RNA Virus Particles Research Articles

An interactome map of the nucleocapsid protein from a highly pathogenic North American porcine reproductive and respiratory syndrome virus strain generated using SILAC‐based quantitative proteomics

Positive strand RNA viruses replicate in the cytoplasm of an infected cell and encode nucleocapsid proteins. These proteins function to promote encapsidation of the RNA genome and virus particle assembly as well as playing potential roles in viral RNA synthesis. Nucleocapsid proteins can also associate with cellular proteins and signaling cascades. The arterivirus nucleocapsid (N) protein is no exception and localizes to both the cytoplasm and the nucleolus in virus‐infected cells. This study generated an interactome map of the N protein from a highly virulent North American strain of porcine reproductive and respiratory syndrome virus (PRRSV). This is a major pathogen of swine resulting in significant morbidity and mortality. Crucial to the study was the use of SILAC coupled to affinity purification using GFP‐traps and LC‐MS/MS. This approach has not been applied before to the investigation of host/viral protein interactomes and this study revealed that the PRRSV N protein interacts with the host cell protein synthesis machinery especially at the level of translation initiation as well as with the RNA post‐transcriptional modification machinery. Applications of the dataset can include studies of virus/host interactions and the design of live attenuated recombinant vaccines.

Role of HIV-1 RNA and protein determinants for the selective packaging of spliced and unspliced viral RNA and host U6 and 7SL RNA in virus particles.

HIV-1 particles contain RNA species other than the unspliced viral RNA genome. For instance, viral spliced RNAs and host 7SL and U6 RNAs are natural components that are non-randomly incorporated. To understand the mechanism of packaging selectivity, we analyzed the content of a large panel of HIV-1 variants mutated either in the 5′UTR structures of the viral RNA or in the Gag-nucleocapsid protein (GagNC). In parallel, we determined whether the selection of host 7SL and U6 RNAs is dependent or not on viral RNA and/or GagNC. Our results reveal that the polyA hairpin in the 5′UTR is a major packaging determinant for both spliced and unspliced viral RNAs. In contrast, 5′UTR RNA structures have little influence on the U6 and 7SL RNAs, indicating that packaging of these host RNAs is independent of viral RNA packaging. Experiments with GagNC mutants indicated that the two zinc-fingers and N-terminal basic residues restrict the incorporation of the spliced RNAs, while favoring unspliced RNA packaging. GagNC through the zinc-finger motifs also restricts the packaging of 7SL and U6 RNAs. Thus, GagNC is a major contributor to the packaging selectivity. Altogether our results provide new molecular insight on how HIV selects distinct RNA species for incorporation into particles.

The Acidic Domain of Hepatitis C Virus NS4A Contributes to RNA Replication and Virus Particle Assembly

Hepatitis C virus NS3-4A is a membrane-bound enzyme complex that exhibits serine protease, RNA helicase, and RNA-stimulated ATPase activities. This enzyme complex is essential for viral genome replication and has been recently implicated in virus particle assembly. To help clarify the role of NS4A in these processes, we conducted alanine scanning mutagenesis on the C-terminal acidic domain of NS4A in the context of a chimeric genotype 2a reporter virus. Of 13 mutants tested, two (Y45A and F48A) had severe defects in replication, while seven (K41A, L44A, D49A, E50A, M51A, E52A, and E53A) efficiently replicated but had severe defects in virus particle assembly. Multiple strategies were used to identify second-site mutations that suppressed these NS4A defects. The replication defect of NS4A F48A was partially suppressed by mutation of NS4B I7F, indicating that a genetic interaction between NS4A and NS4B contributes to RNA replication. Furthermore, the virus assembly defect of NS4A K41A was suppressed by NS3 Q221L, a mutation previously implicated in overcoming other virus assembly defects. We therefore examined the known enzymatic activities of wild-type or mutant forms of NS3-4A but did not detect specific defects in the mutants. Taken together, our data reveal interactions between NS4A and NS4B that control genome replication and between NS3 and NS4A that control virus assembly.

