AcMNPV肌动蛋白核定位基因的亚细胞定位

当前细胞核内肌动蛋白的功能是一个研究热点,核内存在肌动蛋白并参与细胞核内发生的许多生命活动。杆状病毒是迄今唯一报道的利用核内肌动蛋白进行复制增殖的病原微生物,为核内肌动蛋白的结构与功能的研究提供了独特的系统。有报道显示AcMNPV的ie-1,pe38,ac4,he65,ac102和ac152基因与肌动蛋白单体的核转运有关,然而关于这六个基因的亚细胞定位及其在肌动蛋白入核过程中的功能并没有深入的研究。本文首次揭示了这六个基因的亚细胞定位,IE1和AC152是全细胞分布,PE38和AC102也为核质分布,但主要分布在细胞核中,而AC4和HE65定位于细胞质。但AC102和IE1可以分别介导AC4和HE65入核。同时我们发现当使用外源强启动子OpIE2,在转染ie-1或pe38之后表达ac4和he65可以部分招募肌动蛋白单体入核,而时序性共转染这四个基因则可招募最多肌动蛋白单体入核。对肌动蛋白核定位基因在细胞中的定位分析为进一步了解杆状病毒介导肌动蛋白入核的分子机理提供支持。     

Genetic variation and virulence of Autographa californica multiple nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus isolates

To determine the genetic diversity within the baculovirus species Autographa calfornica multiple nucleopolyhedrovirus (AcMNPV; Baculoviridae: Alphabaculovirus), a PCR-based method was used to identify and classify baculoviruses found in virus samples from the lepidopteran host species A. californica, Autographa gamma, Trichoplusia ni, Rachiplusia ou, Anagrapha falcifera, Galleria mellonella, and Heliothis virescens. Alignment and phylogenetic inference from partial nucleotide sequences of three highly conserved genes (lef-8, lef-9, and polh) indicated that 45 of 74 samples contained isolates of AcMNPV, while six samples contained isolates of Rachiplusia ou multiple nucleopolyhedrovirus strain R1 (RoMNPV-R1) and 25 samples contained isolates of the species Trichoplusia ni single nucleopolyhedrovirus (TnSNPV; Alphabaculovirus). One sample from A. californica contained a previously undescribed NPV related to alphabaculoviruses of the armyworm genus Spodoptera. Data from PCR and sequence analysis of the ie-2 gene and a region containing ORF ac86 in samples from the AcMNPV and RoMNPV clades indicated a distinct group of viruses, mostly from G. mellonella, that are characterized by an unusual ie-2 gene previously found in the strain Plutella xylostella multiple nucleopolyhedrovirus CL3 (PlxyMNPV-CL3) and a large deletion within ac86 previously described in the AcMNPV isolate 1.2 and PlxyMNPV-CL3. PCR and sequence analysis of baculovirus repeated ORF (bro) genes revealed that the bro gene ac2 was split into two separate bro genes in some samples from the AcMNPV clade. Comparison of sequences in this region suggests that ac2 was formed by a deletion that fused the two novel bro genes together. In bioassays of a selection of isolates against T. ni, significant differences were observed in the insecticidal properties of individual isolates, but no trends were observed among the AcMNPV, TnSNPV, or RoMNPV groups of isolates. This study expands on what we know about the variation of AcMNPV, AcMNPV-like and TnSNPV viruses, provides novel information on the distinct groups in which AcMNPV isolates occur, and contributes to data useful for the registration, evaluation, and improvement of AcMNPV, AcMNPV-like, and TnSNPV isolates as biological control agents.     

杆状病毒Ac78 C末端保守区域的功能初步分析

杆状病毒模式种苜蓿丫纹夜蛾核多角体病毒(Autographa californica multiple nucleopolyhedrovirus, AcMNPV)的orf78 (即Ac78)是最近被发现的杆状病毒核心基因,在杆状病毒的生活周期中具有重要功能.氨基酸序列分析表明,Ac78的C末端105～108位氨基酸区域在Group I NPVs旁系同源物中高度保守.为研究该保守区域在Ac78功能中的作用,利用Bac-to-Bac杆状病毒表达载体系统成功构建了缺失该保守区域,并且携带绿色荧光蛋白基因和多角体蛋白基因的Ac78截短补回型重组病毒(vAc78:del105-108).荧光显微镜分析和病毒生长曲线测定结果表明,在vAc78:del105-108转染的Sf9细胞中,感染性的芽生型病毒粒子(budded virion,BV)产生量与Ac78全长补回型重组病毒(vAc78:HA)基本一致;电镜观察发现,在vAc78:del105-108转染的细胞中,呈现与vAc78:HA的现象一致的典型的杆状病毒感染特征,多粒包埋型病毒粒子(multiple nucleocapsid enveloped occlusion derived virion,M-ODV)以及包埋有M-ODV的包涵体均能正常形成;免疫荧光实验表明,在vAc78:del105-108感染的Sf9细胞中,从病毒感染细胞24 h时开始,Ac78专一定位于感染细胞的内核膜附近,与vAc78:HA的现象一致.上述结果表明,Ac78的C末端105～108位氨基酸保守区域对于BV和M-ODV的有效产生以及Ac78的亚细胞定位非必需.     

