The Motif of 76KRKCSK in Bm65 Is an Efficient Nuclear Localization Signal Involved in Production of Infectious Virions

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.


INTRODUCTION
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 two phenotypes are budded viruses (BVs) and occlusion-derived viruses (ODVs), which carry identical genetic information but differ structurally and functionally (Slack and Arif, 2007;Cheng and Lynn, 2009). BVs are produced at the initial stage of baculovirus life cycle and are responsible for the spread of infection from cell to cell, while ODVs are produced in the late stage of viral life cycle and mediate the horizontal transmission among insects (Clem and Passarelli, 2013;Rohrmann, 2013).
Bombyx mori nucleopolyhedrovirus (BmNPV) belongs to the Alphabaculovirus genus, Baculoviridae family and infects exclusively silkworms. BmNPV epizootics result in serious losses in silk production. Therefore, 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). Additionally, the mechanism of BmNPV proliferation in silkworm has been gradually elucidated. Like other viruses, 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. Previous research reported that Bm65 is an early gene by transcriptional analysis (Tang et al., 2013), indicating that Bm65 may be involved with viral propagation. Tang et al. (2015Tang et al. ( , 2017 further reported that Bm65 localizes mainly in nucleus and is involved with the repair of UV-damaged DNA. However, the size of Bm65 in BmNPV-infected conditions remains unclear. So, we want to check the expression of Bm65 in BmNPVinfected BmN cells. Meanwhile, the mechanism of nuclear import and the impact of Bm65 on viral propagation are demonstrated in the study. In the current study, a series of transient expression plasmids, including Bm65 truncations and point mutations in Bm65, were fused with enhanced green fluorescent protein (EGFP) respectively. The target DNA fragments were under control of ie1 promoter for expression of fusion protein tagged with EGFP. These plasmids were transfected into BmN cells to examine the intracellular distribution of fluorescent signal. Furthermore, the effect of mutations in Bm65 76 KRKCSK motif on viral propagation was further evaluated by analysis of production of infectious virions.

Bacmid, Virus, Plasmids, Bacterial Strains, and Cells
Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid (Bmbacmid) with a deletion of Bm65 (Bm Bm65KO ) was generated as previously described (Tang et al., 2013), and propagated in Escherichia coli strain DH10B harboring the pMON7124 helper plasmid. vBm (PBm65−Bm65−egfp) was made by Tang et al. (2015) and used as a control of wild type in the study. Plasmid of pFastHTB-P Bm65 -Bm65-egfp was constructed as previously described by Tang et al. (2015). Recombinant plasmid pFastHTB-P ie1 -ns1-egfp constructed by Li et al. (2015) was used to construct serial Bm65 truncations fused with egfp to study intracellular distribution of fluorescence signals in BmN cells. E. coli strains DH5α and DH10B were maintained in our laboratory. BmN cells were cultured at 27 • C in TC-100 medium supplemented with 10% Gibco fetal calf serum (Life Technologies).

C79-R ACGCTTAAGATTGTATTCCATGCGG
Underlined letters indicate restriction enzyme digestion sites.
Frontiers in Microbiology | www.frontiersin.org Bm65M9-F and Bm65-R7, Bm65M10-F and Bm65-R8, and Bm65M11-F and Bm65-R9, respectively. Each mutant Bm65 sequence was isolated from the corresponding recombinant plasmid by digestion, and the resulting DNA fragments were purified and subcloned into vector pFastHTB-P ie1 -ns1-egfp to generate the final plasmids. All primers used in the study are listed in Table 1.

