![]() The GC content of AjGV DNA was found to be 65 +/- 1% and the genome size as determined from reassociation kinetics was 92 kb. Southern hybridization of the restriction profiles of the viral DNA with SalI fragment from pUCTnGV (containing granulin gene) was performed and hybridization was found on single discrete fragments with molecular weights ranging from 12.0 to 2.7 kb. Restriction profiles of viral DNA with seven enzymes were obtained and the average molecular weight was found to be approximately 92 +/- 7 kb. The major polypeptide of the capsids had molecular weight of 34.9 +/- 0.3 kDa. ![]() 5 +/- 0.8 kDa and 12 of these polypeptides were contained in the nucleocapsids. The virus particles were found to contain 16 polypeptides with molecular weights ranging from 106.4 +/- 2.1 to 15. Protein gel electrophoresis of granulin gave degradation products from 27.2 to 15.4 kDa, when protease associated with capsules was not inhibited. SDS-PAGE electrophoresis was carried out and the granulin polypeptide (28.9 +/- 0.5 kDa) was found to contain two other associated polypeptides of molecular weight 58.2 +/- 2.3 and 55.2 +/- 1.3 kDa, respectively. The empty capsids measured 265 +/- 9 x 63 +/- 3 nm. The virus particles (274 +/- 9 x 95 +/- 4 nm) contain nucleocapsids (297 +/- 7 x 52 +/- 2 nm) within an envelope. The granulosis virus isolated from Achaea janata is composed of capsules (463 +/- 25 x 280 +/- 22 nm) which contain single virus particles. Our findings demonstrated that SfMEF interacts with and mediates the nuclear import of Ac34, which is a new nucleocytoplasmic transport pathway used by alphabaculovirus to achieve successful viral replication within the nucleus of the infected cells. Co-immunoprecipitation analysis showed that the above mutations in the potential zinc finger motif disrupted the interaction between Ac34 and SfMEF, and the loss of the interaction resulted in decreased BV production. The mutations of C116 or C119 in a potential CCCH zinc finger motif (C116-X2-C119-X8-C128-X2-H131) of Ac34 led to an exclusive cytoplasmic distribution of Ac34, in consistent with our previous finding of mutations of C128 or H131 in this motif. The deletion of ac34 did not affect the subcellular localization of SfMEF however, knocking down Sfmef prevented the nuclear import of Ac34 in virus-infected cells. ![]() Co-immunoprecipitation assays confirmed that Ac34 could interact with SfMEF in the absence of other baculovirus proteins. The result indicated that Spodoptera frugiperda mRNA export factor (SfMEF) may interact with Ac34 during baculovirus infection. To investigate the mechanism of Ac34 nuclear import, mass spectrometric analysis was performed to identify potential proteins that may be involved in the nuclear import of Ac34. Our previous study showed that the nuclear localization of Ac34 was required for optimal production of budded virions. For successful alphabaculovirus replication, viral proteins must be transported to the nucleus. Autographa californica multiple nucleopolyhedrovirus ORF34 (ac34) is one of the unique genes of alphabaculoviruses. ![]()
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