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Open reading frame Bm21 of Bombyx mori nucleopolyhedrovirus is not essential for virus replication in vitro, but its deletion extends the median survival time of infected larvae In this report, the open reading frame 21 (Bm21) of Bombyx mori nucleopolyhedrovirus (BmNPV), one of the unique genes of group I NPVs, was characterized. Bm21 is predicted to encode a protein of 55.8 kDa and was found to contain imperfectly conserved leucine-rich repeats. 39 Rapid amplification of cDNA ends (39RACE) showed that the transcript of Bm21 was first detected from 6 h post-infection and that it also encompassed the complete Bm20. 59RACE revealed three transcription initiation sites, one of which mapped to the baculovirus early transcription motifs CGTGC and CAGT. Transient-expression and superinfection assays indicated that BM21 localized in the nucleus of infected BmN cells. To study the function of BM21, a Bm21-null virus was constructed using bacmid technology. Viral one-step growth curve analyses showed that the Bm21-null virus had similar budded virus production kinetics to those of the parental virus. Bioassay analyses showed that the median lethal concentration (LC 50 ) of the Bm21-null virus was similar to that of the control virus; however, the median survival time (ST 50 ) of the knockout virus was significantly longer than the control virus. These results indicate that BM21 is not essential for virus replication in vitro, but that deletion of the gene delays the killing of the infected larvae. INTRODUCTIONThe lepidopteran nucleopolyhedroviruses (NPVs) can be divided into two groups, I and II, based on the phylogenetic studies of baculoviruses (Zanotto et al., 1993;Bulach et al., 1999;Herniou et al., 2001). Autographa californica multicapsid NPV (AcMNPV) and Bombyx mori NPV (BmNPV) are examples of group I NPVs, whilst Lymantria dispar NPV, Spodoptera exigua MNPV and Helicoverpa armigera singlenucleocapsid NPV belong to group II. Genomic comparison has revealed that there are unique group I genes that are conserved only in group I NPVs and do not exist in other baculoviruses. Currently, there are 11 unique group I genes: ac1 (protein tyrosine phosphatase 1 gene, ptp1), ac16 (odve26), ac30, ac42 (global transactivator gene, gta), ac72, ac73, ac114, ac124, ac128 (gp64), ac132 and ac151 (ie2) (E. A. Herniou, personal communication; Ikeda et al., 2006). Many of the unique group I genes have been characterized to some extent. For example, three genes encode structural proteins: PTP1 is present in both the budded virus (BV) and occlusion-derived virus (ODV) (Li & Miller, 1995), and can induce enhanced locomotory activity in infected larvae (Kamita et al., 2005); BV/ODV-E26 is a structural protein of the envelope of BV and ODV (Beniya et al., 1998), and is a palmitoylated multifunctional structural protein associated with DNA binding (Burks et al., 2007) and co-localizes with IE1 (Imai et al., 2004); and gp64 encodes a viral envelope fusion protein that is responsible for viral attachment, fusion and egress (Blissard & Wenz, ...

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen in the sericulture industry and enters host cells via macropinocytic endocytosis; however, the current understanding of the BmNPV entry mechanism remains limited. To confirm whether direct membrane fusion (DMF) results in productive BmNPV infection, DMF infectivity induced by low pH during BmNPV infection was investigated, and the infectious viral particle was traced using an eGFP-labeled virion. We found that BmNPV infection efficiently induced fluid uptake, which allowed BmNPV to bypass the cell membrane barrier via macropinocytosis. However, DMF induced by a low pH abolished the infection. While low pH is an essential condition for membrane fusion triggering, it is not sufficient for productive BmNPV infection, and DMF results in failure to transport the nucleocapsid into the nucleus. These results indicate that transport via macropinocytic vesicles facilitates BmNPV entry into the nucleus and contribute to our understanding of the BmNPV entry mechanism.