Preparation of Recombinant Viruses
Construction of Bm65-deleted Bm-bacmid (Bm Bm65KO ) was performed as previously described (Tang et al., 2013). Colonies resistant to chloramphenicol, ampicillin, and kanamycin were selected for PCR confirmation with 65US-F/65US-R, Cm-F/Cm-R, and 65DS-F/65DS-R (data not shown). Deletion of Bm65 and correct insertion of Cm cassette were named Bm Bm65KO . To generate a flag-tagged Bm65 repair Bm-bacmid, the fragment containing Bm65 and its native promoter sequence tagged with flag coding sequence at the 3 end was amplified from Bm-bacmid by PCR with Bm65-F4 and Bm65-flag-R. The DNA fragment was subcloned into pFastHTB-Pie1-egfp-sv40-PH (Li et al., 2014) and pFastHTB (Invitrogen) digested with SnaBI and XhoI to generate pFastHTB-Pie1-egfp-sv40-P Bm65 -Bm65-flag and pFastHTB-P Bm65 -Bm65-flag, respectively. P ie1egfp-sv40-P Bm65 -Bm65-flag was transferred into the polyhedrin locus of the Bm Bm65KO , and the resulting Bm Bm65−Flag−GFP was selected by blue-white screening and further confirmed by PCR with M13 primers.

Transfection and Fluorescence Microscopy
BmN cells (10 6 cells/well) were seeded into six-well culture plates and incubated at 27 • C for 16-24 h before transfection. Recombinant DNA molecules (2 µg/well) and 5 µl Cellfectin (Invitrogen Life Technology) were mixed in 200 µl TC-100 serum free medium and incubated at 27 • C for 45 min. Then, 800 µl serum free medium was added into the DNA-Cellfectin solution, which was finally overlaid onto BmN cells and incubated at 27 • C for 5 h. After the incubated cells were washed with serum free TC-100 medium, 2 ml TC-100 medium containing 10% fetal bovine serum was added into each well for further culture. At 96 h post-transfection (hpt), the BV-enriched culture supernatants were harvested for further study. Fluorescence in BmN cells was observed through fluorescence microscopy (Olympus-IX73-DP80, Japan) at selected time points for further analysis. The fluorescent signal in each picture was counted one by one through observation by naked eye. The fluorescent cells were counted in each of 289 mm 2 figure and 15 field of view were used for counting, which was used to make the statistical analysis using t-test.
The cells were directly observed and photographed using Confocal Laser Scanning Microscopy (Model:TCS SP5 II, Leica corporation of Germany). Laser source is single-laser excitation source for multicolor and fluorescence emission was excited at 488 and 345 nm to detect the fluorescence signal of EGFP and DAPI, respectively. Glycerol objective (× 63) was used for imaging.

Analysis of Viral Growth Curve
To further study whether deletion of Bm65 and alanine mutations in the 76 KRKCSK motif affected production of recombinant virus, a virus growth curve analysis was performed as described previously (Tang et al., 2013). A monolayer of BmN cells (10 6 ) was seeded in six-well plates for transfection with 2.0 µg of bacmid DNA and the experiments were repeated three times. The supernatants of transfected cells containing recombinant viruses were harvested at selected time points. The 50% tissue culture infective dose (TCID 50 ) end-point dilution was carried out to determine the titer of BV as described previously, with modifications (O'Reilly et al., 1994). Viral infections were characterized by observation of fluorescence in BmN cells. It was identified to be positive if green fluorescence was observed in one or more BmN cells by fluorescence microscopy. Statistical analysis was performed using single factor analysis of variance.

Comparison of the Amino Acid Sequences of Bm65 Homologs
Bm65 codes for 104 deduced amino acid residues with a putative molecular mass of 12.2 kDa. Bm65 is not a core gene existing in all baculoviruses. Alignment result revealed that it is a highly conserved gene in baculoviruses of the genera Alphabaculovirus and Betabaculovirus (Figure 1). Ac79 [orf 79 of Autographa californica multiple nucleopolyhedrovirus (AcMNPV)], a homologs of Bm65, has been reported to be an early gene involved in efficient budded virus production (Wu and Passarelli, 2012). Tang et al. (2015) reported that a conserved motif of GIY-YIG nuclease superfamily was found in the sequence of Bm65. Moreover, two possible typical nuclear localization signal (NLS) motifs of 33 RRIK and 76 KRKCSK are contained in the middle part of Bm65 sequence, and alignment result revealed that they are conserved in the Bm65 sequence and its homologs, Additionally, a leucine-rich motif of 92 PLLLHKFLL in the C-terminal part of Bm65 sequence was found to be conserved in the multiple sequences, which was predicted to be a potential nuclear export signal by analysis of the online tool 1 .

