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

Abstract

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...

Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Protein BV/ODV-C42 Mediates the Nuclear Entry of P78/83

“…Deletion analysis demonstrated that c42 is an essential gene for nucleocapsid assembly, whereas no interruption in viral genomic DNA replication was observed (20). Previous reports also provide evidence that C42 binds to P78/83 in a nucleocapsid-independent manner in infected cells (1). Therefore, we hypothesize that one of the pathways through which C42 participates in BV propagation is via interaction with P78/83.…”

“…Multiple sequence comparison showed that the NLS motif in C42, which belongs to the pat4 type, is one of the most conserved sequences in all the homologs of C42 either in group I or group II nucleopolyhedrovirus (1,8). Although the molecular mass of C42 (approximately 41.5 kDa) is very close to the 40-kDa limit, which is the empirical threshold for energy-free diffusion through the nuclear pore (8), the nuclear distribution pattern of C42 is absolutely dependent on the NLS motif at its C terminus.…”

“…BV/ODV-c42 (orf101; c42) is a highly conserved, viral late gene which encodes a 42-kDa viral nucleocapsid protein (1). A putative nuclear localization signal (NLS) ( 357 KRKK 360 ) motif was found at the C terminus of C42 and is thought to be associated with C42's subcellular distribution pattern in AcMNPV-infected cells (1).…”

“…A putative nuclear localization signal (NLS) ( 357 KRKK 360 ) motif was found at the C terminus of C42 and is thought to be associated with C42's subcellular distribution pattern in AcMNPV-infected cells (1). Deletion analysis demonstrated that c42 is an essential gene for nucleocapsid assembly, whereas no interruption in viral genomic DNA replication was observed (20).…”

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

“…A construct of the ODV-EC43 deletion produced BVs, but the virions were non-infectious (Alfonso et al, 2012;Fang et al, 2003Fang et al, , 2009). ODV-EC27 was reported to be a type of viral-encoded cyclin homologue, and could interact with C42 and p78/83 (Belyavskyi et al, 1998;Braunagel et al, 2001). The DNA-binding protein P6.9 was identified in the ODVs of all baculoviruses; the majority of P6.9 was distributed near the inner nuclear membrane, whilst a portion localized in the virogenic stroma throughout the course of infection.…”