Microinjection analysis of envelope-glycoprotein messenger activities of avian leukosis viral RNAs.

Virion RNA from the avian leukosis virus Rous-associated virus 2 (RAV-2) and poly(A)-containing RNAs from RAV-2-infected chick embryo fibroblasts were microinjected into fibroblasts transformed by the Bryan high-titer strain of Rous sarcoma virus (RSV), which is deficient in viral envelope glycoprotein. Production of infectious RSV following these injections depended upon the viral envelope-messenger activity of the injected RNA. This system constituted a sensitive and rigorous assay system for viral envelope-messenger RNA. It was found that 21S mRNA from RAV-2-infected cells expressed the highest activity, while 35S mRNA expressed comparatively little. In addition, RAV-2-virion RNA expressed little messenger activity. The rate of formation of infectious RSV following 21S mRNA injections reached a peak near 9 hr, which was followed by a rapid decline. Evidence has been obtained that a small fraction of both 35S virion RNA and 35S mRNA from virus-infected cells was encapsulated into virus particles following their injection into virus-producing cells.     

The pathogenesis of Rift Valley fever

Rift Valley fever (RVF) is an emerging zoonotic disease distributed in sub-Saharan African countries and the Arabian Peninsula. The disease is caused by the Rift Valley fever virus (RVFV) of the family Bunyaviridae and the genus Phlebovirus. The virus is transmitted by mosquitoes, and virus replication in domestic ruminant results in high rates of mortality and abortion. RVFV infection in humans usually causes a self-limiting, acute and febrile illness; however, a small number of cases progress to neurological disorders, partial or complete blindness, hemorrhagic fever, or thrombosis. This review describes the pathology of RVF in human patients and several animal models, and summarizes the role of viral virulence factors and host factors that affect RVFV pathogenesis.     

Replication and packaging of Norwalk virus RNA in cultured mammalian cells

Human noroviruses, the most common cause of nonbacterial gastroenteritis, are characterized by high infectivity rate, low infectious dose, and unusually high stability outside the host. However, human norovirus research is hindered by the lack of a cell culture system and a small animal model of infection. Norwalk virus (NV) is the prototype strain of human noroviruses. We report here replication of NV viral RNA and its packaging into virus particles in mammalian cells by intracellular expression of native forms of NV viral RNA devoid of extraneous nucleotide sequences derived from the expression vector by the use of replication-deficient vaccinia virus MVA encoding the bacteriophage T7 RNA polymerase (MVA/T7). Expressed genomic RNA was found to replicate; NV subgenomic RNA was transcribed from genomic RNA by use of NV nonstructural proteins expressed from genomic RNA and was subsequently translated into NV capsid protein VP1. Viral genomic RNA was packaged into virus particles generated in mammalian cells. The cesium chloride (CsCl) density gradient profile of virus particles containing genomic RNA was similar to that of NV purified from stool. These observations indicate that the NV cDNA constructed here is a biologically infectious clone, and that mammalian cells have the ability to replicate NV genomic RNA. This work establishes a mammalian cell-based system for analysis of human norovirus replication and, thus, makes it feasible to investigate antiviral agents in mammalian cells.     

RNA Structures and Their Role in Selective Genome Packaging

To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.     

Changes in genome composition of the Friend virus complex in erythroleukemia cells during the course of differentiation induced by dimethyl sulfoxide

The Friend spleen focus-forming virus (SFFV) complex released by Friend virus-transformed erythroid cells has been analyzed with respect to changes in the genome composition that may occur during induction of erythropoiesis with dimethyl sulfoxide. It is shown that: (a) There are three types of virus particles, one with buoyant density 1.20 g/ml, one with density 1.17 g/ml (the density of the cloned lymphatic leukemia virus helper component of the complex), and a major fraction that has a density of 1.14 g/ml. (b) Three RNA subunits-35S, 32S, and 30S-have previously been shown to be detectable in the Friend virus complex. The 1.20-g/ml particles contain only 30S RNA, whilst the 1.14- to 1.17-g/ml particles contain a mixture consisting of predominantly 30S and 32S RNA and about 5-10% 35S RNA. (c) Induction of differentiation results in an increase in the 1.14-g/ml particles and 32S RNA. The amount of 30S RNA does not change. (d) Hybridization of the different genomic viral RNAs with full-length virus cDNA shows that the 30S RNA (of induced and uninduced Friend virus) is more closely related to the 32S RNA of the induced Friend virus than to the 32S RNA of the constitutively released Friend virus. (e) The 30S RNA contains SFFV-specific sequences. (f) A hypothesis is presented in which the induction of the new 32S RNA species is related to the increase of SFFV activity and to a specific function of the SFFV during induction of erythropoiesis.     

