Truncated Hantavirus Nucleocapsid Proteins for Serotyping Hantaan, Seoul, and Dobrava Hantavirus Infections

Truncated recombinant nucleocapsid proteins (rNPs) of Hantaan virus (HTNV), Seoul virus (SEOV), and Dobrava virus (DOBV) were expressed by a baculovirus system. The truncated rNPs, which lacked 49 (rNP50) or 154 (rNP155) N-terminal amino acids of the NPs of HTNV, SEOV, and DOBV, were able to differentiate HTNV-, SEOV-, and DOBV-specific immune sera. Recombinant NP50s retained higher reactivities than rNP155s and were proven useful for enzyme-linked immunosorbent assay (ELISA). The ELISAs based on the rNP50s of HTNV, SEOV, and DOBV successfully differentiated three groups of patient sera, previously defined by neutralization tests: 17 with HTNV infection, 12 with SEOV infection, and 20 with DOBV infection. The entire rNP of Puumala virus (PUUV) distinguished PUUV infection from the other types of hantavirus infection. Serotyping with these rNP50s can be recommended as a rapid and efficient system for hantavirus diagnosis.     

汉坦病毒感染乳大鼠大脑皮层星形胶质细胞

目的 建立汉坦病毒（HTNV）76-118株或汉城病毒（SEOV）L99株感染乳大鼠大脑皮层星形胶质细胞的体外培养体系,并检测其对汉坦病毒的易感性。 方法 通过免疫荧光显微技术、免疫印迹法、反转录聚合酶链反应检测星形胶质细胞对汉坦病毒的易感性,并通过反转录聚合酶链反应检测病毒S片段和GFAP基因表达情况。 结果 HTNV 或SEOV感染星形胶质细胞后病毒核衣壳蛋白（NP）和GFAP的表达增加,且GFAP和NP共定位,两者可能相互作用,同时病毒S片段和GFAP基因表达增加。 结论 HTNV或SEOV能有效感染和激活星形胶质细胞,GFAP可能调节血脑屏障结构或功能的完整性、病毒NP合成及病毒复制。     

Rapid and specific detection of Sin Nombre virus antibodies in patients with hantavirus pulmonary syndrome by a strip immunoblot assay suitable for field diagnosis.

To develop a rapid antibody test for Sin Nombre hantavirus (SNV) infection for diagnosis of hantavirus pulmonary syndrome (HPS) in field settings where advanced instrumentation is not available, a strip immunoblot assay bearing four immobilized antigens for SNV and a recombinant nucleocapsid protein antigen of Seoul hantavirus (SEOV) was prepared. The SNV antigens included a full-length recombinant-expressed nucleocapsid (N) protein (rN), a recombinant-expressed G1 protein (residues 35 to 117), and synthetic peptides derived from N (residues 17 to 59) and G1 (residues 55 to 88). On the basis of the observed reactivities of hantavirus-infected patient and control sera, we determined that a positive assay requires reactivity with SNV or SEOV rN antigen and at least one other antigen. Isolated reactivity to either viral rN antigen is indeterminate, and any pattern of reactivity that does not include reactivity to an rN antigen is considered indeterminate but is unlikely to represent hantavirus infection. Fifty-eight of 59 samples from patients with acute SNV-associated HPS were positive according to these criteria, and one was initially indeterminate. Four of four samples from patients with HPS due to other hantaviruses were positive, as were most samples from patients with SEOV and Puumala virus infections. Of 192 control serum samples, 2 (1%) were positive and 2 were indeterminate. Acute SNV infection was distinguishable from remote SNV infection or infection with hantaviruses other than SNV by the presence of G1 peptide antigen reactivities in the former. The strip immunoblot assay shows promise for the detection of SNV antibodies early in the course of HPS.     

