C and V proteins of Sendai virus target signaling pathways leading to IRF-3 activation for the negative regulation of interferon-beta production

We here report a molecular basis for downregulation of interferon (IFN)-beta production by V and C proteins of Sendai virus (SeV). The infection of HeLa cells with SeV poorly induced IFN-beta even if the expression of C/C' was disrupted. In contrast, when the expression of C/C'/Y1/Y2 or V/W was disrupted, SeV infection strongly induced IFN-beta production and significantly activated the interferon regulatory factor (IRF)-3 pathway. The independent expression of C or V inhibited the double-stranded (ds) RNA- or Newcastle disease virus (NDV)-induced activation of IRF-3 and NF-kappa B, as well as the IFN-beta promoter. This inhibitory effect was also observed when Y1, Y2, or a C-terminal half fragment (aa 85-204) of C was independently expressed. Phosphorylation and homodimer formation of IRF-3 were suppressed not only in cells infected with SeV capable of expressing both C/C'/Y1/Y2 (or Y1/Y2) and V/W, but also in HeLa cells constitutively expressing Y1. These results suggest that C, Y1, Y2, and V block signaling pathways leading to IRF-3 activation to downregulate IFN-beta production.     

Molecular and functional interactions of cat APOBEC3 and feline foamy and immunodeficiency virus proteins: different ways to counteract host-encoded restriction

Defined host-encoded feline APOBEC3 (feA3) cytidine deaminases efficiently restrict the replication and spread of exogenous retroviruses like Feline Immunodeficiency Virus (FIV) and Feline Foamy Virus (FFV) which developed different feA3 counter-acting strategies. Here we characterize the molecular interaction of FFV proteins with the diverse feA3 proteins. The FFV accessory protein Bet is the virus-encoded defense factor which is shown here to bind all feA3 proteins independent of whether they restrict FFV, a feature shared with FIV Vif that induces degradation of all feA3s including those that do not inactivate FIV. In contrast, only some feA3 proteins bind to FFV Gag, a pattern that in part reflects the restriction pattern detected. Additionally, one-domain feA3 proteins can homo- and hetero-dimerize in vitro, but a trans-dominant phenotype of any of the low-activity feA3 forms on FFV restriction by one of the highly-active feA3Z2 proteins was not detectable.     

MAP kinases and NF-kappaB collaborate to induce ICAM-1 gene expression in the early phase of adenovirus infection

Replication-defective adenoviruses (Ad) utilized as vectors for gene transfer are known to induce an inflammatory and immune response upon exposure to respiratory cells in vitro and in vivo. Among the different mediators of inflammation, we recently demonstrated that a replication-defective Ad serotype 5, deleted in the early genes E1 and E3 (Ad.CFTR), induces the proinflammatory intercellular adhesion molecule 1 (ICAM-1) in A549 respiratory cells in vitro and in lung portions of nonhuman primates in vivo, Gene Ther. 5, 131-136). More recently, we described the involvement of the nuclear factor kappaB (NF-kappaB) in the induction of ICAM-1 upon 24 h of exposure of the same Ad5-derived vector, Gene Ther. 8, 1436-1442). Here we investigated whether the early phase of virus-cell interaction is sufficient to stimulate ICAM-1 upregulation. A549 cells were exposed to wild-type Ad5 (Ad5), to Ad.CFTR, and to Ad5 inactivated by incubation at 56 degrees C (Ad5/56 degrees C). Ad5, Ad.CFTR, and Ad5/56 degrees C activated NF-kappaB and increased ICAM-1 mRNA levels within 4 h after exposure. The role of the mitogen-activated protein kinases (MAPKs) on the ICAM-1 mRNA induction was studied. ICAM-1 mRNA upregulation was inhibited upon incubation with several chemicals, namely, the ERK1/2 inhibitors PD98059 and AG1288 (by 98 and 67%, respectively), of the p38/MAPK pathway SB203580 (by 50%), of the JNK pathway dimethylaminopurine (by 83%), and of the NF-kappaB parthenolide (by 96%). Ad5 and Ad5/56 degrees C stimulated ERK1/2, p38/MAPK, and JNK1 starting 10 min and peaking 20-30 min after exposure. The present results indicate a link between the activation of the three major MAPK pathways, NF-kappaB, and the upregulation of ICAM-1 gene expression evoked by Ad5 in the very initial phase of infection.     

