Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp)

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.     

Purification and enzymatic properties of an RNA polymerase-RNA complex from influenza virus

An RNA polymerase-viral RNA complex was purified from influenza A/PR/8 virions by combination of cesium trifluoroacetate centrifugation and phosphocellulose column chromatography. Surface proteins were removed from the detergent-treated virions by the centrifugation. Starting from the M protein-free ribonucleoprotein (RNP) fraction, an RNA polymerase-RNA complex lacking NP protein was isolated by repeated chromatography on phosphocellulose columns. The isolated RNA polymerase-RNA complex, which is composed of PB1, PB2, PA and vRNA, cleaved capped poly(A) endonucleolytically at 10-12 nucleotides from the 5' end and incorporated GMP into the 3' end of the resulting capped fragments. In the presence of all four ribonucleotide triphosphate substrates, the cleaved fragments were elongated to polynucleotides in the absence of exogenous vRNA. The RNA synthesis was primed not only by capped polynucleotides but also dinucleotide ApG. These results indicate that the purified RNA polymerase-RNA complex is as active in viral mRNAs synthesis as native RNP and that NP protein is not required for the catalytic function.     

Expression, purification, and characterization of SARS coronavirus RNA polymerase

The RNA-dependent RNA polymerase (RdRp) of SARS coronavirus (SARS-CoV) is essential for viral replication and a potential target for anti-SARS drugs. We report here the cloning, expression, and purification of the N-terminal GST-fused SARS-CoV RdRp and its polymerase catalytic domain in Escherichia coli. During purification, the full-length GST-RdRp was found to cleave into three main fragments: an N-terminal p12 fragment, a middle p30 fragment, and a C-terminal p64 fragment comprising the catalytic domain, presumably due to bacterial proteases. Biochemical assays show that the full-length GST-RdRp has RdRp activity and the p64 and p12 fragments form a complex that exhibits comparable RdRp activity, whereas the GST-p64 protein has no activity, suggesting that the p12 domain is required for polymerase activity possibly via involvement in template-primer binding. Nonnucleoside HIV-1 RT inhibitors are shown to have no evident inhibitory effect on SARS-CoV RdRp activity. This work provides a basis for biochemical and structural studies of SARS-CoV RdRp and for development of anti-SARS drugs.     

Cell culture and in vivo analyses of cytopathic hepatitis C virus mutants

HCV-JFH1 yields subclones that develop cytopathic plaques (Sekine-Osajima Y, et al., Virology 2008; 371:71). Here, we investigated viral amino acid substitutions in cytopathic mutant HCV-JFH1 clones and their characteristics in vitro and in vivo. The mutant viruses with individual C2441S, P2938S or R2985P signature substitutions, and with all three substitutions, showed significantly higher intracellular replication efficiencies and greater cytopathic effects than the parental JFH1 in vitro. The mutant HCV-inoculated mice showed significantly higher serum HCV RNA and higher level of expression of ER stress-related proteins in early period of infection. At 8 weeks post inoculation, these signature mutations had reverted to the wild type sequences. HCV-induced cytopathogenicity is associated with the level of intracellular viral replication and is determined by certain amino acid substitutions in HCV-NS5A and NS5B regions. The cytopathic HCV clones exhibit high replication competence in vivo but may be eliminated during the early stages of infection.     

Interactions between p27 and p88 replicase proteins of Red clover necrotic mosaic virus play an essential role in viral RNA replication and suppression of RNA silencing via the 480-kDa viral replicase complex assembly

Red clover necrotic mosaic virus (RCNMV), a positive-sense RNA virus with a bipartite genome, encodes p27 and p88 replicase proteins that are required for viral RNA replication and suppression of RNA silencing. In this study, we indentified domains in p27 and p88 responsible for their protein-protein interactions using in vitro pull-down assays with the purified recombinant proteins. Coimmunoprecipitation analysis in combination with blue-native polyacrylamide gel electrophoresis using mutated p27 proteins showed that both p27-p27 and p27-p88 interactions are essential for the formation of the 480-kDa complex, which has RCNMV-specific RNA-dependent RNA polymerase activity. Furthermore, we found a good correlation between the accumulated levels of the 480-kDa complex and replication levels and the suppression of RNA silencing activity. Our results indicate that interactions between RCNMV replicase proteins play an essential role in viral RNA replication and in suppressing RNA silencing via the 480-kDa replicase complex assembly.     

Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity

HCV culture in vitro results in massive cell death, which suggests the presence of HCV-induced cytopathic effects. Therefore, we investigated its mechanisms and viral nucleotide sequences involved in this effect using HCV-JFH1 cell culture and a newly developed HCV plaque assay technique. The plaque assay developed cytopathic plaques, depending on the titer of the inoculum. In the virus-infected cells, the ER stress markers, GRP78 and phosphorylated eIF2-alpha, were overexpressed. Cells in the plaques were strongly positive for an apoptosis marker, annexin V. Isolated virus subclones from individual plaque showed greater replication efficiency and cytopathogenicity than the parental virus. The plaque-purified virus had 9 amino acid substitutions, of which 5 were clustered in the C terminal of the NS5B region. Taken together, the cytopathic effect of HCV infection involves ER-stress-induced apoptotic cell death. Certain HCV genomic structures may determine the viral replication capacity and cytopathogenicity.     

Complete Nucleotide Sequence and Phylogenetic Analyses of Hepatitis B Virus Isolated from Two Pileated Gibbons

Virus-like particles (VLPs, named HmTV1-17), about 40nm in diameter were found in the violet root rot fungus Helicobasidium mompa Tanaka strain No. 17, which had been isolated from an apple tree. Purified preparations of HmTV1-17 contained two species of double-stranded RNA (dsRNA), designated 17L and 17S. cDNAs were constructed from HmTV1-17 genomic dsRNAs purified using CF-11 cellulose column chromatography. The sequences of 17L and 17S cDNA comprised 5207 and 2096bp, respectively. Although 17S has no large open reading flame (ORF) on either strand, 17L has two large overlapping ORFs. The 5 located ORF1 encodes the coat protein (CP, 788 amino acids), whereas the gene product of ORF2, which is in the –1 frame relative to ORF1, shows the typical features of a RNA dependent RNA polymerase (RDRP, 845 amino acids). Phylogenetic analysis based on RDRP showed that HmTV1-17 is closely related to Sphaeropsis sapinea SsRV1, a member of the genus Totivirus from filamentous fungus S. sapinea.     

n-Butyrate Mediated Inhibition of Papovavirus DNA Replication In Vivo and in Cell Culture: A Mechanistic Approach

n-Butyrate, an inhibitor of G₁-to-S transition inhibits papovavirus DNA replication in cell culture. To explore the efficacy of n-butyrate in vivo and to better understand its mechanism, we studied the effect of n-butyrate on viral DNA replication in mice acutely infected with polyomavirus and in the papovavirus-infected cells in culture. Newborn mice treated with n-butyrate stop growing and become runted. When infected with polyomavirus, these mice show a strong overall inhibition of viral DNA. However, a notable exception to this was the continued viral DNA replication in the differentiated mouse keratinocytes and renal epithelial cells as determined by in situ hybridization. n-Butyrate significantly inhibited viral DNA replication in the cultured IDL cells, and in polyomavirus-infected C2C12 myoblasts based on Southern blot analysis and in situ hybridization. DNA polymerase alpha (but not DNA polymerase beta) and the characteristic nuclear expression of PCNA were both inhibited in the n-butyrate treated IDL and C2C12 cells. n-Butyrate, therefore, inhibited host and viral DNA synthesis in the undifferentiated cells.     

Caraparu virus (group C <Emphasis Type="Italic">Orthobunyavirus</Emphasis>): sequencing and phylogenetic analysis based on the conserved region 3 of the RNA polymerase gene

Here, for the first time, we report the nucleotide sequence of Caraparu virus (CARV) L segment and the analysis of the RNA polymerase region 3 encoded by this segment. The 1,404 bp nucleotide sequence shares the highest identity with Bunyamwera, La Crosse, Oropouche, and Akabane virus sequences. The amino acid sequence was deduced and aligned with sequences from members of the Bunyaviridae family and used for phylogenetic analysis. The CARV clustered in the Orthobunyavirus genus. The premotif A and motifs A–E are present in the region 3 of the Bunyaviridae family, were also conserved in CARV L protein, as well as other conserved regions among Orthobunyavirus genus. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database: EF122411     

基于创新的四维密码方法分析共调控与蛋白互作关系

【摘要】目的 基于创新的四维密码子的方法挖掘共调控且互作的基因对，研究共调控与蛋白互作之间的关联趋势。方法 我们提出了创新的基于四维密码子的关联分析方法，并把此方法应用于与结肠癌相关的2000个疾病基因。结果 发现共调控的互作蛋白对比随机情况下显著；随着共调控强度的增强，蛋白间互作趋势越来越明显。结论 基于创新的四维密码子的关联分析方法，能够研究共调控与蛋白互作的关联趋势。     