Nucleocapsid protein of cell culture-adapted Seoul virus strain 80–39: Analysis of its encoding sequence,expression in yeast and immuno-reactivity

Seoul virus (SEOV) is a hantavirus causing a mild to moderate form of hemorrhagic fever with renal syndrome that is distributed mainly in Asia. The nucleocapsid (N) protein-encoding sequence of SEOV (strain 80-39) was RT-PCR-amplified and cloned into a yeast expression vector containing a galactose-inducible promoter. A survey of the pattern of synonymous codon preferences for a total of 22 N protein-encoding hantavirus genes including 13 of SEOV strains revealed that there is minor variation in codon usage by the same gene in different viral genomes. Introduction of the expression plasmid into yeast Saccharomyces cerevisiae resulted in the high-level expression of a hexahistidine-tagged N protein derivative. The nickel-chelation chromatography purified, yeast-expressed SEOV N protein reacted in the immunoblot with a SEOV-specific monoclonal antibody and certain HTNV- and PUUV-cross-reactive monoclonal antibodies. The immunization of a rabbit with the recombinant N protein resulted in the induction of a high-titered antibody response. In ELISA studies, the N protein was able to detect antibodies in sera of experimentally infected laboratory rats and in human anti-hantavirus-positive sera or serum pools of patients from different geographical origin. The yeast-expressed SEOV N protein represents a promising antigen for development of diagnostic tools in serology, sero prevalence studies and vaccine development.     

酵母双杂交筛选与单剪接型2.2 kb乙型肝炎病毒剪接特异性新蛋白相互作用的肝细胞蛋白

目的 筛选与单剪接型2.2 kb乙型肝炎病毒(HBV)剪接特异性新蛋白相互作用的肝细胞蛋白.方法 PCR扩增单剪接型2.2 kb HBV剪接特异性新基因TPss并克隆于诱饵载体pGBKT7,在证实TPss蛋白不具有自激活作用的前提下,以酵母双杂交系统筛查与TPss蛋白相互作用的肝细胞蛋白,进而通过哺乳动物细胞双杂交实验验证候选肝细胞蛋白与TPss蛋白在Huh7和HepG2肝细胞中的相互作用.结果 构建酵母双杂交诱饵载体pGBKT7-TPss,Western blot显示其在酵母中表达TPss蛋白.酵母双杂交筛选及哺乳动物细胞双杂交证实TPss蛋白可与4种肝细胞蛋白相互作用,即组织蛋白酶B、微粒体环氧化物水解酶、组织蛋白酶D与纤维蛋白原γ链.结论 TPss可与多种肝细胞蛋白相互作用.     

Cross-reactive and serospecific epitopes of nucleocapsid proteins of three hantaviruses: prospects for new diagnostic tools

The diagnosis of infectious diseases is sometimes difficult because of extensive immunological cross-reactivity between related viral antigens. On the path of constructing sero-specific antigens, we have identified residues involved in sero-specific and cross-reactive recognition of the nucleocapsid proteins (NPs) of Puumala virus (PUUV), Seoul virus (SEOV), and Sin Nombre virus (SNV) using serum samples from 17 Nephropathia epidemica patients. The mapping was performed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis on a panel of N protein derivatives and alanine-substitution mutants in the three different hantavirus backgrounds. Four regions with different serological profiles were identified encompassing the amino acids (aa) 14-17, 22-24, 26, and 35-38. One of the regions showed strong cross-reactivity and was important for the recognition of SEOV and SNV antigens, but not the PUUV antigen (aa 35-38). Two regions displayed perceivable SEOV characteristics (aa 14-17 and aa 22-24 and 26) and the combined result of the alanine replacements resulted in a synergetic effect against the PUUV antigen (aa 14-17, 22-24, 26).     

Identification of phosphoprotein:phosphoprotein and phosphoprotein:nucleocapsid protein interaction domains of the Newcastle disease virus

Summary. The yeast two-hybrid system has been used to identify domains of the Newcastle disease virus (NDV) phosphoprotein (P) involved in self-association and interaction with the nucleocapsid protein (NP). Deletion analysis was used to map the domain(s) of the P protein involved in P:P and P:NP interactions. The C-terminal 45 amino acids (residues 247–291) were shown to play a major role in both of the interactions. Comparison of these findings with other reports suggests that paramyxoviruses are different with respect to interaction domain(s) between these two essential viral proteins involved in genome replication.     

