Baculovirus VP80 protein and the F-actin cytoskeleton interact and connect the viral replication factory with the nuclear periphery

Recently, we showed that the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) VP80 protein is essential for the formation of both virion types, budded virus (BV) and occlusion-derived virus (ODV). Deletion of the vp80 gene did not affect assembly of nucleocapsids. However, these nucleocapsids were not able to migrate from the virogenic stroma to the nuclear periphery. In the current paper, we constructed a baculovirus recombinant with enhanced-green fluorescent protein (EGFP)-tagged VP80, allowing visualization of the VP80 distribution pattern during infection. In baculovirus-infected cells, the EGFP-VP80 protein is entirely localized in nuclei, adjacent to the virus-triggered F-actin scaffold that forms a highly organized three-dimensional network connecting the virogenic stroma physically with the nuclear envelope. Interaction between VP80 and host actin was confirmed by coimmunoprecipitation. We further showed that VP80 is associated with the nucleocapsid fraction of both BVs and ODVs, typically at one end of the nucleocapsids. In addition, the presence of sequence motifs with homology to invertebrate paramyosin proteins strongly supports a role for VP80 in the polar transport of nucleocapsids to the periphery of the nucleus on their way to the plasma membrane to form BVs and for assembly in the nuclear periphery to form ODVs for embedding in viral occlusion bodies.     

Identification of domains in Autographa californica multiple nucleopolyhedrovirus late expression factor 3 required for nuclear transport of P143

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 3 (LEF-3) is an essential protein for DNA replication in transient assays. P143, a large DNA-binding protein with DNA-unwinding activity, is also essential for viral DNA replication in vivo. Both LEF-3 and P143 are found in the nucleus of AcMNPV-infected cells, but only LEF-3 localizes to the nucleus when expressed in transfected cells on its own from a plasmid expression vector. P143 requires LEF-3 as a transporter to enter the nucleus. To investigate the possibility that LEF-3 carries a nuclear localization signal domain, we constructed a series of LEF-3 deletion mutants and examined the intracellular localization of the products in plasmid-transfected cells. We discovered that the N-terminal 56 amino acid residues of LEF-3 were sufficient for nuclear localization and that this domain, when fused with either the green fluorescent protein reporter gene or P143, was able to direct these proteins to the nucleus. Transient DNA replication assays demonstrated that fusing the LEF-3 nuclear localization signal domain to P143 did not alter the function of P143 in supporting DNA replication but was not sufficient to substitute for whole LEF-3. These data show that although one role for LEF-3 during virus infection is to transport P143 to the nucleus, LEF-3 performs other essential replication functions once inside the nucleus.     

hhLIM与actin相互作用依赖其C端LIM结构域

hhLIM是LIM蛋白家族成员之一,该蛋白质含有两个LIM结构域,在基因表达调节、细胞骨架组构及细胞肥大过程中发挥重要作用.构建hhLIM不同LIM结构域的突变体,探讨其两个LIM结构域在与actin相互结合中的作用及其可能机制.GST-pull down和hhLIM及其突变体与actin细胞定位关系的免疫荧光分析结果表明,C端的LIM结构域2是hhLIM与actin结合所必需的,该结构域中的两个Cys置换为Ser后可使hhLIM结合actin的功能完全丧失,N端的LIM结构域1突变使hhLIM结合actin的能力下降.F-actin交联实验结果显示,hhLIM通过LIM结构域2与actin直接结合并起到交联F-actin的作用.结果表明,LIM结构域2在hhLIM与actin相互作用及调节actin细胞骨架组构中起决定性作用.     

Mechanisms for flexibility in DNA sequence recognition and VP16-induced complex formation by the Oct-1 POU domain.

DNA binding by the Oct-1 protein is directed by its POU domain, a bipartite DNA-binding domain made up of a POU-specific (POUS) domain and a POU-homeo (POUH) domain, two helix-turn-helix-containing DNA-binding modules that cooperate in DNA recognition. Although the best-characterized DNA target for Oct-1 binding is the octamer sequence ATGCAAAT, Oct-1 also binds a number of different DNA sequence elements. For example, Oct-1 recognizes a form of the herpes simplex virus VP16-responsive TAATGARAT element, called the (OCTA-)TAATGARAT site, that lacks octamer site similarity. Our studies suggest two mechanisms by which Oct-1 achieves flexible DNA sequence recognition. First, an important arginine found in the Oct-1 POUS domain tolerates substitutions of its base contacts within the octamer site. Second, on the (OCTA-)TAATGARAT site, the POUS domain is located on the side of the POUH domain opposite from where it is located on an octamer site. This flexibility of the Oct-1 POU domain in DNA binding also has an impact on its participation in a multiprotein-DNA complex with VP16. We show that Oct-1 POUS domain residues that contact DNA have different effects on VP16-induced complex formation depending on whether the VP16-responsive element involved has overlapping octamer similarity or not.     

HCF-dependent nuclear import of VP16.

Transactivation by VP16 requires the formation of a multicomponent complex, the TAATGAAAT recognition factor complex (TRF.C), that contains in addition to VP16, two cellular proteins, Oct-1 and HCF. HCF binds directly to VP16 and this promotes subsequent interaction of the VP16-HCF complex with the POU DNA-binding domain of Oct-1 and selective assembly onto target sites. Here we demonstrate a novel role of HCF in the intracellular compartmentalization of VP16. We show that while VP16 does not contain a consensus nuclear localization signal (NLS) and is largely cytoplasmic, co-expression with HCF resulted in VP16 nuclear accumulation. A candidate NLS within the C-terminus of HCF was identified and insertion of this motif into green fluorescent protein (GFP) promoted nuclear accumulation. Conversely, removal of this signal from HCF (HCFDeltaNLS) resulted in its cytoplasmic accumulation. Co-expression of HCFDeltaNLS with wild-type (wt) VP16, or of wt HCF with VP16 mutants lacking HCF-binding activity failed to promote the nuclear enrichment of VP16. These results indicate that in addition to its role in stabilizing TRF.C, HCF acts as a nuclear import factor for VP16.     

The three-dimensional structure of the C-terminal DNA-binding domain of human Ku70.

The proteins Ku70 (69.8 kDa) and Ku80 (82.7 kDa) form a heterodimeric complex that is an essential component of the nonhomologous end joining DNA double-strand break repair pathway in mammalian cells. Interaction of Ku with DNA is central for the functions of Ku. Ku70, which is mainly responsible for the DNA binding activity of the Ku heterodimer, contains two DNA-binding domains. We have solved the solution structure of the Ku80-independent DNA-binding domain of Ku70 encompassing residues 536-609 using nuclear magnetic resonance spectroscopy. Residues 536-560 are highly flexible and have a random structure but form specific interactions with DNA. Residues 561-609 of Ku70 form a well defined structure with 3 alpha-helices and also interact with DNA. The three-dimensional structure indicates that all conserved hydrophobic residues are in the hydrophobic core and therefore may be important for structural integrity. Most of the conserved positively charged residues are likely to be critical for DNA recognition. The C-terminal DNA-binding domain of Ku70 contains a helix-extended strand-helix motif, which occurs in other nucleic acid-binding proteins and may represent a common nucleic acid binding motif.