The insect baculovirus chitinase (CHIA) and cathepsin protease (V-CATH) enzymes cause terminal host insect liquefaction, enhancing the dissemination of progeny virions away from the host cadavers. Regulated and delayed cellular release of these host tissue-degrading enzymes ensures that liquefaction starts only after optimal viral replication has occurred. Baculoviral CHIA remains intracellular due to its C-terminal KDEL endoplasmic reticulum (ER) retention motif. However, the mechanism for cellular retention of the inactive V-CATH progenitor (proV-CATH) has not yet been determined. Signal peptide cleavage occurs upon cotranslational ER import of the v-cath -expressed protein, and ER-resident CHIA is needed for the folding of proV-CATH. Although this implies that CHIA and proV-CATH bind each other in the ER, the putative CHIA–proV-CATH interaction has not been experimentally verified. We demonstrate that the amino-terminal 22 amino acids (aa) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) preproV-CATH are responsible for the entry of proV-CATH into the ER. Furthermore, the CHIA–green fluorescent protein (GFP) and proV-CATH-red fluorescent protein (RFP) fusion proteins colocalize in the ER. Using monomeric RFP (mRFP)-based bimolecular fluorescence complementation (BiFC), we determined that CHIA and proV-CATH interact directly with each other in the ER during virus replication. Moreover, reciprocal Ni/His pulldowns of His-tagged proteins confirmed the CHIA–proV-CATH interaction biochemically. The reciprocal copurification of CHIA and proV-CATH suggests a specific CHIA–proV-CATH interaction and corroborates our BiFC data. Deletion of the CHIA KDEL motif allowed for premature CHIA secretion from cells, and proV-CATH was similarly prematurely secreted from cells along with ΔKDEL-CHIA. These data suggest that CHIA and proV-CATH interact directly with each other and that this interaction aids the cellular retention of proV-CATH.

me53 is an immediate-early/late gene found in all lepidopteran baculoviruses sequenced to date. Deletion of me53 results in a greater-than-1,000-fold reduction in budded-virus production in tissue culture (J. de Jong, B. M. Arif, D. A. Theilmann, and P. J. Krell, J. Virol. 83:7440-7448, 2009). We investigated the localization of ME53 using an ME53 construct fused to green fluorescent protein (GFP). ME53:GFP adopted a primarily cytoplasmic distribution at early times postinfection and a primarily nuclear distribution at late times postinfection. Additionally, at late times ME53:GFP formed distinct foci at the cell periphery. These foci colocalized with the major envelope fusion protein GP64 and frequently with VP39 capsid protein, suggesting that these cell membrane regions may represent viral budding sites. Deletion of vp39 did not influence the distribution of ME53:GFP; however, deletion of gp64 abolished ME53:GFP foci at the cell periphery, implying an association between ME53 and GP64. Despite the association of ME53 and GP64, ME53 fractionated with the nucleocapsid only after budded-virus fractionation. Together these findings suggest that ME53 may be providing a scaffold that bridges the viral envelope and nucleocapsid.

me53 is a highly conserved baculovirus gene found in all lepidopteran baculoviruses that have been fully sequenced to date. The putative ME53 protein contains a zinc finger domain and has been previously described as a major early transcript. We generated an me53 -null bacmid (AcΔme53GFP), as well as a repair virus (AcRepME53:HA-GFP) carrying me53 with a C-terminal hemagglutinin (HA) tag, under the control of its native early and late promoter elements. Sf9 and BTI-Tn- 5b1 cells transfected with AcΔme53GFP resulted in a 3-log reduction in budded-virus (BV) production compared to both the parental Autographa californica multiple nucleopolyhedrosis virus and the repair bacmids, demonstrating that although me53 is not essential for replication, replication is compromised in its absence. Our data also suggest that me53 does not affect DNA replication. Cell fractionation showed that ME53 is found in both the nucleus and the cytoplasm as early as 6 h postinfection. Deletion of the early transcriptional start site resulted in a 10- to 360-fold reduction of BV yield; however, deletion of the late promoter (ATAAG) resulted in a 160- to 1,000-fold reduction, suggesting that, in the context of BV production, ME53 is required both early and late in the infection cycle. Additional Western blot analysis of purified virions from the repair virus revealed that ME53:HA is associated with both BV and occlusion-derived virions. Together, these results indicate that me53 , although not essential for viral replication, is required for efficient BV production.

Fowl adenovirus 4 (FAdV-4) is associated with economically important poultry diseases. Recent studies of fully sequenced genomes of FAdV-4 isolates suggest potential genomic regions associated with virulence and amenable for manipulation and vector development. Direct manipulation of viral genomes is cumbersome, as opposed to that of infectious clones—viral genomes cloned into plasmid or cosmid vectors. In this work, we generated an infectious clone, pFAdV-4 ON1, containing the entire viral genome of a nonpathogenic FAdV-4 (ON1 isolate). pFAdV-4 ON1 was used for targeted deletion of open reading frames (ORFs) 16 and 17 and replacement with the enhanced green fluorescence protein (EGFP) expression cassette to generate recombinant viruses. These viruses were viable, and EGFP was expressed in infected cells. Their replication, however, was significantly reduced with respect to that of the wild-type virus. These observations suggest the potential utility of FAdV-4 as a vaccine vector and the importance of ORFs 16 and 17 for virus replication at wild-type levels. To our knowledge, this is the first report of an infectious clone based on the FAdV-4 genome, and our results demonstrate its utility for studies of virulence determinants and as a platform for either vaccine or gene delivery vectors.

Serratia marcescens phage eta is a temperate unclassified member of the Siphoviridae which had been reported as containing hypermodified guanine residues.

The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol -null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production. IMPORTANCE The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are insufficient for viral DNA synthesis and virus replication. Rather, we identified three features, including two nuclear localization signals and a highly conserved 10-amino-acid sequence in the AcMNPV DNApol C terminus, all three of which are important for both nuclear localization of DNApol and for DNApol activity, as measured by viral DNA synthesis and virus replication.