The identification and characterization of a monoclonal antibody to the vaccinia virus E3 protein

Monoclonal antibodies with reactivity to vaccinia virus specific proteins are useful reagents to study the proteins as well as to help understand aspects of the poxvirus life cycle. Using a vaccinia virus proteomics microarray, we found a hybridoma (MAb 3015B2) from a vaccinia virus vaccinated mouse that reacted with the product of the E3L gene. The specificity to the E3 protein was confirmed by Western blotting and immunofluorescence of cells infected with either wild-type vaccinia virus or a mutant virus with the E3L gene deleted. Antibody reactivity with E3 was also seen in cells transfected with a plasmid expressing the E3 protein. A panel of mutated vaccinia viruses with truncations in the E3L gene revealed that while MAb 3015B2 reacted with E3 lacking the C-terminal 7 amino acids, it lost reactivity with a mutant E3 lacking the C-terminal 26 amino acids. This indicates that the antigenic site recognized by 3015B2 is on the C-terminus, somewhere between amino acids 164 through 183. The antibody also recognizes the E3 protein encoded by other orthopoxviruses. This antibody will be useful for further investigations of the E3 protein as well as a useful reagent to indicate vaccinia virus early protein expression.     

Measles virus protein-specific IgM, IgA, and IgG subclass responses during the acute and convalescent phase of infection

The availability of new generation serological assays allowed re-evaluation of the antibody response to measles virus. IgM, IgA, total IgG, and IgG subclass responses were studied to the three major immunogenic measles virus proteins: the fusion protein (F), haemagglutinin (H), and nucleoprotein (N). Plasma samples were obtained from clinically diagnosed measles cases (n = 146) in Khartoum (Sudan) within a week after onset of the rash. Convalescent phase samples were collected from 32 of 117 laboratory-confirmed measles cases at different time points after onset of rash. Glycoprotein-specific IgM, IgG, and IgA antibody levels correlated well to the N-specific response. For IgG and IgA, responses to F were higher than to H. IgA antibody levels were undetectable in about one third of the laboratory-confirmed cases during the acute phase, but positive in all patients tested 1-4 weeks after infection. IgM levels declined rapidly and were lost 3-6 months after infection. IgA levels declined slowly during the first year but did not return to background levels during the subsequent 2 years. IgG avidity maturation was detected during a 3-6 month period after infection. The predominant IgG subclasses during the acute phase were IgG(1) and IgG(3). The latter was lost in the convalescent phase, while the IgG(4) isotype showed a slight rise afterwards. Interestingly, acute phase IgG(3) and IgA responses were associated, and were only detected in samples with high IgG. This study provides a comprehensive perspective on the antibody response to wild-type measles virus infection.     

Analysis of the human serological immune response to a variable region of the attachment (G) protein of respiratory syncytial virus during primary infection

The serum antibody responses of babies to the variable carboxy-terminal region of the attachment (G) protein of respiratory syncytial virus (RSV) have been analysed using paired acute and convalescent sera from infants experiencing their first RSV infection with viruses of known genotype. The variable 84–85 carboxy-terminal amino acids of the G protein of six recent isolates of group A RSV were expressed in Escherichia coli as fusion proteins with glutathione S-transferase. About half the infants developed antibodies which recognised these fusion proteins. The patterns of response obtained in enzyme linked immunosorbant assays and immunoblotting assays were closely related to the infecting genotype. © 1996 Wiley-Liss, Inc.     

甲型H3N2流感病毒截短型PB1-F2蛋白与宿主蛋白的相互作用的初步研究

目的利用酵母双杂交技术研究甲型H3N2流感病毒截短型PB1-F2蛋白与人类宿主蛋白的相互作用,为该病毒蛋白的功能研究和致病机制提供理论依据。方法以本实验室分离和鉴定的甲型H3N2流感病毒A／Guangdong／7028／2010为模版,构建pGBKT7-PB1-F2重组载体,利用Y2HGold酵母双杂交系统,从人类通用cDNA文库中筛选与其相互作用的蛋白。结果成功构建含诱饵蛋白基因的pGBKT7-PB1-F2重组载体,转化酵母自激活和毒性实验显示为阴性：酵母双杂交实验显示Y2HGold和Y187酵母的结合率为5．22％,符合实验要求;经筛选和验证后,得到3个与截短型PB1-F2蛋白有相互作用的阳性克隆,分别为钾／钠ATP酶B1亚基、热休克蛋白40和白介素-2受体1亚基。结论初步推断截短型H3N2流感病毒PB1-F2蛋白可能影响流感病毒在宿主细胞中的复制功能和凋亡调控。     