SUMO-1 modification regulates the protein stability of the large regulatory protein Rep78 of adeno associated virus type 2 (AAV-2)

The large Rep proteins Rep78 and Rep68 of the helper-dependent adeno associated virus type 2 (AAV-2) are essential for both site-specific integration of AAV DNA in the absence of helpervirus and productive AAV replication in the presence of helpervirus. We have identified UBC9, the E2 conjugating enzyme for the small ubiquitin-related polypeptide SUMO-1, as binding partner of the large Rep proteins in yeast two-hybrid analysis and in GST pulldown assays. Modification of the large Rep proteins with SUMO-1 could be demonstrated in immunoblot analysis and in immunoprecipitations, with the lysine residue at amino acid position 84 serving as the major attachment site. The largely sumolation-deficient Rep78 lysine to arginine point mutant showed a strongly reduced half-life as compared to the wild-type protein. This finding implicates a role for sumolation in the regulation of Rep78 protein stability that is assumed to be critical for the establishment and maintenance of AAV latency.     

PIV5 M protein interaction with host protein angiomotin-like 1

Paramyxovirus matrix (M) proteins organize virus assembly, functioning as adapters that link together viral ribonucleoprotein complexes and viral glycoproteins at infected cell plasma membranes. M proteins may also function to recruit and manipulate host factors to assist virus budding, similar to retroviral Gag proteins. By yeast two-hybrid screening, angiomotin-like 1 (AmotL1) was identified as a host factor that interacts with the M protein of parainfluenza virus 5 (PIV5). AmotL1-M protein interaction was observed in yeast, in transfected mammalian cells, and in virus-infected cells. Binding was mapped to a 83-amino acid region derived from the C-terminal portion of AmotL1. Overexpression of M-binding AmotL1-derived polypeptides potently inhibited production of PIV5 VLPs and impaired virus budding. Expression of these polypeptides moderately inhibited production of mumps VLPs, but had no effect on production of Nipah VLPs. siRNA-mediated depletion of AmotL1 protein reduced PIV5 budding, suggesting that this interaction is beneficial to paramyxovirus infection.     

The Tat protein of the human immunodeficiency virus type 1 (HIV-1) interacts with the EGF-like repeats of the Notch proteins and the EGF precursor

Employing the yeast two-hybrid system, the Tat protein of the human immunodeficiency virus (HIV) was shown to interact with a region spanning the EGF-like repeats 1-6 of the mouse Notch1, the human Notch2 and the Drosophila Notch. This observation was confirmed in mammalian cells by demonstrating an interaction between the HIV Tat and the EGF-like repeats 1-6 of the various Notch proteins. The HIV Tat protein interacted also with the full-length mouse Notch1 receptor when co-expressed in mammalian cells. Moreover, the HIV Tat protein interacted also with the EGF-like repeats 1-4-spanning domain of the human EGF precursor. The ability of the HIV Tat protein to interact with the Notch proteins and possibly with other EGF-like repeats-bearing proteins, suggests that such interactions might modulate their physiological functions, thus affecting various AIDS-associated pathologies.     

Two distinct regions of the BPV1 E1 replication protein interact with the activation domain of E2

Papillomavirus E1 and E2 proteins co-operation in viral DNA replication is mediated by protein-protein interactions that lead to formation of an E1-E2 complex. To identify the domains involved, portions of the two proteins were expressed as fusions to the DNA-binding protein LexA or the transactivation domain of VP16 and analyzed by the yeast two-hybrid system. The C-terminal 266 amino acids of BPV1 E1 (E1C266) interacted strongly with E2 in the yeast system and in a mammalian two-hybrid assay. VP16-E1C266 interacted with a region encompassing amino acids 1-200 of the transactivation domain of E2 that was fused to LexA. The interaction between E1 full length and E2 was clearly observed only when E1 was expressed as LexA-E1 chimera. In addition, we found that in the LexA context also the N-terminal region encompassing the first 340 amino acids of E1 (E1N340) interacted with E2 full length. The interactions of E1N340 and E1C266 with E2 were confirmed also by in vitro binding studies. These observations demonstrate that two distinct regions of E1 mediate the interaction with E2 in vivo.     

Comparison of the protective efficacy of naked DNA, DNA-based Sindbis replicon, and packaged Sindbis replicon vectors expressing Hantavirus structural genes in hamsters.