Analysis of Recombinant Bm65 Expressed in BmNPV-Infected BmN Cells
Construction of recombinant virus for expression of Bm65 is described as Figure 2A. Flag-tagged Bm65 was examined from 3 to 72 hpi under the control of its natural promoter (Figures 2B-D). Western blotting analysis of total protein from BmN cells infected with vBm Bm65−flag−GFP or vBm Bm65(M1)−Flag−GFP was carried out. A specific protein band was examined from 3 to 72 hpi ( Figure 2B). However, the experimentally determined mass of Bm65 was about four times as large as that of the predicted size of Bm65, indicating that Bm65 exists with a mainly tetrameric form in BmNPV-infected BmN cells. Protein lysate from BmN cells infected with vBm-bacmid Bm65−Flag−GFP was treated with β-mercaptoethanol, which was subsequently subjected to Western blotting analysis. Only a Bm65 specific protein band migrating at only a size of approx 12 kDa was observed from 3 to 72 hpi (Figure 2C), showing that treatment of β-mercaptoethanol transformed the tetrameric form of Bm65 into a monomer. To explore the formative mechanism of Bm65 tetramer, three cysteine residues at positions 12, 46, and 79 of Bm65 were substituted by alanine residues and the mutant name fusion with flag was defined as Bm65(M1)-flag. The total protein from BmN cells infected with recombinant virus for expression of Bm65 mutant was determined by Western blotting. Only the tetrameric form of Bm65 was detected from 3 to 96 hpi (Figure 2D), indicating that the mutations did not Further experiments would be required to characterize this 26 kDa band. In conclusion, these results revealed that Bm65 is an early protein with a tetrameric form existing in BmNPVinfected BmN cells, but the formative mechanism requires further research.

Subcellular Localization of Bm65 Truncations Fusion With EGFP
Bm65 is an early protein correlated with the repair of damaged DNA in nucleus of host cell (Tang et al., , 2017. Therefore, it is indispensable for Bm65 to enter the nucleus of target cell for DNA repair. However, the mechanism of Bm65 translocation into nucleus remains unknown. Two possible NLS motifs of 33 RRIK and 76 KRKCSK, and a possible nuclear export signal of 92 PLLLHKFLL are contained in the Bm65 sequence. To identify whether these putative motifs are functional, four truncations with the C-terminal and N-terminal deletions of Bm65 fusion with EGFP under control of ie1 promoter were constructed to study intracellular distribution of Bm65, by observation of fluorescence signal. BmN cells were not infected in this assay. Transient expression assay showed that Bm65 was located mainly in the nuclei and only a little in the cytoplasm, but the intracellular distribution of truncated forms of Bm65 was correlated with the deletions of Bm65 (Figure 3). The truncated form of Bm65 (aa 1-84) accumulated almost exclusively in the nuclei of BmN cells. Compared with the truncated form of Bm65 (aa 1-84), a small part of green fluorescence was found in the cytoplasm except for the majority of green fluorescence accumulated in nucleus from the panel 2 of Figure 3 when BmN cells were transfected with the complete Bm65. The result indicated that the 92 PLLLHKFLL motif may be involved with nuclear export of Bm65. The motif of 92 PLLLHKFLL may be a functional nuclear export signal for transport of Bm65 from nucleus to cytoplasm. Further research is required to demonstrate the role of the 92 PLLLHKFLL motif on nuclear export of Bm65. However, the truncated form (aa 1-75) of Bm65 was uniformly distributed within BmN cells when Bm65 with the C-terminal deletion of 29 aa, containing the motifs of 92 PLLLHKFLL and 76 KRKCSK, was expressed in BmN cells, indicating that 76 KRKCSK may be a functional NLS for the active transport of Bm65 from cytoplasm to nucleus. Additionally, the truncated forms (aa 37-104 and aa 71-104) of Bm65 were accumulated mainly in the nucleus and only a little in the cytoplasm. The truncated form (aa 1-36) of Bm65 was evenly distributed in transfected BmN cells. The results indicated that the motif of 33 RRIK has no effect on the nuclear import of Bm65, and the nuclear import of Bm65 is strictly dependent of the 76 KRKCSK motif.