Characterization of antibody responses to the Sj23 antigen of Schistosoma japonicum after infection and immunization

Sj23, a member of the tetraspanin protein family, is a 23-kDa surface-exposed protein of Schistosoma japonicum and expressed in all infective parasite stages, which has been regarded as a potential candidate in vaccine development for schistosomiasis. In this study, we found that, in the BALB/c mouse model, Sj23 elicited rapid humoral responses after parasite infection and the dominant antibodies were of IgG2a subclass. Immunization with Sj23 by priming with recombinant SFV RNA virus particles followed by a boost with recombinant protein also generated strong IgG2a responses which did not provide protection against challenge with cercariae. Our data indicated that one of the functions of Sj23 of S. japonicum is to facilitate parasite immune regulation. Sj23 antigen-based vaccine may require strong adjuvant that can drive IgG1 responses which are more critical in resistance to helminth infection.

Caulimoviridae Tubule-Guided Transport Is Dictated by Movement Protein Properties

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway.

Rhabdovirus Evasion of the Interferon System

The family Rhabdoviridae contains important pathogens of humans, livestock, and crops, including the insect-transmitted vesicular stomatitis virus (VSV) and the neurotropic rabies virus (RV), which is directly transmitted between mammals. In spite of a highly similar organization of RNA genomes, proteins, and virus particles, cell biology of VSV and RV is divergent in several aspects, particularly with respect to their interplay with the cellular host defense. While infection with both rhabdoviruses is recognized via viral triphosphate RNAs by the cytoplasmic RNA helicase/translocase RIG-I, the viral counteractions to limit the response are contrasting. VSV infection is characterized by a rapid general shutdown of host gene expression and severe cytopathic effects, due to multiple activities of the matrix (M) protein affecting host polymerase functions and mRNA nuclear export, and by rapid and high-level virus replication. In contrast, RV spread and transmission relies on preserving the integrity of host cells, particularly of neurons. While a general cell shutdown by RV M is not observed, RV phosphoprotein (P) has developed independent functions to interfere with activation of IRFs and with STAT signaling. The molecular mechanisms employed are different from those of the paramyxovirus P gene products serving similar functions, and illustrate evolution of IFN antagonists to specifically support virus survival in the natural niches.

The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry

Double-stranded (ds) RNA virus particles are organized around a central icosahedral core capsid made of 120 identical subunits. This core capsid is unable to invade cells from outside, and animal dsRNA viruses have acquired surrounding capsid layers that are used to deliver a transcriptionally active core particle across the membrane during cell entry. In contrast, dsRNA viruses infecting primitive eukaryotes have only a simple core capsid, and as a consequence are transmitted only vertically. Here, we report the 3.4 A X-ray structure of a picobirnavirus--an animal dsRNA virus associated with diarrhoea and gastroenteritis in humans. The structure shows a simple core capsid with a distinctive icosahedral arrangement, displaying 60 two-fold symmetric dimers of a coat protein (CP) with a new 3D-fold. We show that, as many non-enveloped animal viruses, CP undergoes an autoproteolytic cleavage, releasing a post-translationally modified peptide that remains associated with nucleic acid within the capsid. Our data also show that picobirnavirus particles are capable of disrupting biological membranes in vitro, indicating that its simple 120-subunits capsid has evolved animal cell invasion properties.

