Capsid Protein Open Reading Frame Research Articles

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream.

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein.IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed.

Molecular epidemiology of genogroup II norovirus infections in acute gastroenteritis patients during 2014\u20132016 in Pudong New Area, Shanghai, China

BackgroundNorovirus (NoV), a member of the Caliciviridae, is now recognized as the leading cause of acute gastroenteritis (AGE) worldwide. Globally, the GII.4 Sydney_2012 variant has predominated in NoV-related AGE since 2012, although the novel variant GII.17 has also been reported as responsible for gastroenteritis outbreaks in East Asia since 2014. This study aimed to disclose the recent genotype patterns of NoV genogroup II (GII) presenting in AGE patients in Pudong New Area of Shanghai through a laboratory-based syndromic surveillance system. The study further aimed to delineate the predominant strains circulating in the population.MethodsPudong New Area is located in eastern Shanghai and covers 20.89% of the Shanghai population. The laboratory-based syndromic surveillance system is composed of 12 sentinel hospitals among the 68 general hospitals in this area. AGE patients who sought medical care were sampled following an AGE surveillance protocol. Stool samples were collected from participating patients, and a standardized questionnaire was given to each patient by trained nurses to gain information on the disease profiles and demographics of the patients. Real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to screen the GI nd GII NoV and RT-PCR was used to amplify NoV GII partial capsid protein open reading frame 2 (ORF2). NoV Genotyping Tool (version 1.0, RIVM, MA Bilthoven, Netherlands) was used for genotyping, and a phylogenetic analysis was conducted by MEGA 7.0.ResultsDuring 2014–2016, among the 2069 virus-infected AGE cases, 65.88% were caused by NoV. NoV-AGE occurred most frequently in the periods from October to March. The patients with more severe diarrheal symptoms and vomiting were more likely to be infected by NoV. The main genotypes were GII.17 (44.69%) and GII.4 (39.26%), which dominated the NoV-AGE epidemics jointly or in turn, whereas a slight increase in GII.2 was observed beginning in May 2016. The GII.17 strains tended to cluster more with the Hu/JP/2014/GII.P17_GII.17/Kawasaki323 variants, representing novel prevalent strains. Among the GII.4 strains, the GII.4 Sydney_2012 variant was still the predominant strain.ConclusionsNoV GII has become the main cause of virus-infected AGE in Pudong New Area, Shanghai. The predominant genotypes of NoV GII were GII.17 and GII.4. Comprehensive laboratory-based surveillance is important for clinical diagnosis and treatment. Identification of emerging new genotypes is also crucial for the prevention and control of NoV-infected AGE.

Whole-Genome Synthesis and Characterization of Viable S13-Like Bacteriophages

BackgroundUnprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template.Methodology/Principal FindingsThe sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl2, the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition.Conclusions/SignificanceThe bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.

A Local, Interactive Network of 3′ RNA Elements Supports Translation and Replication of Turnip Crinkle Virus

The majority of the 3' untranslated region (UTR) of Turnip crinkle virus (TCV) was previously identified as forming a highly interactive structure with a ribosome-binding tRNA-shaped structure (TSS) acting as a scaffold and undergoing a widespread conformational shift upon binding to RNA-dependent RNA polymerase (RdRp). Tertiary interactions in the region were explored by identifying two highly detrimental mutations within and adjacent to a hairpin H4 upstream of the TSS that reduce translation in vivo and cause identical structural changes in the loop of the 3' terminal hairpin Pr. Second-site changes that compensate for defects in translation/accumulation and reverse the structural differences in the Pr loop were found in the Pr stem, as well as in a specific stem within the TSS and within the capsid protein (CP) coding region, suggesting that the second-site changes were correcting a conformational defect and not restoring specific base pairing. The RdRp-mediated conformational shift extended upstream through this CP open reading frame (ORF) region after bypassing much of an intervening, largely unstructured region, supporting a connection between 3' elements and coding region elements. These data suggest that the Pr loop, TSS, and H4 are central elements in the regulation of translation and replication in TCV and allow for development of an RNA interactome that maps the higher-order structure of a postulated RNA domain within the 3' region of a plus-strand RNA virus.

Hepatitis E Virus ORF2 Protein Activates the Pro-Apoptotic Gene CHOP and Anti-Apoptotic Heat Shock Proteins

BackgroundHepatitis E virus (HEV) is a non-enveloped plus-strand RNA virus that causes acute hepatitis. The capsid protein open reading frame 2 (ORF2) is known to induce endoplasmic reticulum stress in ORF2 expressing cells.Methodology/Principal FindingsIn this study we found that HEV ORF2 activates the expression of the pro-apoptotic gene C/EBP homologous protein (CHOP). ORF2 stimulates the CHOP promoter mainly through AARE (amino acid response elements) and to a minor extent the ERSE (endoplasmic reticulum stress response elements). Activating transcription factor 4 (ATF4) protein binds and activates the AARE regulatory sites of the CHOP promoter. ORF2 expression also leads to increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) that in turn initiates the translation of ATF4 mRNA. The pro-apoptotic gene CHOP is an important trigger to initiate endoplasmic reticulum stress induced apoptosis. However, the activation of CHOP by ORF2 in this study did not induce apoptosis, nor did BCL2-associated X protein (Bax) translocate to mitochondria. Microarray analysis revealed an ORF2 specific increased expression of chaperones Hsp72, Hsp70B', and co-chaperone Hsp40. Co-immunoprecipitation (Co-IP) and in silico molecular docking analysis suggests that HEV ORF2 interacts with Hsp72. In addition, Hsp72 shows nuclear accumulation in ORF2 expressing cells.Conclusions/SignificanceThese data provide new insight into simultaneously occurring counter-acting effects of HEV ORF2 that may be part of a strategy to prevent host suicide before completion of the viral replication cycle.