肺癌患者血清中游离DNA含量与肿瘤标志物相关性分析

通过对52例肺癌患者、8例肺部良性疾病患者和10名健康人血清游离DNA含量与肿瘤标志物(TM)CA19-9、CA125、CYFRA21-1、CEA和NSE的联合检测,对两种检测结果作相关性分析。将表皮生长因子受体(EGFR)作为肿瘤基因标志物,运用荧光定量RT-PCR方法检测血清游离DNA含量。结果显示,10名健康人血清游离DNA含量平均为18.81ng/mL(范围:0.17~54.64ng/mL)。若以19ng/mL作为血清游离DNA含量的阳性临界值,则87.5%的健康人低于此水平。8例肺部良性疾病患者游离DNA含量均值为76.86ng/mL(范围:5.33~189.7ng/mL),其中4例(50%)高于阳性临界值。52例肺癌患者游离DNA均值为107.6ng/mL(范围:6.39~1617ng/mL),37例(71.2%)的肺癌患者血清游离DNA含量高于正常值。血清游离DNA含量诊断肺癌的价值等同于肿瘤标志物五项指标联合检测。其灵敏度、特异性、准确性分别为:71.15%,50%,68.3%,提示血清游离DNA含量作为一项新颖肿瘤标志物具有潜在的临床诊断价值。     

Principal host relationships and evolutionary history of the North American arenaviruses

A previous study suggested that the genomes of the arenaviruses native to North America are a product of genetic recombination between New World arenaviruses with significantly different phylogenetic histories. The purpose of this study was to extend our knowledge of the principal host relationships and evolutionary history of the North American arenaviruses. The results of this study suggest that the large-eared woodrat (Neotoma macrotis) is a principal host of Bear Canyon virus and that the present-day association of Bear Canyon virus with the California mouse (Peromyscus californicus) in southern California represents a successful host-jumping event from the large-eared woodrat to the California mouse. Together, the results of analyses of viral gene sequence data in this study and our knowledge of the phylogeography of the rodents that serve as principal hosts of the New World arenaviruses suggest that genetic recombination between arenaviruses with significantly different phylogenetic histories did not play a role in the evolution of the North American arenaviruses.     

Crystal structure of the serine protease domain of Sesbania mosaic virus polyprotein and mutational analysis of residues forming the S1-binding pocket

Sesbania mosaic virus (SeMV) polyprotein is processed by its N-terminal serine protease domain. The crystal structure of the protease domain was determined to a resolution of 2.4 A using multiple isomorphous replacement and anomalous scattering. The SeMV protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. The residues of the S1-binding pocket, H298, T279 and N308 were mutated to alanine in the DeltaN70-Protease-VPg polyprotein, and the cis-cleavage activity was examined. The H298A and T279A mutants were inactive, while the N308A mutant was partially active, suggesting that the interactions of H298 and T279 with P1-glutamate are crucial for the E-T/S cleavage. A region of exposed aromatic amino acids, probably essential for interaction with VPg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease.     

Vascular lesion in a patient of chronic active Epstein-Barr virus infection with hypersensitivity to mosquito bites: vasculitis induced by mosquito bite with the infiltration of nonneoplastic Epstein-Barr virus-positive cells and subsequent development of natural killer/T-cell lymphoma with angiodestruction

This report describes a vasculitis and subsequently developing angiodestructive lymphoma in an 11-year-old Japanese-Filipino girl exhibiting mosquito allergy with the background of chronic active Epstein-Barr virus (EBV) infection. She developed necrotic skin ulcer at the site of mosquito bite, and histopathological examination revealed EBV-positive mononuclear cell infiltration throughout the wall of small-sized muscular artery. These EBV-positive lymphoid cells were oligoclonal in Southern blot analysis for EBV terminal repeats. Effectiveness of steroid therapy also supports the nonneoplastic nature. Approximately 1 year later, she developed progressive large skin ulcer without mosquito bites. Microscopically, the angiocentric or angiodestructive pattern of EBV-positive atypical cells supported the diagnosis of extranodal natural killer/T-cell lymphoma. Southern blot analysis revealed the monoclonal neoplastic nature of EBV-positive cells. In contrast to the primary mosquito bite lesion, natural killer/T-cell lymphoma cells exhibited the higher expression of EBV latent membrane protein 1 mRNA and the apparent protein expression detected by immunohistochemistry.