Effect of Alanine Mutations in the Motifs of 33 RRIK and 76 KRKCSK on the Nuclear Import of Bm65
The truncated form (aa 37-104) of Bm65 with deletion of the 33 RRIK motif was found to be accumulated mainly in the nucleus, which is in line with the subcellular localization of Bm65 in BmN cells. The result indicated that the deletion did not affect the trafficking of Bm65 from cytoplasm to nucleus. To confirm this, alanine mutations were introduced into the 33 RRIK motif of Bm65 to study the subcellular distribution of Bm65 through fluorescence observation. The 33 AAAA (mutant 1) was accumulated mainly in the nucleus and some fluorescent aggregates were also present (Figure 4). However, the 76 AAACSA (mutant 2) altered the pattern of subcellular localization of Bm65 in BmN cells, and the mutations severely blocked the transport of Bm65 from cytoplasm to nucleus. Fluorescence was accumulated exclusively in cytoplasm, and there were some fluorescent aggregates in the cytoplasm (Figure 4). According to the above results, it is believed that the motif of 33 RRIK is not involved with the nuclear import of Bm65, but the motif of 76 KRKCSK is an efficient NLS for transport of Bm65 from cytoplasm to nucleus.
To define the importance of basic amino acid residues in the motif of 76 KRKCSK, a series of single point mutations, including 76 ARKCSK (mutant 3), 76 KAKCSK (mutant 4), 76 KRACSK (mutant 5) and 76 KRKCSA (mutant 6), were made to examine the subcellular localization of Bm65 by fluorescence microscopy. Mutant 3 did not block the nuclear import of Bm65, but other mutations impaired or blocked the nuclear import of Bm65 (Figure 3). Mutant 4 obviously impaired the nuclear import of Bm65, because some fluorescence was accumulated in the cytoplasm of some cells, and some fluorescence was found to be only in nucleus of some cells. The fluorescence of typically accumulated in cytoplasm for the mutation of mutant 4 was selected to describe the phenomenon of reduced nuclear entry (Figure 4). The result indicated that the residue 77 R is involved with the efficiency of nuclear import. Mutant 5 and mutant 6 severely blocked the nuclear import of Bm65, and all fluorescence was accumulated dominantly in the cytoplasm. In conclusion, these mutations resulted in the varied distribution pattern of green fluorescence in BmN cells compared with that of wild type Bm65. The intracellular distribution of fluorescence is summarized in Table 2. The results indicated that the 76 KRKCSK motif is an efficient NLS for the final destination of Bm65, and the residues of 78 K and 81 K are essential for nuclear import of Bm65.

The 76 AAACSA Mutant Blocked the Nuclear Import of Bm65
To further study the effect of the mutant 2 on the subcellular localization of Bm65 in virus-infected BmN cells, recombinant virus of vBm Bm65(M2)−GFP (Figure 5A) was used to infect BmN cells for expression of mutant Bm65 EGFP fusion. Bm65 labeled EGFP was under the control of ie1 promoter. The fluorescence signal was observed exclusively in the cytoplasm of virus-infected BmN cells at 3 hpi, and the fluorescence continued to be observed in cytoplasm from 6 to 72 hpi. Additionally, Bm65(M2)-GFP showed fluorescent aggregates in the form of punctate dots in the cytoplasm of virusinfected cells (Figure 5B). Together, these results demonstrated that alanine mutations including K76A, R77A, K78A, and K81A completely blocked the nuclear import of Bm65 in virus-infected BmN cells, and the motif of 76 KKKCSK was confirmed to be an efficient NLS for transport of Bm65 from cytoplasm to nucleus. vBm Bm65−GFP was used as the control of wild type ( Figure 5C). The result indicated that Bm65-GFP was accumulated mainly in the nucleus of virus-infected BmN cells, and some fulorescent aggregates were found to be accumulated in nucleus of virus-infected BmN cells ( Figure 5D). Additionally, Tang et al. (2015) have examined the subcellular localization of wild type Bm65-EGFP by the observation of confocal microscopy. The results also confirmed that fluorescence signal and some fluorescenct aggregates were accumulated mainly in nucleus of virusinfected BmN cells. Compared with the localization of wild type Bm65, the mutant 2 was accumulated mainly in cytoplasm of virus-infected BmN cells. The results indicated that the motif of 76 KKKCSK was an efficient NLS for the nuclear import of Bm65.