B591 Infectious RNA: A Novel Therapeutic Modality for Myeloma

Introduction and Aims: Coxsackievirus A21 (CVA21) selectively propagates in myeloma cells leading to eradication of myeloma x enografts in SCID mice. However, repeat intravenous administration of this agent in a clinical setting will be problematic since CVA21 viral particles are neutralized by antiviral antibodies whose circulating titers will increase between treatments. Also, because of the high mutation rates of picornaviral polymerases (3 r 10-3), picornavirus preparations are heavily contaminated with ‘quasispecies’ whose genome sequences do not match the product specification. On average, each picornavirus has one or two randomly mutated bases in its genome. Picornaviruses therefore present substantial manufacturing challenges. To address these problems, we sought to determine whether infectious RNA could be used in place of virus particles to initiate an oncolytic CVA21 infection in vivo. Materials and Methods: Infectious RNA encoding CVA21 was transcribed from plasmid DNA using T7 polymerase (err or rate 3 r 10-6) and purified by gel-extraction. Varying doses of RNA (1, 2, 4, 8, 16 or 32 micrograms) or CVA21 virus particles were injected directly into subcutaneous KAS6/1 myeloma xenografts (0.5 cm average diameter) in tumor-bearing SCID mice. Animals were monitored for tumor regression, signs of CVA21-induced myositis (a known toxicity of the virus in this model), and CVA21 viremia. In ongoing studies, RNA corresponding to a retargeted CVA21 that is nontoxic to muscle is also being administered to KAS6/1 tumorbearing animals. Results and Conclusions: Within 48 hours of injecting the RNA into subcutaneous KAS6/1 xenografts, high titers of infectious CVA21 virions wer e detected in the bloodstream. Tumors regressed rapidly thereafter and mice developed signs of myositis. At euthanasia, CVA21 was recovered from regressing tumors and from skeletal muscles. Treatment outcomes did not differ between animals treated by intratumoral injection of RNA or fully infectious CVA21 virus particles. Dose-response studies showed that an effective oncolytic infection could be established by intratumoral injection of as little as 1 mg of infectious RNA. The oncolytic infection could also be initiated by intravenous injection of infectious RNA. Our study demonstrates that INA is a highly promising alternative drug formulation for oncolytic CVA21 virotherapy of multiple myeloma.

The NS3 Helicase and NS5B-to-3′X Regions Are Important for Efficient Hepatitis C Virus Strain JFH-1 Replication in Huh7 Cells

The JFH-1 strain of hepatitis C virus (HCV) is a genotype 2a strain that can replicate autonomously in Huh7 cells. The J6 strain is also a genotype 2a strain, but its full genomic RNA does not replicate in Huh7 cells. However, chimeric J6/JFH-1 RNA that has J6 structural-protein-coding regions and JFH-1 nonstructural-protein-coding regions can replicate autonomously and produce infectious HCV particles. In order to determine the mechanisms underlying JFH-1 RNA replication, we constructed various J6/JFH-1 chimeras and tested their RNA replication and virus particle production abilities in Huh7 cells. Via subgenomic-RNA-replication assays, we found that both the JFH-1 NS5B-to-3'X (N5BX) and the NS3 helicase (N3H) regions are important for the replication of the J6CF replicon. We applied these results to full-length genomic RNA replication and analyzed replication using Northern blotting. We found that a chimeric J6 clone with JFH-1 N3H and N5BX could replicate autonomously but that a chimeric J6 clone with only JFH-1 N5BX had no replication ability. Finally, we tested the virus production abilities of these clones and found that a chimeric J6 clone with JFH-1 N3H and N5BX could produce infectious HCV particles. In conclusion, the JFH-1 NS3 helicase and NS5B-to-3'X regions are important for efficient replication and virus particle formation of HCV genotype 2a strains.

Retrotransposons: Metaparasites and Agents of Genome Evolution

Transposable elements comprise the bulk of higher plant genomes. The majority of these elements are the Class I LTR retrotransposons, which transpose via an RNA intermediate in a "Copy-and-Paste" mechanism. Because retrotransposons use cellular resources and their own enzymes to replicate independently of the genome as a whole, and have thereby become in many cases more predominant than the cellular genes, they have been considered "selfish DNA" and nuclear parasites. They are thought to share many features of the internal life cycle of retroviruses such as HIV (lentiviruses). However, whereas at least some of the retroviruses arriving in an organism during an infection must be functional in order for the infection to proceed, some LTR retrotransposon families appear to completely lack active members even though they remain mobile. Furthermore, the process of retrotransposition is inherently error-prone and mutagenic, giving rise to "pseudospecies," or clusters of imperfect copies. The non-autonomous retrotransposons are able to cis- and trans-parasitize host retrotransposons to gain mobility, much as do defective interfering particles of RNA viruses. Hence, a complex dynamic is set up, whereby the impact of retrotransposons on genomes can be under selection on the organismal level; the impact of non-autonomous retrotransposons on autonomous ones can likewise be under selection if there is selection on the autonomous elements themselves. We are exploring the retrotransposon life cycle and the causes and possible consequences of non-autonomy at each stage regarding genome evolution.