In vivo mapping of a sequence required for interference with the yeast killer virus.

The Saccharomyces cerevisiae viruses are noninfectious double-stranded RNA viruses whose segments are separately encapsidated. A large viral double-stranded RNA (L1; 4580 base pairs) encodes all required viral functions. M1, a double-stranded RNA of 1.9 kilobases, encodes an extracellular toxin (killer toxin) and cellular immunity to that toxin. Some strains contain smaller, S, double-stranded RNAs, derived from M1 by internal deletion. Particles containing these defective interfering RNAs can displace M1 particles by faster replication and thus convert the host strain to a nonkiller phenotype. In this work, we report the development of an assay in which the expression of S plus-strand from an inducible plasmid causes the loss of M1 particles. This assay provides a convenient method for identifying in vivo cis-acting sequences important in viral replication and packaging. We have mapped the sequence involved in interference to a region of 132 base pairs that includes two sequences similar to the viral binding site sequence previously identified in L1 by in vitro experiments.     

Inactivation of Venezuelan Equine Encephalitis Virus Genome Using Two Methods

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses.     

3D聚合酶突变对肠道病毒A组71型复制影响的研究

目的 研究3D聚合酶(3D polymerase,3D^pol)突变对肠道病毒A组71型(Enterovirus A71,EV-A71)复制的影响。方法 扩增EV-A71病毒基因组并克隆至TOPO-XL2-PCR载体;应用体外点突变方法在病毒3D^pol区引入N16S和I256V突变;采用T7聚合酶转录病毒基因组RNA并转染RD细胞,获得N16S和I256V病毒突变株。观察亲本病毒与突变病毒株的增殖特征以及病毒RNA含量。结果 RNA转染RD细胞后可获得具有感染性的病毒。体外增殖试验显示,正常培养条件下(36.5 ℃)3株病毒增殖趋势一致, 感染细胞72 h后病毒滴度和RNA拷贝数均达到峰值且组间差异无统计学意义。温度敏感性试验显示,与正常培养条件比较,病毒在高温(39.5 ℃)的滴度和RNA拷贝数均大幅下降,并且高温对N16S和I256V突变株的增殖抑制较亲本株更为显著。结论 成功构建EV-A71感染性克隆并获得病毒突变株,3D^pol的N16S和I256V突变在高温条件下可能进一步降低病毒复制能力。     

An Association Between Globin Messenger RNA and 60S RNA Derived from Friend Leukemia Virus

The association between certain cellular RNAs and purified RNA tumor viruses prompted us to examine the possibility that specific host messenger RNAs might also be incorporated into RNA tumor viruses. Using a mouse cell line infected with Friend leukemia virus, T-3-Cl-2, which can be induced to accumulate mouse-globin messenger RNA, we show that mouse-globin messenger RNA sequences are present in viral particles purified from the culture medium of globin-producing cells. These globin messenger RNA sequences are absent from viral particles derived from T-3-Cl-2 cells that are not producing globin messenger RNA. Virus-associated globin messenger RNA sequences sediment in association with the 60S viral RNA complex as well as in free, 9S form. However, under mild denaturing conditions which result in the conversion of viral 60S RNA to 30S and smaller forms, all the globin sequences sediment as 9S RNA. Appropriate control experiments indicate that the virus-associated globin messenger RNA is resistant to degradation by exogenous ribonuclease; that exogenously added globin messenger RNA does not become associated with the 60S viral RNA complex; and that globin messenger RNA can be detected in virions derived from cells both induced for and constitutively synthesizing globin messenger RNA.     