The Orf virus E3L homologue is able to complement deletion of the vaccinia virus E3L gene in vitro but not in vivo

Orf virus (OV), the prototypic parapoxvirus, is resistant to the effects of interferon (IFN) and this function of OV has been mapped to the OV20.0L gene. The protein product of this gene shares 31% amino acid identity to the E3L-encoded protein of vaccinia virus (VV) that is required for the broad host range and IFN-resistant phenotype of VV in cells in culture and for virulence of the virus in vivo. In this study we investigated whether the distantly related OV E3L homologue could complement the deletion of E3L in VV. The recombinant VV (VV/ORF-E3L) expressing the OV E3L homologue in place of VV E3L was indistinguishable from wt VV in its cell-culture phenotype. But VV/ORF-E3L was over a 1000-fold less pathogenic than wt VV (LD(50) > 5 x 10(6) PFU, compared to LD(50) of wtVV = 4 x 10(3) PFU) following intranasal infection of mice. While wt VV spread to the lungs and brain and replicated to high titers in the brain of infected mice, VV/ORF-E3L could not be detected in the lungs or brain following intranasal infection. VV/ORF-E3L was at least 100,000-fold less pathogenic than wt VV on intracranial injection. Domain swap experiments demonstrate that the difference in pathogenesis maps to the C-terminal domain of these proteins. This domain has been shown to be required for the dsRNA binding function of the VV E3L.     

Genetic and phylogenetic analysis of the non-structural proteins NS1, NS2 and NS3 of epizootic haemorrhagic disease virus (EHDV)

Three unique non-structural (NS) proteins are produced by Epizootic haemorrhagic disease virus (EHDV) during infection of a host cell; NS1, NS2 and NS3. This study presents a complete genetic and phylogenetic analysis of these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each. Accession numbers, gene and protein sizes, ORF positions, G + C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. Unlike the core, or outer-coat proteins, there are no characteristic genetic or phylogenetic traits common to all of the EHDV NS proteins; indicating that each is evolving under different selection pressures. These differences are discussed. Evidence of genetic recombination in genome segment 8 (coding for NS2) is also presented, together with evidence of gene duplication and mutation, suggesting the EHDV genome may have evolved using mechanisms such as these.     

The cysteine-histidine-rich region of the movement protein of Cucumber mosaic virus contributes to plasmodesmal targeting, zinc binding and pathogenesis

Viral movement proteins (MPs) are central to the establishment of viral pathogenesis, and yet relatively little is understood about the structural and functional aspects of MPs or about the host factors on which they depend. Through chemical mutagenesis of transgenic Arabidopsis expressing Cucumber mosaic virus (CMV) MP fused with the green fluorescent protein, we have studied the function of a central region of the MP, defined by a number of conserved cysteine and histidine residues (Cys-His-rich region), which potentially functions as a zinc-binding domain. Transient expression of mutant MPs identified through an in planta screen for altered MP function or constructed with altered putative zinc ligands through site-directed mutagenesis showed that mutations in the Cys-His-rich region affected localization to and trafficking through plasmodesmata. In vitro zinc-binding analysis revealed that wild type (wt) CMV MP had the ability to bind zinc and that movement-defective mutants bound zinc with less affinity than wt MP. Furthermore, a correlation between the association of the MP with plasmodesmata and virus pathogenesis was shown. We discuss roles of the Cys-His region in biochemical and biological functions of the MP during virus movement.     

