Abstract
orf65 (Bm65) of Bombyx mori nucleopolyhedrovirus (BmNPV) codes for a putative 104-amino-acid protein containing three cysteine residues with a putative molecular mass of 12.2 kDa. Previous studies have showed that Bm65 accumulates mainly in nucleus and involved in the repair of UV-damaged DNA. However, the mechanism of nuclear import of Bm65 remains unclear. In this study, a SDS-stable Bm65 tetramer was found in BmNPV-infected BmN cells, and alanine substitutions for the three cysteine residues did not affect the formation of Bm65 tetramer. Additionally, a basic amino acid cluster of the Bm65 protein was identified as an efficient nuclear localization signal (NLS). Firstly, transient expression of GFP-fused truncated Bm65 variants revealed that the 76KRKCSK motif functions as the NLS. This was also confirmed by alanine substitution in the 76KRKCSK motif, which caused attenuated nuclear localization of Bm65. Next, the 76KRKCSK motif-mutated bacmid was generated and the 76KRKCSK motif was also found to be important for nuclear localization of Bm65 in BmNPV-infected conditions. Lastly, analyses of flag-tagged Bm65 expressing bacmids revealed that the mutations in 76KRKCSK motif did not affect the synthesis of Bm65 tetramer, but severely impaired production levels of infectious virions. In conclusion, Bm65 exists in mainly a tetrameric form in virus-infected cells, which may be involved with production levels of infectious virions.
Highlights
Baculoviruses are within a group of enveloped, double-stranded DNA insect viruses with large, closed and circular genomes ranging in size from 80 to 180 kbp (Herniou et al, 2003; van Oers and Vlak, 2007; Yin et al, 2015), which are characterized by a biphasic infection phase with production of two types of progeny virions during the viral life cycle
The cells were transfected with 2 μg recombinant plasmids, which can transiently express Bm65 and Bm65 truncations fused with enhanced green fluorescent protein (EGFP) under the control of ie1 promoter
The results indicated that Bm65-flag was accumulated mainly in the nucleus of Bombyx mori nucleopolyhedrovirus (BmNPV)-infected BmN cells, and some fluorescent aggregates were found to be accumulated in nucleus of virus-infected BmN cells (Figure 7B)
Summary
Baculoviruses are within a group of enveloped, double-stranded DNA insect viruses with large, closed and circular genomes ranging in size from 80 to 180 kbp (Herniou et al, 2003; van Oers and Vlak, 2007; Yin et al, 2015), which are characterized by a biphasic infection phase with production of two types of progeny virions during the viral life cycle. It is necessary to clarify the mechanism of BmNPV infection at the molecular level, which is helpful to control viral spread among silkworms. The functions of most viral genes in the process of BmNPV propagation, interactions between BmNPV and silkworm, and the innate response against BmNPV invasion have been extensively studied since Gomi et al (1999) published the sequence of BmNPV genome (Ono et al, 2012; Qin et al, 2012; Xue et al, 2012). The propagation of BmNPV in host cells is inevitably involved with an important number of virus-encoded proteins that are required to generate progeny virions. The mechanism of nuclear import and the impact of Bm65 on viral propagation are demonstrated in the study
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