Disruption of Intrinsic Stability of the Tetramer for Bm65 Fusion With EGFP
Due to some mutations in the 76 KRKCSK NLS, Bm65 mutants were found to be accumulated exclusively in cytoplasm. To determine the effect of alanine mutations in the motif 76 KRKCSK on the tetrameric form of Bm65, recombinant virus vBm Bm65(M2)−Flag−GFP (Figure 6A) for expression of Bm65 mutant was made and used to infect BmN cells for Western blot analysis. Only a tetramer specific band of Bm65 was detected from 24 to 72 hpi (Figure 6B), and alanine mutations in the motif 76 KRKCSK did not affect the formation of Bm65 tetramer. So, the tetrameric form of Bm65 can be produced in cytoplasmic and nuclear sites, but it was not required for the nuclear import of Bm65 because the monomer of Bm65-GFP also entered nucleus. According to the above results, it is regarded that the introduced mutations were not involved with the tetramer formation and/or on protein stability.
To further determine whether a fusion protein labeled with Bm65 produced the tetrameric form in BmN cells, recombinant viruses for expression of the N-terminal or C-terminal extension of Bm65 labeled with EGFP was under the control of ie1 promoter, which were prepared and used to infect BmN cells for Western blotting analysis. A band migrating at 40 kDa, the size expected for a Bm65:EGFP fusion was detected from 24 to 72 hpi (Figures 6C,D) using antibodies against EGFP, and only the monomer form of Bm65 fusion, regardless of N-terminal or C-terminal, was formed. According to the result, it is regarded that EGFP fusion affect the stability the tetramer of Bm65.

Immunocytochemistry Assay for Distribution of Bm65-Flag and Bm65(M2)-Flag
Although GFP-tagged Bm65 and its variants are sufficient to just prove nuclear localization ability of the NLS, Western N(++)/C, N(+)/C, N/C, and C indicated that fluorescence signal was predominantly but not mainly in the nucleus, in the nucleus was heavier than that of in the cytoplasm, equally between the nucleus and the cytoplasm, and mainly in the cytoplasm (C), respectively.
blotting revealed that the recombinant virus expressing Bm65-GFP also expressed a large amount of extra proteins detected by anti-GFP antibody (Figures 6C,D). To preclude the possibility of fluorescent aggregates from the huge amount of truncated GFP and the mislocalization of Bm65 caused by GFP-tag, the immunocytochemistry assay was made in vBm Bm65(M2)−Flag−GFP -infected BmN cells using antibodies against flag tag ( Figure 7C). Meanwhile, vBm Bm65−Flag−GFP was used as a control ( Figure 7A). The immunocytochemistry assay of Bm65-flag was made in vBm Bm65−Flag−GFP -infected BmN cells using antibodies against flag, which was used as a control of wild type. The subcellular localization of Bm65 and its variant was judged from the distribution of red fluorescent signal observed by confocal microscopy.
The results indicated that Bm65-flag was accumulated mainly in the nucleus of BmNPV-infected BmN cells, and some fluorescent aggregates were found to be accumulated in nucleus of virus-infected BmN cells ( Figure 7B). However, the mutant 2 was accumulated mainly in cytoplasm of virus-infected BmN cells and some fluorescent aggregates was found to be accumulated in cytoplasm (Figure 7D).