The role of viral protein Ac34 in nuclear relocation of subunits of the actin-related protein 2/3 complex

The actin nucleator actin-related protein complex(Arp2/3) is composed of seven subunits: Arp2,Arp3, p40/ARPC1(P40), p34/ARPC2(P34), p21/ARPC3(P21), p20/ARPC4(P20), and p16/ARPC5(P16). Arp2/3 plays crucial roles in a variety of cellular activities through regulation of actin polymerization. Autographa californica multiple nucleopolyhedrovirus(Ac MNPV), one of the beststudied alphabaculoviruses, induces Arp2/3 nuclear relocation and mediates nuclear actin polymerization to assist in virus replication. We have demonstrated that Ac34, a viral late-gene product, induces translocation of the P40 subunit of Arp2/3 to the nucleus during Ac MNPV infection. However, it remains unknown whether Ac34 could relocate other Arp2/3 subunits to the nucleus. In this study, the effects of the viral protein Ac34 on the distribution of these subunits were studied by an immunofluorescence assay. Arp2, P34, P21, and P20 cloned from Spodoptera frugiperda(Sf9) cells showed mainly cytoplasmic localization and were relocated to the nucleus in the presence of Ac34. In addition, Arp3 was localized in the cytoplasm in both the presence and absence of Ac34, and P16 showed whole-cell localization. In contrast to Sf9 cells, all subunits of mammalian Arp2/3 showed no nuclear relocation in the presence of Ac34. Co-immunoprecipitation analysis of the interaction between Ac34 and Arp2/3 subunits revealed that Ac34 bound to P40,P34, and P20 of Sf9 cells. However, none of the subunits of mammalian Arp2/3 interacted with Ac34, indicating that protein-protein interaction is essential for Ac34 to relocate Arp2/3 subunits to the nucleus.     

Autographa californica multiple nucleopolyhedrovirus nucleocapsid protein BV/ODV-C42 mediates the nuclear entry of P78/83

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-c42 (orf101; c42), which encodes a 41.5-kDa viral nucleocapsid protein with a putative nuclear localization signal (NLS) motif at the C terminus, is a highly conserved gene among members of the Baculoviridae family. C42 is demonstrated to be essential for AcMNPV propagation and can bind to nucleocapsid protein P78/83, a viral activator for the actin-related protein 2/3 (ARP2/3) complex to initiate nuclear actin polymerization, which is essential for viral nucleocapsid morphogenesis during AcMNPV infection. Here, we report the identification of a novel pathway through which c42 functions in nucleocapsid assembly. Cotransfection of Sf9 cells with c42 and p78/83 plasmids demonstrated that C42 was capable of recruiting P78/83 to the nuclei of uninfected cells and that the NLS motif of C42 was essential for this process. To validate this nuclear relocation mode in bacmid-transfected cells, a c42-disrupted bacmid (vAc(c42ko-gfp)) and rescued bacmids with wild-type c42 (vAc(c42res-gfp)) or with NLS coding sequence-mutated c42 (vAc(c42nls-gfp)) were prepared. By immuno-staining, P78/83 was found to be localized in the cytoplasm of either vAc(c42ko-gfp)- or vAc(c42nls-gfp)-transfected cells, whereas P78/83 was relocated to the nuclei of vAc(c42res-gfp)-transfected cells. Furthermore, F-actin-specific staining confirmed that there was no actin polymerization activity in the nuclei of either vAc(c42ko-gfp)- or vAc(c42nls-gfp)-transfected cells, which might be attributed to the absence of nuclear P78/83, an activator of the ARP2/3 complex to initiate nuclear actin polymerization. We therefore hypothesize a mode of action where C42 binds to P78/83 in the cytoplasm to form a protein complex and cotransports to the nucleus under the direction of the NLS motif in C42 during AcMNPV infection.     

Differential activity of two non-hr origins during replication of the baculovirus Autographa californica nuclear polyhedrosis virus genome

The identification of potential baculovirus origins of replication (ori) has involved the generation and characterization of defective interfering particles that contain major genomic deletions yet retain their capability to replicate by testing the replication ability of transiently transfected plasmids carrying viral sequences in infected cells. So far, there has not been any evidence to demonstrate the actual utilization of these putative origins in Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) replication. By using the method of origin mapping by competitive PCR, we have obtained quantitative data for the ori activity of the HindIII-K region and the ie-1 promoter sequence in AcMNPV. We also provide evidence for differential activity of the two ori in the context of the viral genome through the replication phase of viral infection. Comparison of the number of molecules representing the HindIII-K and ie-1 origins vis-à-vis the non-ori polH region in a size-selected nascent DNA preparation revealed that the HindIII-K ori is utilized approximately 14 times more efficiently than the ie-1 region during the late phase of infection. HindIII-K also remains the more active ori through the early and middle replication phases. Our results provide in vivo evidence in support of the view that AcMNPV replication involves multiple ori that are activated with vastly different efficiencies during the viral infection cycle.     