Relationships between infectious titer, capsid protein levels, and reverse transcriptase activities of diverse human immunodeficiency virus type 1 isolates.

Most studies on human immunodeficiency virus type 1 (HIV-1) replication kinetics or fitness must rely on a particular assay to initially standardize inocula from virus stocks. The most accurate measure of infectious HIV-1 titers involves a limiting dilution-infection assay and a calculation of the dose required for 50% infectivity of susceptible cells in tissue culture (TCID(50)). Surrogate assays are now commonly used to measure the amount of p24 capsid, the endogenous reverse transcriptase (RT) activity, or the amount of viral genomic RNA in virus particles. However, a direct comparison of these surrogate assays and actual infectious HIV-1 titers from TCID(50) assays has not been performed with even the most conserved laboratory strains, let alone the highly divergent primary HIV-1 isolates of different subtypes. This study indicates that endogenous RT activity, not p24 content or viral RNA load, is the best surrogate measure of infectious HIV-1 titer in both cell-free supernatants and viruses purified on sucrose cushions. Sequence variation between HIV-1 subtypes did not appear to affect the function or activity of the RT enzyme in this endogenous assay but did affect the detection of p24 capsid by both enzyme immunoassays and Western blots. Clear groupings of non-syncytium-inducing (NSI), CCR5-tropic (R5), and SI/CXCR4-tropic (X4) HIV-1 isolates were observed when we compared the slopes derived from correlations of RT activity with infectious titers. Finally, the replication efficiency or fitness of both the NSI/R5 and SI/X4 HIV-1 isolates was not linked to the titers of the virus stocks.

Escaping from the cell: assembly and budding of negative-strand RNA viruses.

Negative-strand RNA virus particles are formed by a process that includes the assembly of viral components at the plasma membranes of infected cells and the subsequent release of particles by budding. Here, we review recent progress that has been made in understanding the mechanisms of negative-strand RNA virus assembly and bud- ding. Important topics for discussion include the key role played by the viral matrix proteins in assembly of viruses and viruslike particles, as well as roles played by additional viral components such as the viral glycoproteins. Various interactions that contribute to virus assembly are discussed, including interactions between matrix proteins and membranes, interactions between matrix proteins and glycoproteins, interactions between matrix proteins and nucleocapsids, and interactions that lead to matrix protein self-assembly. Selection of specific sites on plasma membranes to be used for virus assembly and budding is described, including the asymmetric budding of some viruses in polarized epithelial cells and assembly of viral components in lipid raft microdomains. Evidence for the involvement of cellular proteins in the late stages of rhabdovirus and filovirus budding is discussed as well as the possible involvement of similar host factors in the late stages of budding of other negative-strand RNA viruses.

Duplication of the primary encapsidation and dimer linkage region of human immunodeficiency virus type 1 RNA results in the appearance of monomeric RNA in virions.

The dimerization initiation site (DIS) and the dimer linkage sequences (DLS) of human immunodeficiency virus type 1 have been shown to mediate in vitro dimerization of genomic RNA. However, the precise role of the DIS-DLS region in virion assembly and RNA dimerization in virus particles has not been fully elucidated, since deletion or mutation of the DIS-DLS region also abolishes the packaging ability of genomic RNA. To characterize the DIS-DLS region without altering packaging ability, we generated mutant constructs carrying a duplication of approximately 1,000 bases including the encapsidation signal and DIS-DLS (E/DLS) region. We found that duplication of the E/DLS region resulted in the appearance of monomeric RNA in virus particles. No monomers were observed in virions of mutants carrying the E/DLS region only at ectopic positions. Monomers were not observed when pol or env regions were duplicated, indicating an absolute need for two intact E/DLS regions on the same RNA for generating particles with monomeric RNA. These monomeric RNAs were most likely generated by intramolecular interaction between two E/DLS regions on one genome. Moreover, incomplete genome dimerization did not affect RNA packaging and virion formation. Examination of intramolecular interaction between E/DLS regions could be a convenient tool for characterizing the E/DLS region in virion assembly and RNA dimerization within virus particles.