N-Glycosylation contributes to the limited cross-reactivity between hemagglutinin neuraminidase proteins of human parainfluenza virus type 4A and 4B

cDNAs encoding human parainfluenza virus type 4B (hPIV-4B) hemagglutinin neuraminidase (HN) protein were cloned and the nucleotide sequences were determined. A high degree of identity (81.4%) was observed between the nucleotide sequences of hPIV-4A and -4B HN proteins, and an 87.3% identity was found between the deduced amino acid sequences. This degree of identity is considered to be greater than immunological similarity between hPIV-4A and -4B HN proteins determined using monoclonal antibodies. To elucidate the causes of the antigenic difference between HN proteins of hPIV-4A and -4B, we constructed three cDNAs of hPIV-4B HN whose potential N-glycosylation sites were partially or completely the same as in hPIV-4A HN cDNA. We compared the antigenicity of the expressed wild-type and mutant proteins, and found that the antigenicities of the mutant hPIV-4B HN proteins were more similar to the hPIV-4A HN protein than to the non-mutant hPIV-4B HN protein. This study indicated that the antigenic diversity between hPIV-4A and -4B was partly caused by deletion or creation of glycosylation sites, showing that the point mutations resulting in deletion or creation of glycosylation sites is one of the initial steps leading to the division of virus into subtypes. Received: 21 January 2000     

The baculovirus P10 protein of Autographa californica nucleopolyhedrovirus forms two distinct cytoskeletal-like structures and associates with polyhedral occlusion bodies during infection

The role of the microtubule-associated P10 protein of baculoviruses is not yet understood. P10 has previously been linked with the formation of a number of cytoskeletal-like or cytoskeleton-associated structures in the nucleus and cytoplasm, thought to be involved in the morphogenesis of virus polyhedral occlusion bodies. The formation of these structures was studied by immunofluorescence laser scanning confocal microscopy in TN368 cells, a model system amenable to the study of virus interaction with the host cell cytoskeleton. We show that the Autographa californica nucleopolyhedrovirus P10 protein forms two distinct cytoskeletal-like structures, microtubule-associated filaments and perinuclear tubular aggregates. P10 also associates with polyhedral occlusion bodies. Depolymerisation of the microtubule network with colchicine prevents formation of P10 filaments but not of P10 tubules. Colchicine treatment enhances the association of P10 with occlusion bodies. Transient expression of P10 showed that both filaments and tubules can form in the absence of other viral proteins. We postulate a number of possible roles of the P10 protein during virus infection and morphogenesis.     

Mosquito La protein binds to the 3' untranslated region of the positive and negative polarity dengue virus RNAs and relocates to the cytoplasm of infected cells

The untranslated regions (UTRs) of the positive and negative strand RNAs of several viruses are major binding sites for cellular and viral proteins. Human La autoantigen is one of the cellular proteins that interacts with various positive strand RNA viral genomes including that of dengue virus (DEN) within the 5'- and 3'-UTRs of positive (+) and the 3'-UTR of negative strand (-) RNA, and with the nonstructural proteins NS3 and NS5, that form DEN replicase complex. Since DEN replicates in human and mosquito cells, some functional interactions have to be conserved in both hosts. In the present report, we demonstrate that mosquito La protein interacts with the 3'-UTRs of (+) and (-) polarity viral RNAs. The localization of La protein, examined by confocal microscopy, indicates that La protein is redistributed in DEN-infected cells. Furthermore, the presence of La protein in an in vitro replication system inhibited RNA synthesis in a dose-dependent manner, suggesting that La protein plays an important role in dengue virus replicative cycle.     

The fourth genus in the Orthomyxoviridae: sequence analyses of two Thogoto virus polymerase proteins and comparison with influenza viruses

The tick-borne Thogoto virus (THOV) is the type species of a newly recognized fourth genus, Thogotovirus, in the family Orthomyxoviridae. Because of the distant relationship of THOV with the influenza viruses, determination of its genomic information can potentially be used to identify important domains in influenza virus proteins. We have determined the complete nucleotide sequence of the second longest RNA segment of THOV. The molecule comprises 2212 nucleotides with a single large open reading frame encoding a protein of 710 amino acids, estimated M_r 81 284. The protein shares 77% amino acid similarity with the PB1-like protein of Dhori virus, a related tick-borne virus, and 50–53% with the PB1 polymerase proteins of influenza virus A, B and C. All the motifs characteristic of RNA-dependent polymerases were identified including the SSDD motif common to all RNA-dependant RNA polymerases, indicating that the THOV protein is functionally analogous to the influenza virus PB1 proteins and involved in chain elongation. We also report the corrected sequence of the third longest RNA segment of THOV, encoding a protein which shares 44–47% amino acid similarity with the PA-like polymerase proteins of influenza virus A, B and C. The biological significance of conserved domains in these orthomyxovirid proteins is discussed.     