SUPPRESSION OF AcMNPV REPLICATION BY ADF AND THYMOSIN PROTEIN UP‐REGULATION IN A NEW TESTIS CELL LINE,Ha‐shl‐t

Host cytoskeletons facilitate the entry, replication, and egress of viruses because cytoskeletons are essential for viral survival. One mechanism of resisting viral infections involves regulating cytoskeletal polymerization/depolymerization. However, the molecular mechanisms of regulating these changes in cytoskeleton to suppress viral replication remain unclear. We established a cell line (named Ha‐shl‐t) from the pupal testis of Helicoverpa armigera (Lepidoptera: Noctuidae). The new testis cell line suppresses Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) replication via disassembly of cytoskeleton. Up‐regulation of thymosin (actin disassembling factor) and adf (actin depolymerizing factor) reduces F‐actin. Silencing thymosin or adf or treating cells with the F‐actin stabilizer phalloidin led to increased AcMNPV replication, while treating cells with an F‐actin assembly inhibitor cytochalasin B decreased viral replication. We infer that Ha‐shl‐t cells utilize F‐actin depolymerization to suppress AcMNPV replication by up‐regulating thymosin and adf. We propose Ha‐shl‐t as a model system for investigating cytoskeletal regulation in antiviral action and testicular biology generally.     

Autographa Californica Multiple Nucleopolyhedrovirus P48 (Ac103) Is Required for the Efficient Formation of Virus-Induced Intranuclear Microvesicles

Our previous study has shown that the Autographa californica multiple nucleopolyhedrovirus(AcMNPV) p48(ac103)gene is essential for the nuclear egress of nucleocapsids and the formation of occlusion-derived virions(ODVs). However,the exact role of p48 in the morphogenesis of ODVs remains unknown. In this study, we demonstrated that p48 was required for the efficient formation of intranuclear microvesicles. To further understand its functional role in intranuclear microvesicle formation, we characterized the distribution of the P48 protein, which was found to be associated with the nucleocapsid and envelope fractions of both budded virions and ODVs. In Ac MNPV-infected cells, P48 was predominantly localized to nucleocapsids in the virogenic stroma and the nucleocapsids enveloped in ODVs, with a limited but discernible distribution in the plasma membrane, nuclear envelope, intranuclear microvesicles, and ODV envelope. Furthermore,coimmunoprecipitation assays showed that among the viral proteins required for intranuclear microvesicle formation, P48 associated with Ac93 in the absence of viral infection.     

Baculovirus infection triggers a shift from amino acid starvation-induced autophagy to apoptosis

Autophagy plays a central role in regulating important cellular functions such as cell survival during starvation and control of infectious pathogens. On the other hand, many pathogens have evolved mechanisms of inhibition of autophagy such as blockage of the formation of autophagosomes or the fusion of autophagosomes with lysosomes. Baculoviruses are important insect pathogens for pest control, and autophagy activity increases significantly during insect metamorphosis. However, it is not clear whether baculovirus infection has effects on the increased autophagy. In the present study, we investigated the effects of the Autographa californica nucleopolyhedrovirus (AcMNPV) infection on autophagy in SL-HP cell line from Spodoptera litura induced under amino acid deprivation. The results revealed that AcMNPV infection did not inhibit autophagy but triggered apoptosis under starvation pressure. In the early stage of infection under starvation, mitochondrial dysfunction was detected, suggesting the organelles might be involved in cell apoptosis. The semi-quantitative PCR assay revealed that the expression of both p35 and ie-1 genes of AcMNPV had no significant difference between the starved and unstarved SL-HP cells. The western blot analysis showed that no cleavage of endogenous Atg6 occurred during the process of apoptosis in SL-HP cells. These data demonstrated that some permissive insect cells may defend baculovirus infection via apoptosis under starvation and apoptosis is independent of the cleavage of Atg6 in SL-HP cells.     

Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115

Per os infectivity factors PIF1 (Ac119) and PIF2 (Ac022), like P74, are essential for oral infection of lepidopteran larval hosts of Autographa californica M nucleopolyhedrovirus (AcMNPV). Here we show that Ac115 also is a PIF (PIF3) and that, unlike PIF1 and PIF2, it does not mediate specific binding of AcMNPV occlusion-derived virus (ODV) to midgut target cells. We used an improved in vivo fluorescence dequenching assay to compare binding, fusion, and competition among control AcMNPV ODV and the ODVs of AcMNPV PIF1, PIF2, and PIF3 deletion mutants. Our results showed that binding and fusion of PIF1 and PIF2 mutants, but not the PIF3 mutant, were both qualitatively and quantitatively different from those of control ODV. Unlike control and PIF3-deficient ODV, an excess of PIF1- or PIF2-deficient ODV failed to compete effectively with control ODV's binding to specific receptors on midgut epithelial cells. Moreover, the levels of PIF1- and PIF2-deficient ODV binding were depressed threefold compared to control levels. Binding, fusion, and competition by PIF3-deficient ODV, however, were all indistinguishable from those of control ODV. These results implicated PIF1 and PIF2 as ODV envelope attachment proteins that mediate specific binding to primary target cells within the midgut. In contrast, PIF3 mediates another unidentified, but critical, early event during primary infection.     

Ac23, an envelope fusion protein homolog in the baculovirus Autographa californica multicapsid nucleopolyhedrovirus,is a viral pathogenicity factor

Viral envelope fusion proteins are important structural proteins that mediate viral entry and may affect or determine the host range of a virus. The acquisition, exchange, and evolution of such envelope proteins may dramatically affect the success and evolutionary divergence of viruses. In the family Baculoviridae, two very different envelope fusion proteins have been identified. Budded virions of group I nucleopolyhedroviruses (NPVs) such as the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), contain the essential GP64 envelope fusion protein. In contrast group II NPVs and granuloviruses have no gp64 gene but instead encode a different envelope protein called F. F proteins from group II NPVs can functionally substitute for GP64 in gp64null AcMNPV viruses, indicating that GP64 and these F proteins serve a similar functional role. Interestingly, AcMNPV (and other gp64-containing group I NPVs) also contain an F gene homolog (Ac23) but the AcMNPV F homolog cannot compensate for the loss of gp64. In the present study, we show that Ac23 is expressed and is found in budded virions. To examine the function of F protein homologs from the gp64-containing baculoviruses, we generated an Ac23null AcMNPV genome by homologous recombination in E. coli. We found that Ac23 was not required for viral replication or pathogenesis in cell culture or infected animals. However, Ac23 accelerated the mortality of infected insect hosts by approximately 28% or 26 h. Thus, Ac23 represents an important viral pathogenicity factor in larvae infected with AcMNPV.     

棉铃虫单核衣壳核多角体病毒(HaSNPV)Hind III-L片段的序列分析

本文报道了棉铃虫单核衣壳核多角体病毒 (Helicoverpaarmigerasingle nucleocapsidnucleopolyhedrovirus,HaSNPV)基因组的HindIII L片段的全序列。该片段全长 2 6 35bp ,包括 5个有意义的开放阅读框 :HaSNPVORF2 2 7,晚期表达因子 10基因 (lef10 ) ,vp10 5 4基因 ,Ac5 5 (AcMNPVORF5 5的同源基因 ) ,Ac5 6 (AcMNPVORF5 6的同源基因 )。与其它 6种杆状病毒的氨基酸序列比较表明 ,HaSNPV的lef10基因与甜菜夜蛾核型多角体病毒 (SeMNPV)的同源性最高 ,为6 4 % ,与冷杉毒蛾核型多角体病毒 (OpMNPV)的同源性最低 ,为 4 3% ;HaSNPV的vp10 5 4基因与SeMNPV的同源性最高 ,为 6 5 % ,与OpMNPV的同源性最低 ,为 4 9%。序列比较表明 ,HaSNPV的LEF10与VP10 5 4蛋白与其它 6种杆状病毒具有相同的保守区和亮氨酸拉链 (leucinezipper)     

P74 mediates specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to primary cellular targets in the midgut epithelia of Heliothis virescens Larvae

P74, an envelope protein of the occlusion-derived virus (ODV) of Autographa californica M nucleopolyhedrovirus (AcMNPV), is critical for oral infection of Trichoplusia ni larvae. The role of P74 during primary infection, however, is unknown. Here we provide evidence that P74 facilitates binding of AcMNPV ODV to a specific receptor within the larval midgut epithelia of another host species, Heliothis virescens. We adapted a fluorescence dequenching assay to compare binding, fusion, and competition of wild-type AcMNPV ODV in vivo with itself and with the ODV of a p74-deficient AcMNPV mutant. We found that relative to wild-type ODV, binding and fusion of ODV deficient in P74 were both qualitatively and quantitatively different. Unlike wild-type ODV, an excess of P74-deficient ODV failed to compete effectively with wild-type ODV binding, and the overall binding level of the mutant ODV was one-third that of the wild type. These results implicated P74 as an ODV attachment protein that binds to a specific receptor on primary target cells within the midgut.