In recent years, the number of studies of the function of conserved baculovirus genes has increased with the development of bacmid technology, facilitating the generation of viruses null for genes of interest and evaluations of their phenotypes in infected or bacmid DNA-transfected cells. We sought to characterize ac79 since it is conserved in many baculoviruses, and orthologs are found in other viruses and bacteria. ac79 is not an essential gene for virus replication, and viruses lacking ac79 were able to produce infectious BVs () and ODVs that were infectious in insects (data not shown). However, it is required for efficient levels of infectious BV production, since the deletion of ac79 resulted in about a 10-fold decrease in levels of infectious BV. Since ac79 is not conserved in all baculovirus genomes, its function may be partially redundant with that of other viral or host genes, or it may provide an advantage to virus replication in some hosts.
ac79 is an early gene based on its expression being independent of DNA replication, its timing of expression, and its transcription initiation being at a site other than a late or very late consensus start site. ac79 does not appear to affect late or very late protein accumulation, although we did not test if the onset of gene expression at any phase was affected. Ac79 was detected from 6 to 96 h p.i. (A). It localized to the nucleus and cytoplasm (C) but accumulated in the nucleus as the time course of infection progressed, suggesting a nuclear-specific role during virus replication. The appearance of tubular structures containing capsid proteins in the nucleus of cells infected with Ac79KO-PG supports a nuclear role.
Viruses lacking ac79 produced reduced levels (about 10-fold) of infectious BV; however, viral DNA replication and late and very late gene expression appeared to be unaffected, suggesting the completion of the replication cycle. In addition, the timing of the onset of infectious BV production was similar for all viruses. Together, these observations suggest that Ac79 may have a role in facilitating events after viral DNA replication, such as proper capsid assembly or virion maturation, the efficient transport of nucleocapsids from the nucleus to the cytoplasm, or virus budding from the cell.
Previous studies showed that the deletion of the AcMNPV gp64
, or pp31
gene results in a reduction in the level of BV production but that viral DNA replication remains unaffected (7
). Although these mutants share similar phenotypes, the specific functions of each gene in the process of BV production may differ. Defects in BV production could be due to defective virus egress; impaired nucleocapsid structure integrity; or alterations in the transport of nucleocapsids within the cell, packaging, or virus assembly. The lack of ac79
did not appear to affect the egress of BV from the cell or the transport of nucleocapsids from the nucleus to the cytoplasm, since infectious and noninfectious virions were released ( and ). In addition, consistent with the finding that Ac79 was not a structural component of the BV or ODV (D and E), we found that the lack of ac79
did not affect the gross morphology of budded virions released from the cells (C). This is supported by previous studies that did not identify Ac79 as a component of BV by proteomic methods (28
Ac79KO-PG-infected cells produced tubular VP39-containing structures along the inner nuclear membrane. Interestingly, previous studies that described mutations in the ac53
, and alkaline nuclease genes also described similar tubular sheaths (18
). This phenotype has been associated with nucleocapsid transport, nucleic acid resolution, or packaging defects. In addition, capsid protein-containing tubular structures have been observed following treatment with cytochalasin D, a microfilament elongation inhibitor, even though viral DNA synthesis was not affected (27
). The interference of cytochalasin D with capsid assembly indicated that microfilaments were involved in this nuclear process (27
). Given that defects in different genes result in similar phenotypes, it makes it difficult to determine if ac79
functions in any of these processes or has another function.
A previous study suggested that Ac79 may be related to bacterial DNA repair UvrC excision endonucleases and intron-encoded endonucleases, based on the presence of the Uri motif (1
). To explore this further, we compared the Ac79 peptide sequence to sequences in protein structure databases using HHpred v 2.0 (4
). The results showed significant predicted structural similarities between Ac79 and UvrC, bacteriophage T4 endonuclease II, and the I-TevI intron-encoded endonucleases with the GIY-YIG domain (data not shown), indicating structural parallels between these endonucleases and Ac79. It is not known if Ac79 binds DNA, a function that is localized at the C terminus of GIY-YIG endonucleases (25
), or if it has endonuclease function; however, structural similarities, along with the presence of key residues important for nuclease function, suggest that Ac79 is related to these endonucleases.
To test the importance of residues conserved between Ac79 and GIY-YIG-containing endonucleases, we constructed viruses with mutations in the conserved GIY-YIG-corresponding motif (Ac79 amino acids Y24 and G26) or in residues predicted to participate in endonucleolytic catalysis (Ac79 amino acids R34 and E72). None of the mutants showed tubular capsid-like structures similar to those observed in Ac79KO-PG-infected cells. It is possible that the elongated structures were caused by the presence of the undeleted N-terminal fragment of Ac79 (amino acids 1 to 38) in Ac79KO-PG, which contained the conserved tyrosines and the RX3H sequence, which may have hindered the activity of cellular or viral proteins necessary for proper nucleocapsid formation. However, this N-terminal peptide does not have a dominant negative function, since it is also present in the amino acid point mutants. Among the viruses with point mutations in Ac79, only the virus with the conservative E72D change showed reduced BV production. This glutamic acid was also deleted in Ac79KO-PG, suggesting a role for this amino acid in productive virus infection. Curiously, we did not observe reduced BV production when Y24 and G26 or R34 were mutated. It is possible that these mutations were repaired by recombination events, with the N-terminal 38 amino acids remaining at the ac79 locus, even though our experiments were carried out with transfected bacmid DNA to minimize homologous recombination events during reiterative virus replication cycles. Further work is needed to determine the requirement of Y24, G26, and R34 in infectious BV production. It appears that the tubular capsid protein-containing structures observed in Ac79KO-PG-infected cells are not required for the defect in infectious BV production. Although the E72D mutation suggests that endonucleolytic activity is important for infectious BV production, additional experiments will be required to further define this role.
Mutations in baculovirus DNA replication and processing genes result in altered capsid protein-containing structures, suggesting that viral DNA affects the nucleocapsid architecture (23
). We tested whether there is an interaction between Ac79 and VLF-1, which is involved in viral genome processing, hypothesizing that Ac79 may provide the endonuclease activity needed during genome processing and packaging, but we could not detect an interaction by coimmunoprecipitation (our unpublished data). Although Ac79KO-PG produces elongated capsid protein-containing structures, it also produces normal nucleocapsids in both occluded and budded virions. It is premature to speculate whether Ac79 functions similarly to UvrC, cleaving phosphodiester bonds during DNA repair, or to T4 endonuclease II, degrading host DNA to reutilize nucleotides for its DNA synthesis, or whether it functions in creating double-strand breaks characteristic of a homing endonuclease like I-TevI endonuclease. Further work is needed to determine if Ac79 has endonucleolytic activity and to further define its role during BV production.