Alanine Mutations in the Motif 76 KRKCSK Sharply Impaired the Production of Infectious Virions
To analyze whether Bm65 is required for viral production, four recombinant Bm-Bacmids, including fully deleted-type (Bm Bm65KO−GFP ), wild-type (Bm WT−GFP ), repair-type 1 (Bm Bm65−Flag−GFP ), and repair-type 2 (Bm Bm65(M2)−Flag−GFP ) were generated and further transfected into BmN cells. Green fluorescence was observed by fluorescence microscopy in individual cultured cells as early as 24 hpt. However, in cells  transfected with the Bm65KO and BM65(M2) constructs, the level of fluorescence was significantly lower than that of the control viruses ( Figure 8B). Additionally, the transfected supernatants were harvested at 96 hpt, and used to infect BmN cells at a multiplicity of infection of 5. No difference was observed between the control viruses vBm Bm65−Flag−GFP and vBm WT−GFP , but a 90% reduction on the fluorescence level was detected for vBm Bm65KO−GFP and vBm Bm65(M2)−Flag−GFP , compared with the controls (data not shown). To confirm the role of Bm65 during viral propagation, the supernatants containing infectious virions were harvested from BmN cells transfected with each of the four bacmids at 24, 48, 72, and 96 hpt, and were used to infect BmN cells to compare the infectivity of vBm WT−GFP , Bm Bm65KO−GFP , vBm Bm65−Flag−GFP , and vBm Bm65(M2)−Flag−GFP by analysis of the growth curves. The assay of TCID 50 endpoint dilution on BmN cells was performed to determine BV titer. The results showed a steady increase in virus production and the same kinetics of growth for vBm WT−GFP and vBm Bm65−Flag−GFP . In contrast, recombinant viruses of vBm Bm65KO−GFP and vBm Bm65(M2)−Flag−GFP showed reduced viral growth. According to the viral growth curve, the four viruses all showed an increased tendency in the slope of the growth curves, but the proliferation rate of vBm Bm65KO−GFP and vBm Bm65(M2)−Flag−GFP are obviously slower than that of the vBm WT−GFP or vBm Bm65−Flag−GFP (Figure 8C). Statistical analysis revealed that there was a significant difference of the viral titers between Bm65KO or BM65(M2) and Bm65 wild type virus from 48 to 96 hpi. Additionally, the viruses expressing GFP-tagged Bm65 and GFP-tagged Bm65 mutant 1 ( 33 RRIK/AAAA) were constructed in our lab and they grew about as well as the wild type (Data not shown). The results confirmed that Bm65 is not essential for viral replication, but deletion of Bm65 and alanine mutations in the motif 76 KRKCSK significantly decreased the levels of infectious virions. As is well-known, the generation of progeny virions are involved with some steps, including virion assembly within nucleus, nuclear egress of assembled virions and budding from host cells. However, it is still unclear in which step was affected by the Bm65 mutant for the impairment of progeny production.

