小菜蛾颗粒体病毒晚期表达因子基因的功能分析

小菜蛾颗粒体病毒(Plutella xylostella granulovirus,PlxyGV)基因组含有15个杆状病毒晚期表达因子(Lateexpression factor,lef)基因同源物。PlxyGV的14个lef基因(不包括ie-0)预期编码产物与苜蓿银纹夜蛾核多角体病毒(Autographa californica multicapsid nucleopolyhedrovirus,AcMNPV)LEF蛋白的序列相似度为13%～53%。其中,LEF-8、LEF-9和P47在两种病毒之间的相似度较高,分别为49%、53%和46%。为了研究不同杆状病毒种间lef基因的功能相关性,验证PlxyGVlef基因的功能,本文利用AcMNPV的一个瞬时表达实验系统测试PlxyGVlef基因在Sf9细胞中激活AcMNPV晚期启动子控制的报告基因表达的能力。实验结果显示,在其它AcMNPVlef基因都存在的情况下,PlxyGVlef-2能够部分替代AcMNPVlef-2的活性。序列对比结果显示Plx-yGV LEF-2的C端末比其它GV和鳞翅目NPV的LEF-2分别多出约100aa和70aa。     

Differential requirements for baculovirus late expression factor genes in two cell lines.

A plasmid library of 18 late expression factor (LEF) genes (LEF library) from the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) supports transient expression from a late viral promoter in the SF-21 cell line, derived from Spodoptera frugiperda. We found, however, that this LEF library was unable to support expression from the same promoter in the TN-368 cell line, derived from Trichoplusia ni, which is also permissive for AcMNPV replication. To identify the additional factor(s) required for expression in TN-368 cells, we cotransfected the LEF library with clones representing portions of the AcMNPV genome not represented in the LEF library. A single additional gene was identified; this gene corresponded to ORF70 of the complete AcMNPV sequence and potentially encodes a 34-kDa cysteine-rich polypeptide. Because of its differential effect on late gene expression in the two cell lines, we renamed ORF70 hcf-1 (for host cell-specific factor 1). hcf-1 was involved in expression from reporter plasmids under late and very late but not early promoter control, indicating that it was also a LEF gene. Plasmid DNA replication assays indicated that HCF-1 was involved in virus origin-specific DNA replication in TN-368 cells. Three LEF genes, ie-2, lef-7, and p35, required for optimal virus origin-specific plasmid DNA replication or stability in SF-21 cells had little or no influence in TN-368 cells. Thus, as determined by transient-expression assays, cell line-specific and potentially host-specific factors are required for origin-specific DNA replication or stability.     

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.     

Genetic requirements for homologous recombination in Autographa californica nucleopolyhedrovirus

It is known that baculovirus infection promotes high-frequency recombination between its genomes and plasmid DNA during the construction of recombinant viruses for foreign gene expression. However, little is known about the viral genes necessary to promote homologous recombination (HR). We developed an assay to identify viral genes that are necessary to stimulate HR. In this assay, we used two plasmids containing extensive sequence homology that yielded a visible and quantifiable phenotype if HR occurred. The plasmids contained the green fluorescent protein gene (gfp) that was mutated at either the N or the C terminus and a viral origin of DNA replication. When the plasmids containing these mutant gfp genes were transfected into insect cells alone or together, few green fluorescent protein (GFP)-positive cells were observed, confirming that the host cell machinery alone was not able to promote high levels of HR. However, if viral DNA or viral genes involved in DNA replication were cotransfected into cells along with the mutant gfp-containing plasmids, a dramatic increase in GFP-positive cells was observed. The viral genes ie-1, ie-2, lef-7, and p35 were found to be important for efficient HR in the presence of all other DNA replication genes. However, ie-1 and ie-2 were sufficient to promote HR in the absence of other viral genes. Recombination substrates lacking a viral origin of replication had similar genetic requirements for recombination but were less dependent on ie-1. Interestingly, even though HR was stimulated by the presence of a viral origin of DNA replication, virally stimulated HR could proceed in the presence of the DNA synthesis inhibitor aphidicolin.     

Baculovirus GP64-mediated entry into mammalian cells

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.     

昆虫杆状病毒应用于哺乳动物基因治疗的研究进展

杆状病毒是一类宿主特异性的昆虫病毒。昆虫杆状病毒表达系统是一个高效的真核表达系统,被广泛用于在昆虫细胞或昆虫幼虫中生产外源蛋白质。杆状病毒不能感染哺乳动物,却可以进入不同物种和组织来源的多种哺乳动物细胞,并在合适的哺乳动物启动子控制下表达外源基因。杆状病毒在哺乳动物细胞中不能复制,对细胞没有毒性,加上杆状病毒本身具有基因组大、可操作性好等优点,作为哺乳动物基因治疗的载体,将治疗基因传递给哺乳动物细胞已受到了广泛关注。在此就杆状病毒作为基因治疗载体的最新研究进展进行了阐述并探讨其发展趋势。