Prokaryotic expression and application of human herpes virus 8 small capsid protein encoded by open reading frame 65

目的 构建人疱疹病毒8型(HHV8)小衣壳蛋白ORF65编码基因的原核表达载体,并进行原核表达.方法 提取BCBL-1细胞(HHV8阳性而EBV阴性)DNA,PCR技术扩增HHV8ORF65基因,目的 基因与原核表达载体pThioHis A连接后,转化宿主菌Escherichia coli BL21,异丙基硫代-D-乳糖苷(IPTG)诱导融合蛋白表达,经带有6个组氨酸标签的亲和层析柱纯化获得重组抗原.重组蛋白电泳后转至聚偏氟乙烯(PVDF)膜,与HHV8阳性血清进行免疫印迹反应.对相关患者进行检测,并与美国Biotrin公司生产的HHV8 IgM和HHV8 IgG抗体间接荧光抗体试验(IFA)检测试剂盒和德国Qiagen公司ELISA检测试剂盒进行比较.结果 DNA序列分析表明目的 基因序列与HHV8标准毒株相应序列完全一致,十二烷基硫酸钠-聚丙烯酸胺凝胶电泳(SDS-PAGE)电泳可观察到相对分子质量为31 500的融合蛋白的表达,免疫印迹反应显示重组抗原具有较高的特异性.基因工程抗原的各项技术指标与Biotrin公司和Qiagen公司检测试剂盒有较好的符合率,对临床标本的检测表明该试剂盒简单、实用、结果稳定可靠.结论 HHV8重组抗原具有较好的抗原性,进一步完善后可用于HHV8抗体的检测。

Heterologous RNA encapsidated in Pariacoto virus-like particles forms a dodecahedral cage similar to genomic RNA in wild-type virions.

The genome of some icosahedral RNA viruses plays an essential role in capsid assembly and structure. In T=3 particles of the nodavirus Pariacoto virus (PaV), a remarkable 35% of the single-stranded RNA genome is icosahedrally ordered. This ordered RNA can be visualized at high resolution by X-ray crystallography as a dodecahedral cage consisting of 30 24-nucleotide A-form RNA duplex segments that each underlie a twofold icosahedral axis of the virus particle and interact extensively with the basic N-terminal region of 60 subunits of the capsid protein. To examine whether the PaV genome is a specific determinant of the RNA structure, we produced virus-like particles (VLPs) by expressing the wild-type capsid protein open reading frame from a recombinant baculovirus. VLPs produced by this system encapsidated similar total amounts of RNA as authentic virus particles, but only about 6% of this RNA was PaV specific, the rest being of cellular or baculovirus origin. Examination of the VLPs by electron cryomicroscopy and image reconstruction at 15.4-A resolution showed that the encapsidated RNA formed a dodecahedral cage similar to that of wild-type particles. These results demonstrate that the specific nucleotide sequence of the PaV genome is not required to form the dodecahedral cage of ordered RNA.

Virions of Pariacoto virus contain a minor protein translated from the second AUG codon of the capsid protein open reading frame.

Virions of the alphanodavirus Pariacoto virus (PaV) have T=3 icosahedral symmetry and are assembled from multiple copies of a precursor protein that is cleaved into two mature capsid proteins after assembly. The crystal structure of PaV shows that the N-terminal approximately 30 amino acid residues of the subunits surrounding the 5-fold axes interact extensively with icosahedrally ordered regions of the encapsidated positive-sense genomic RNAs. We found that wild-type PaV particles also contain a minor capsid protein that is truncated by 24 residues at its N terminus. Reverse genetic experiments showed that translation of this protein initiated at the second AUG of the capsid protein open reading frame. When either the longer or shorter version of the capsid protein was expressed independently of the other, it assembled into virus particles and underwent maturational cleavage. Virions that lacked the shorter capsid protein retained infectivity for cultured insect cells and Galleria mellonella larvae.

Analyses of genotypic diversity among North, South, and Central American isolates of sugarcane yellow leaf virus: evidence for Colombian origins and for intraspecific spatial phylogenetic variation.

We have analyzed the genotypic diversity of sugarcane yellow leaf virus (SCYLV) collected from North, South, and Central America by fingerprinting assays and selective cDNA cloning and sequencing. One group of isolates from Colombia, designated the C-population, has been identified as residing at the root node between a separable superpopulation structure of SCYLV and other members of the family Luteoviridae, indicating that the progenitor viruses of the North, South, and Central American isolates of the SCYLV superpopulation most likely arose from a C-population structure. From a model of intrafamilial evolution (F. Moonan et al., Virology 269:156-171, 2000), a prediction could be made that within the SCYLV species, the capacity of genomic sequence divergence would range from lowest in the capsid protein open reading frame 3 (ORF 3) to highest in a region spanning across the carboxy-terminal end of the RNA-dependent RNA polymerase ORF. We have demonstrated the validity and applicability of this intrafamilial model for the prediction of intraspecies SCYLV diversity. Analysis of spatial phylogenetic variation (SPV) within the SCYLV isolates could not be assessed by application of a "partial likelihoods assessed through optimization" (PLATO)-derived intraspecies model alone. However, application of a PLATO-derived intrafamilial model with the intraspecies-derived model allowed distinction of three forms of SPV. Two of the SPV forms identified correspond to the extremes in a continuum of sequence evolution displayed in a SCYLV superpopulation structure, and the third form was diagnostic of a C-population structure. The application of these types of models has value in terms of predicting the types of SCYLV intraspecies diversity that may exist worldwide, and in general, may be useful in application for more informed design of transgenes for use in the elicitation of homology-dependent virus resistance mechanisms in transgenic plants.