DISCUSSION
Nuclear proteins are translated from mRNA in cytoplasm and must be transported to nucleus where they exert their biological functions. It is generally accepted that a protein is targeted to nucleus by classical NLSs, which can be divided into two categories of monopartite and bipartite sequences. For example, the PKKKRKV sequence of simian virus 40 (SV40) large T antigen is a monopartite sequence (Kalderon et al., 1984), and the KRPAATKKAGQAKKKK sequence of nucleophosmin is a bipartite sequence (Robbins et al., 1991). The classical NLSs are directed to form a heterotrimeric complex including the cargo protein and two importins, which further mediates transport of the heterotrimer into nucleus by its affinity with nucleoporins (Ben-Efraim and Gerace, 2001). Some baculovirus proteins have also been reported to contain the classical NLSs. For example, the monopartite motif of classical NLSs was discovered in baculovirus polyhedrin and AcMNPV BV/ODV-c42 (Jarvis et al., 1991;Wang et al., 2008), and residues 804-827 of AcMNPV DNApol are a typical bipartite NLS motif (Feng and Krell, 2014). Additionally, some non-canonical NLSs (nNLSs) have also been identified in baculovirus proteins, which do not correspond with the prototypical NLS consensus sequence. For example, Guo et al. (2010) reported that two basic residue clusters at positions 117-120 ( 117 RKRR) and 144-148 ( 144 RKR-K) constituted an authentic NLS for mediating nuclear localization of Bm47.
A novel localization signal containing K(75)/R(76), H(81), K(83)/R(84), R(87), and K(100) was scattered in the different positions of BmNPV LEF-11, which was identified to be critical for the nuclear localization of target protein (Zhang et al., 2014). The residues KKIREN of LEF-3 have been identified as a core NLS required for nuclear transport, which was further improved by other adjacent positively charged residues (Au et al., 2009).
Previous studies revealed that Bm65 accumulated mainly within the nucleus, and was involved with the repair of UVCinduced DNA damage (Tang et al., , 2017. Moreover, Ac79, a homolog of Bm65, is responsible to encode an early gene product that are structurally similar to UvrC and intronencoded endonucleases, which was required for efficient BV production (Wu and Passarelli, 2012). Alignment result showed 99% sequence identity with the amino acid of Bm65. The high similarity of sequence implied that the role of Bm65 in viral propagation may be similar to Ac79. However, the mechanism by which Bm65 is transported into the nucleus of virus-infected cells and the failure of nuclear import of Bm65 on viral production are still unclear. In the study, flag-labeled Bm65 is under the control of Bm65 native promoter in the study (Figures 2, 8), and it was detected from 3 to 72 hpi (Figures 2B-D). So, we think that Bm65 native promoter is similar to ie1 promoter, and it can function like the ie1 promoter. To accelerate the progress on Bm65 research, some plasmids containing ie1 promoter such as pFastHTB-P ie1 -ns1-egfp have been constructed and conserved in our laboratory, which can be used for the transient expression analysis of Bm65 in BmN cells. So, ie1 promoter was chosen to drive the expression based on the convenience and economic benefits in the study. A series of truncations and arginine/lysine-to-alanine mutations severely impaired the nuclear import of Bm65. The 76 KRKCSK motif of Bm65 was identified as a novel NLS required for nuclear import of Bm65, but another basic cluster of 33 RRIK was identified as unnecessary for the nuclear import of Bm65. Furthermore, the leucine-rich cluster of 92 PLLLHKFLL may function as a nuclear export signal for maintaining the dynamic balance of Bm65 between nucleus and cytoplasm.
Previous research has revealed that Bm65 is not essential for viral propagation (Ono et al., 2012). However, Tang et al. (2013) reported that Bm65 was an essential gene for viral propagation. In the present study, Bm65 was identified as an unnecessary gene for viral propagation. In Tang et al. (2013), Bm65 was regarded to be an essential gene for viral propagation, which may be resulted from the failure of transfection of BmN cells. As we know, donor plasmid of pFB-ieGP-Bm65 was transformed into electrocompetent DH10B cells containing Bm Bm65KO , which was used for the generation of Bm Bm65KO−GP by transposition. Furthermore, the recombinant Bm Bm65KO−GP extracted from DH10B cells was used for transfection of BmN cells. However, the pFB-ieGP-Bm65 was also extracted from DH10B cells besides the Bm Bm65KO−GP , and the abundance of pFB-ieGP-Bm65 is more than that of Bm Bm65KO−GP in the extracted DNA. Fluorescence signal can be observed in BmN cells transfected with pFB-ieGP-Bm65, but no infectious virions were produced in the transfection supernatant. When the transfection supernatant was collected and further used for infection of BmN cells, it can not initiate the second infection of BmN cells and no fluorescence signal was observed in BmN cells. So, Tang et al. (2013) regarded that Bm65 was an essential gene for viral propagation by mistake. In fact, infectious virions can be produced in BmN cells transfected with Bm Bm65KO−GP . Furthermore, the recombinant virus with deletion of Bm65 was confirmed to be infectious by passage analysis in BmN cells, but the analysis of viral titer showed that the deletion significantly decreased the production of infectious virions ( Figure 8C). So, Bm65 was not an essential gene for viral propagation in the study, but it can regulate the production of viral propagation.