Inhibitory effect of dibenzofuran and dibenzosuberol derivatives on rhinovirus replication in vitro; effective prevention of viral entry by dibenzosuberenone

A series of derivatives of dibenzofuran and dibenzosuberol block rhinovirus replication in vitro as judged by their ability to hinder the cytopathic effect in cells infected with HRV14 or HRV16. Both the number and the size of viral plaques were reduced effectively by treatment with these compounds in a dose-dependent fashion, thus affecting viral spread. The compound 2-hydroxy-3-dibenzofuran carboxylic acid was equally effective against HRV16 and HRV14, with IC 50 values of 25 μM in cytopathy assays. Dibenzosuberenone showed minor differences in selectivity, with IC 50 values of 10 and 30 μM for HRV16 and HRV14 cytopathy, respectively. Likewise, dibenzosuberenone effectively prevented the production of HRV16 proteins, viral RNA, and infectious virus particles when present at concentrations above 30 μM. Time-of-addition experiments show that compounds must be administered before or during the viral adsorption step in order to be effective antivirals. Dibenzosuberenone can block the adsorption of viral particles on to cells, preventing further steps in the replication cycle, but is not effective as a direct inactivating agent. These compounds likely interact with viral capsid proteins, affecting receptor interactions required for attachment and subsequent entry into cells.

Foot-and-mouth disease virus and poliovirus particles contain proteins of the replication complex.

Nonstructural proteins 2C, 3CD, 3C, and 3D, and the cellular protein actin, are present in highly purified preparations of foot-and-mouth disease virus (FMDV) and poliovirus. They remain bound in variable amounts to the RNAs when the RNAs are extracted from the viruses with phenol or phenol-sodium dodecyl sulfate (SDS) and, for FMDV, when the RNA is released from the particles by a lowering of the pH below 7. RNA prepared by these methods is rapidly degraded at 37 degrees C, particularly in the presence of NH4+ ions, but hydrolysis can be prevented by antibody against Escherichia coli-expressed 3D, indicating that it is the RNA polymerase that has nuclease activity. In contrast, virion RNA from which the nonstructural proteins and actin have been removed by extraction with guanidine thiocyanate-phenol-chloroform or proteinase K-phenol is stable at 37 degrees C, although its specific infectivity is lower than that of the RNA extracted with phenol or phenol-SDS. The possible implications of the close association of replication complex proteins with the RNA in virus particles are discussed.

Effects of nucleocapsid mutations on human immunodeficiency virus assembly and RNA encapsidation.

The human immunodeficiency virus (HIV) Pr55Gag precursor proteins direct virus particle assembly. While Gag-Gag protein interactions which affect HIV assembly occur in the capsid (CA) domain of Pr55Gag, the nucleocapsid (NC) domain, which functions in viral RNA encapsidation, also appears to participate in virus assembly. In order to dissect the roles of the NC domain and the p6 domain, the C-terminal Gag protein domain, we examined the effects of NC and p6 mutations on virus assembly and RNA encapsidation. In our experimental system, the p6 domain did not appear to affect virus release efficiency but p6 deletions and truncations reduced the specificity of genomic HIV-1 RNA encapsidation. Mutations in the nucleocapsid region reduced particle release, especially when the p2 interdomain peptide or the amino-terminal portion of the NC region was mutated, and NC mutations also reduced both the specificity and the efficiency of HIV-1 RNA encapsidation. These results implicated a linkage between RNA encapsidation and virus particle assembly or release. However, we found that the mutant ApoMTRB, in which the nucleocapsid and p6 domains of HIV-1 Pr55Gag were replaced with the Bacillus subtilis MtrB protein domain, released particles efficiently but packaged no detectable RNA. These results suggest that, for the purposes of virus-like particle assembly and release, NC can be replaced by a protein that does not appear to encapsidate RNA.