Cell-to-cell movement of Alfalfa mosaic virus can be mediated by the movement proteins of Ilar-, bromo-, cucumo-, tobamo- and comoviruses and does not require virion formation

RNA 3 of Alfalfa mosaic virus (AMV) encodes the movement protein (MP) and coat protein (CP). Chimeric RNA 3 with the AMV MP gene replaced by the corresponding MP gene of Prunus necrotic ringspot virus, Brome mosaic virus, Cucumber mosaic virus or Cowpea mosaic virus efficiently moved from cell-to-cell only when the expressed MP was extended at its C-terminus with the C-terminal 44 amino acids of AMV MP. MP of Tobacco mosaic virus supported the movement of the chimeric RNA 3 whether or not the MP was extended with the C-terminal AMV MP sequence. The replacement of the CP gene in RNA 3 by a mutant gene encoding a CP defective in virion formation did not affect cell-to-cell transport of the chimera's with a functional MP. A GST pull-down technique was used to demonstrate for the first time that the C-terminal 44 amino acids of the MP of a virus belonging to the family Bromoviridae interact specifically with AMV virus particles. Together, these results demonstrate that AMV RNA 3 can be transported from cell-to-cell by both tubule-forming and non-tubule-forming MPs if a specific MP-CP interaction occurs.     

The outcome of cross‐priming during virus infection is not directly linked to the ability of the antigen to be cross‐presented

The initiation of CD8⁺ T cell (CTL) immune responses can occur via cross‐priming. Recent data suggested a relationship between cross‐presentation and immunodominance of epitope‐specific T cells. To test this association, we evaluated the efficacy of cross‐presentation for several virus epitopes in vitro and examined if this can be extrapolated in vivo. Employing lymphocytic choriomeningitis virus (LCMV), we demonstrate that the cross‐presentation and cross‐priming of LCMV antigens were dominated by NP396, but not NP205 when analyzing the LCMV‐NP. Although with LCMV‐GP, cross‐presentation was dominated by GP276, and cross‐priming was dominated by GP33. Importantly, although NP396 was significantly more efficient than GP33 in cross‐presentation, cross‐priming of their specific CTL was comparable. In a subsequent virus challenge after cross‐priming, GP33‐specific CTL dominated the response. Accordingly, based on our data, the ability of viral epitopes to be cross‐presented in vitro does not entirely reflect what would occur in cross‐priming. Thus, weak cross‐presenting antigens may still cross‐prime an efficient CTL response depending on other in vivo elements such as the naïve T‐cell precursor frequencies.     

深圳市首例本地登革热3型病例的病原学检测与分析

目的对2016年深圳市报告的首例本地疑似登革热病例查明病因.分离鉴定病原体,从分子水平分析分离株的生物学特征.方法对疑似患者血清标本采用ELISA、胶体金免疫层析法和荧光RT-PCR方法分别检测登革病毒抗体、NS1抗原和病毒核酸,并用C6/36细胞分离登革病毒.采用RT-PCR方法扩增病毒PrM/M-E基因后进行序列测定,并与不同国家和地区的登革毒株进行同源性比较和进化树分析.结果从患者血清标本中检测到登革病毒IgM抗体、NS1抗原和登革3型病毒核酸,并成功分离到登革3型病毒,将其命名为DENV3-SZ1648.深圳市登革3型病毒分离株SZ1648与登革3型国际标准株H87株、国内外流行株80-2、GWL-25株在PrM/M-E基因上核苷酸同源性分别为92.0％、91.8％和90.3％,而与登革1、2、4型国际标准株HAWAII、NGC、H241同源性分别为68.7％、64.2％和63.2％.进化树显示SZ1648株与2007年印度尼西亚分离株MKS-0098亲缘关系最近,在进化树的同一分支上,和85-159株(Indonedia 1985)、2167株(Tahiti 1989)、29472株(Fiji1992)等同属基因Ⅰ亚型.患者发病前1个月在深圳居住,无输血史、无外出史.结论从病原学、血清学和分子生物学特征上均证实该本地病例是由登革3型病毒引起,这也是深圳市首次报道存在本地登革3型病毒的疫情,该毒株最有可能来源于印度尼西亚.