Furthermore, mutations in the motif of 76 KRKCSK resulted in the cytoplasmic accumulation of Bm65. Compared with wildtype Bm65, the mutations greatly decreased the production of infectious virions. Bm65 was first identified to form a stable sodium dodecyl sulfate (SDS)-resistant tetramer in the natural state, and there was some fluorescent aggregates in cytoplasm and nucleus when alanine mutations were made in the motif of 76 KRKCSK. Compared with wild type of Bm65 (Figure 5D), fluorescent aggregates caused by the mutant type of Bm65 are accumulated exclusively in the cytoplasm of virusinfected BmN cells (Figure 5B), and the fluorescent aggregates observed in wild type and mutant Bm65 virus-infected cells vary in the sizes and levels. It is supposed that Bm65 tetramer may increase the possibility of the formation of fluorescent aggregates. Of course, the misfolding of Bm65 is an important factor for the formation of protein aggregates. To the best of our knowledge, some baculovirus proteins such as LEF-10 and LEF-11 form protein aggregates (Dong et al., 2015;Xu et al., 2016). Moreover, amyloid β-protein (Walsh et al., 1999;Rangachari et al., 2007), sup35 (Kryndushkin et al., 2003), and α-synuclein (Rekas et al., 2010) are able to form SDS-resistant aggregates. Protein aggregation is a natural phenomenon that occurs both in vitro and in vivo. However, it is generally regarded to be harmful and has been implicated in a wide variety of diseases such as neurodegenerative diseases, and the possible correlations between protein aggregates and diseases deserves our deep consideration. Besides the tetrameric form from 3 to 96 hpi, a major lower molecular weight band at about 26 kDa was also examined in Figure 2D from 48 to 72 hpi, indicating that it may be a Bm65 dimer. The mutations of cysteine residues at positions 12, 46, and 79 did not affect the formation of Bm65 tetramer, but they directly resulted in increased instability of Bm65 tetramer in late infection. Additionally, some unexpected bands around 26 kDa and below 26 kDa were examined in Figures 6C,D. The abundance of the lower molecular weight proteins is so rich, but the synthesis mechanism is still unclear. Therefore, the possibility of Bm65 tetramer by disulfide linkage formation was precluded by the mutational analysis.
Additionally, recombinant plasmid of pET-30a-Bm65 was constructed by Tang et al. (2015) and transformed into BL21 cells for expression of 6 × His-Bm65. Western blotting result indicated that only 6 × His-Bm65 monomer was present in the soluble supernatant from the lysates of BL21 cells. Moreover, a lot of Bm65 inclusion body was found to be produced in E. coli. Therefore, we think that it is unfavorable for the formation of Bm65 tetramer in E. coli strains. Compared with the monomer in E. coli, Bm65 tetramer was examined in vBm Bm65−Flag−GFP -infected BmN cells, but GFP-tagged Bm65 monomer was examined in virus-infected BmN cells in the study (Figures 6C,D). It was regarded that flag tagged Bm65 is equivalent to the Bm65 native state, but the GFP tag affected the multimerization of Bm65. Moreover, the intermediate band about 26 kDa in Figure 2D from 48 to 72 hpi may be Bm65 dimer or Bm65 protein complex. The replacement of cysteine with alanine may decreased the stability of Bm65 tetramer, which was involved with the generation of the intermediate. Additionally, the formation of Bm65 tetramer may need the assistance from other proteins in BmNPVinfected BmN cells. Tang et al. (2015) reported the monomersonly, which was obtained in the soluble supernatant from the lysates of BL21 cells. In the study, Bm65 tetramer was easily produced in BmNPV-infected BmN cells, which has nothing to do with disulfide linkage. Some mutations in the motif of 76 KRKCSK resulted in failure of the nuclear import of Bm65, which was involved with the decreased production of infectious virions. Moreover, we found that there are a lot of hydrophobic amino acids scattered in the sequence of Bm65, which may be the direct reason for the formation of Bm65 tetramer. Further research is required to disclose the formative mechanism of Bm65 tetramer. Meanwhile, it is also an interesting scientific issue for further investigation whether these hydrophobic amino acids are positively selected during virus/host coevolution.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.