Evidence that the packaging signal for nodaviral RNA2 is a bulged stem-loop.

Flock house virus is an insect virus belonging to the family Nodaviridae; members of this family are characterized by a small bipartite positive-stranded RNA genome. The larger genomic segment, RNA1, encodes viral replication proteins, whereas the smaller one, RNA2, encodes coat protein. Both RNAs are packaged in a single particle. A defective-interfering RNA (DI-634), isolated from a line of Drosophila cells persistently infected with Flock house virus, was used to show that a 32-base region of RNA2 (bases 186-217) is required for packaging into virions. RNA folding analysis predicted that this region forms a stem-loop structure with a 5-base loop and a 13-base-pair bulged stem.     

One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis

Influenza A virus possesses a segmented genome of eight negative-sense, single-stranded RNAs. The eight segments have been shown to be represented in approximately equal molar ratios in a virus population; however, the exact copy number of each viral RNA segment per individual virus particles has not been determined. We have established an experimental approach based on multicolor single-molecule fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Colocalization analysis showed that a high percentage of virus particles package all eight different segments of viral RNAs. To determine the copy number of each RNA segment within individual virus particles, we measured the photobleaching steps of individual virus particles hybridized with fluorescent probes targeting a specific viral RNA. By comparing the photobleaching profiles of probes against the HA RNA segment for the wild-type influenza A/Puerto Rico/8/34 (PR8) and a recombinant PR8 virus carrying two copies of the HA segment, we concluded that only one copy of HA segment is packaged into a wild type virus particle. Our results showed similar photobleaching behaviors for other RNA segments, suggesting that for the majority of the virus particles, only one copy of each RNA segment is packaged into one virus particle. Together, our results support that the packaging of influenza viral genome is a selective process.     

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs--only four nucleotides per genomic RNA--reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.     

Paramyxovirus Glycoprotein Incorporation,Assembly and Budding: A Three Way Dance for Infectious Particle Production

Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout.     

Characterization of Hantaan virions, the prototype virus of hemorrhagic fever with renal syndrome

Hantaan virus, strain 76-118, was propagated to high titer in a clone of Vero cells, and infectious virions were successfully concentrated and purified. Infectivity and virus antigenic activity were closely associated with a virus particle that exhibited a sedimentation rate indistinguishable from a representative member of the Bunyaviridae. Purified virions sedimented to a density of 1.16-1.17 in sucrose and 1.20-1.21 in cesium chloride. Detergent disruption of virions resulted in a nucleocapsid structure (density, 1.18 in sucrose and 1.25 in cesium chloride) and soluble protein antigens. Three separate nucleocapsids were resolved by rate-zonal centrifugation and contained a single but common polypeptide of 50,000 daltons. Electrophoresis of radiolabeled RNA extracted from purified virions yielded a profile of three RNA species with apparent molecular weights of 2.7, 1.2, and 0.6 X 10(6). These data support earlier electron microscopy reports which suggested that Hantaan virus has characteristics similar to some members of the virus family Bunyaviridae.     

Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs

In response to infection, invertebrates process replicating viral RNA genomes into siRNAs of discrete sizes to guide virus clearance by RNA interference. Here, we show that viral siRNAs sequenced from fruit fly, mosquito, and nematode cells were all overlapping in sequence, suggesting a possibility of using siRNAs for viral genome assembly and virus discovery. To test this idea, we examined contigs assembled from published small RNA libraries and discovered five previously undescribed viruses from cultured Drosophila cells and adult mosquitoes, including three with a positive-strand RNA genome and two with a dsRNA genome. Notably, four of the identified viruses exhibited only low sequence similarities to known viruses, such that none could be assigned into an existing virus genus. We also report detection of virus-derived PIWI-interacting RNAs (piRNAs) in Drosophila melanogaster that have not been previously described in any other host species and demonstrate viral genome assembly from viral piRNAs in the absence of viral siRNAs. Thus, this study provides a powerful culture-independent approach for virus discovery in invertebrates by assembling viral genomes directly from host immune response products without prior virus enrichment or amplification. We propose that invertebrate viruses discovered by this approach may include previously undescribed human and vertebrate viral pathogens that are transmitted by arthropod vectors.