Seoul virus (SEOV) is a member of the Hantavirus genus (family Bunyaviridae) and an etiological agent of hemorrhagic fever with renal syndrome. The medium (M) and small (S) gene segments of SEOV encode the viral envelope glycoproteins and nucleocapsid protein, respectively. We compared the immunogenicity and protective efficacy of naked DNA (pWRG7077), DNA-based Sindbis replicon (pSIN2.5), and packaged Sindbis replicon vectors (pSINrep5), containing either the M or S gene segment of SEOV in Syrian hamsters. All of the vectors elicited an anti-SEOV immune response to the expressed SEOV gene products. Vaccinated hamsters were challenged with SEOV and monitored for evidence of infection. Protection from infection was strongly associated with M-gene vaccination. A small number of S-gene-vaccinated animals also were protected. Hamsters vaccinated with the pWRG7077 vector expressing the M gene demonstrated the most consistent protection from SEOV infection and also were protected from heterologous hantavirus (Hantaan virus) infection.     

Multiple interactions among proteins encoded by the mite-transmitted wheat streak mosaic tritimovirus

The genome organization of the mite-transmitted wheat streak mosaic virus (WSMV) appears to parallel that of members of the Potyviridae with monopartite genomes, but there are substantial amino acid dissimilarities with other potyviral polyproteins. To initiate studies on the functions of WSMV-encoded proteins, a protein interaction map was generated using a yeast two-hybrid system. Because the pathway of proteolytic maturation of the WSMV polyprotein has not been experimentally determined, random libraries of WSMV cDNA were made both in DNA-binding domain and activation domain plasmid vectors and introduced into yeast. Sequence analysis of multiple interacting pairs revealed that interactions largely occurred between domains within two groups of proteins. The first involved interactions among nuclear inclusion protein a, nuclear inclusion protein b, and coat protein (CP), and the second involved helper component-proteinase (HC-Pro) and cylindrical inclusion protein (CI). Further immunoblot and deletion mapping analyses of the interactions suggest that subdomains of CI, HC-Pro, and P1 interact with one another. The two-hybrid assay was then performed using full-length genes of CI, HC-Pro, P1, P3, and CP, but no heterologous interactions were detected. In vitro binding assay using glutathione-S-transferase fusion proteins and in vitro translation products, however, revealed mutual interactions among CI, HC-Pro, P1, and P3. The failure to detect interactions between full-length proteins by the two-hybrid assay might be due to adverse effects of expression of viral proteins in yeast cells. The capacity to participate in multiple homomeric and heteromeric molecular interactions is consistent with the pleiotropic nature of many potyviral gene mutants and suggests mechanisms for regulation of various viral processes via a network of viral protein complexes.     

Self-interaction of ORF II protein through the leucine zipper is essential for Soybean chlorotic mottle virus infectivity

The ORF II protein (PII) of Soybean chlorotic mottle virus (SbCMV) is essential for the virus life cycle. We investigated the interactions of SbCMV PII with itself and with other essential virus proteins using a Gal4-based yeast two-hybrid system. PII interacted only with itself and not with any other virus proteins. The PII-PII interaction was confirmed by a Sos-based yeast two-hybrid system and a far-western analysis. Deletion mutagenesis mapped the self-interacting domain to the C-terminal 48 amino acids (amino acids 154-201), which contain two putative leucine zipper motifs. Introduction of amino acid substitutions to leucine/isoleucine in zipper sequences prevented the PII-PII interaction and abolished the infectivity of SbCMV. These results revealed that the self-interaction of PII through a leucine zipper is necessary for virus infection.     

A densely overlapping gene fragmentation approach improves yeast two-hybrid screens for Plasmodium falciparum proteins

Use of the yeast two-hybrid assay to study Plasmodium falciparum protein-protein interactions is limited by poor expression of P. falciparum genes in yeast and lack of easily implemented assays to confirm the results. We report here two methods to create gene fragments - random fragmentation by partial DNAse I digestion and generation of densely overlapping fragments by PCR - that enable most portions of P. falciparum genes to be expressed and screened in the yeast two-hybrid assay. The PCR-based method is less technically challenging and facilitates fine-scale mapping of protein interaction domains. Both approaches revealed a putative interaction between PfMyb2 (PF10_0327) and PFC0365w. We developed new plasmids to express the proteins in wheat germ extracts and confirmed the interaction in both the split-luciferase assay and in co-purification experiments with glutathione-S-transferase and HA-tagged proteins. The combination of improved yeast two-hybrid screening approaches and convenient systems to validate interactions enhances the utility of yeast two-hybrid assays for P. falciparum.