /M phase, where they remain arrested throughout infection (4). A second viral strategy to regulate the host cell cycle is to present, as structural components of the virus, a protein(s) or protein complexes that interact with cellular proteins immediately upon infection. For AcMNPV, the structural protein ODV-EC27 (EC27) is a candidate for such a strategy: it has amino acid similarity with cellular cyclins, and it coprecipitates with cellular cyclin kinases (1, 4). Its introduction into the cell at the time of infection may allow it to interact immediately with cellular cyclin kinases or function as a cyclin homologue in a manner analogous to that of other viral cyclin homologues. In our continuing study of the function of AcMNPV structural proteins and their interactions with host cell proteins, we have identified a new structural protein of budded virus (BV) and ODV, BV/ODV-C42 (C42; or...

“…Sequence analysis confirmed that the construct was in frame. The fusion protein was expressed, isolated, and used for inducing rabbit polyclonal antiserum as previously described (35). The immune serum used in this report was collected 8 days after the fifth boost.…”

Section: Methodsmentioning

confidence: 99%

Identification of the Lymantria dispar Nucleopolyhedrovirus Envelope Fusion Protein Provides Evidence for a Phylogenetic Division of the Baculoviridae

Pearson

Groten

Rohrmann

2000

144

132

The complete genome sequences of a number of diverse members of the Baculoviridae including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of Autographa californica multinucleocapsid NPV (AcMNPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses, Lymantria dispar MNPV The Baculoviridae are a large family of occluded, rod-shaped viruses with circular, supercoiled, double-stranded DNA genomes of 100 to 180 kb depending on the virus strain. Two baculovirus genera have been described; the nucleopolyhedroviruses (NPVs) (34) have multiple virions present in large polyhedron-shaped occlusion bodies, whereas the granuloviruses (GVs) (42) normally display a single nucleocapsid embedded in a small granular occlusion body. NPVs are characterized by the production of two virion forms, or phenotypes (40). Occlusion-derived virions are found in occlusion bodies and initiate infection in midgut cells upon ingestion by susceptible hosts. In contrast, the budded virus (BV) form is not occluded, is produced early in infection, and spreads the infection within the infected insect prior to occlusion body production. Although the nucleocapsids for the two virion forms appear to have similar polypeptide contents, the compositions and structures of their envelopes appear to be distinct (7). BV infection by both Autographa californica multinucleocapsid NPV (AcMNPV) and Orgyia pseudotsugata MNPV (OpMNPV) has been investigated, and a glycoprotein called GP64 has been found to be associated with the BV form of both these viruses (3, 39, 41). GP64 is expressed both early and late in infection and is transported to and incorporated into the cell membrane. As nucleocapsids bud through the cell membrane and exit the cell, they become enveloped in the GP64-modified cell membrane. GP64 appears to be required for the spread of the infection to other cells and for the virus to exit from an infected cell (23,26,28). BV enters cells via an endocytic pathway, and, upon acidification of the endocytic vesicle, the structure of GP64 is altered and fusion of the viral and endosomal membranes occurs (17,20,22). This results in the release of the nucleocapsid into the cytoplasm. Surprisingly, GP64 is closely related to the envelope fusion protein of the Thogoto virus genus of the Orthomyxoviridae, which are minus strand RNA viruses (24,25).Recently the sequences of the 161-kb Lymantria dispar MNPV (LdMNPV) (18), 136-kb Spodoptera exigua MNPV (15), and 178-kb Xestia c-Nigrum GV (XcGV) (11) genomes have been reported. A striking feature of these genomes is the lack of an identifiable homolog to gp64. The fact that the life cycle of LdMNPV is similar to those of AcMNPV and OpMNPV suggested that it must express a different envelope fusion protein. Computer-assisted analyses of the LdMNPV genome identified a single 676-amino-acid product of LdMNPV open reading frame (ORF) 130 (ld130), which was predicted to have both a ...