Function of a unique sequence motif in the long terminal repeat of feline leukemia virus isolated from an unusual set of naturally occurring tumors

Feline leukemia virus (FeLV) proviruses have been characterized from naturally occurring non-B-cell, non-T-cell tumors occurring in the spleens of infected cats. These proviruses exhibit a unique sequence motif in the long terminal repeat (LTR), namely, a 21-bp tandem triplication beginning 25 bp downstream of the enhancer. The repeated finding of the triplication-containing LTR in non-B-cell, non-T-cell lymphomas of the spleen suggests that the unique LTR is an essential participant in the development of tumors of this particular phenotype. The nucleotide sequence of the triplication-containing LTR most closely resembles that of FeLV subgroup C. Studies performed to measure the ability of the triplication-containing LTR to modulate gene expression indicate that the 21-bp triplication provides transcriptional enhancer function to the LTR that contains it and that it substitutes at least in part for the duplication of the enhancer. The 21-bp triplication confers a bona fide enhancer function upon LTR-directed reporter gene expression; however, the possibility of a spacer function was not eliminated. The studies demonstrate further that the triplication-containing LTR acts preferentially in a cell-type-specific manner, i.e., it is 12-fold more active in K-562 cells than is an LTR lacking the triplication. A recombinant, infectious FeLV bearing the 21-bp triplication in U3 was constructed. Cells infected with the recombinant were shown to accumulate higher levels of viral RNA transcripts and virus particles in culture supernatants than did cells infected with the parental type. The triplication-containing LTR is implicated in the induction of tumors of a particular phenotype, perhaps through transcriptional regulation of the virus and/or adjacent cellular genes, in the appropriate target cell.

"Rattlesnake" structure of a filamentous plant RNA virus built of two capsid proteins.

Elongated particles of simple RNA viruses of plants are composed of an RNA molecule coated with numerous identical capsid protein subunits to form a regular helical structure, of which tobacco mosaic virus is the archetype. Filamentous particles of the closterovirus beet yellow virus (BYV) reportedly contain approximately 4000 identical 22-kDa (p22) capsid protein subunits. The BYV genome encodes a 24-kDa protein (p24) that is structurally related to the p22. We searched for the p24 in BYV particles by using immunoelectron microscopy with specific antibodies against the recombinant p24 protein and its N-terminal peptide. A 75-nm segment at one end of the 1370-nm filamentous viral particle was found to be consistently labeled with both types of antibodies, thus indicating that p24 is indeed the second capsid protein and that the closterovirus particle, unlike those of other plant viruses with helical symmetry, has a "rattlesnake" rather than uniform structure.

Many-trillionfold amplification of single RNA virus particles fails to overcome the Muller's ratchet effect.

We showed earlier that transfers of large populations of RNA viruses lead to fitness gains and that repeated genetic bottleneck transfers result in fitness losses due to Muller's ratchet. In the present study, we examined the effects of genetic bottleneck passages intervening between population passages, a process akin to some natural viral transmissions, using vesicular stomatitis virus as a model. Our findings show that the pronounced fitness increases that occur during two successive population passages cannot overcome the fitness decreases caused by a single intervening genetic bottleneck passage. The implications for natural transmissions of RNA viruses are discussed.

Viral Polypeptides and RNA of Defective Interfering Particles of IPN Virus Generated by Serial Undiluted Passaging in TO-2 Cells.

HB-1 virus, a strain of infectious pancreatic necrosis virus (IPNV), was isolated from hard clam, Meretrix lusoria, with TO-2 cell line derived from tilapia ovary. The defective interfering (DI) particles of HB-1 virus could be generated by serial undiluted passaging. The polypeptides and RNA in virus particles of diluted and undiluted virus preparations were compared in order to investigate the nature of defective interfering particles involved in viral interference of HB-1 virus. Cesium chloride density gradient centrifugation was applied to purify virus particles of diluted and undiluted virus preparations. By comparing the polypeptides and RNA in virus particles of diluted and undiluted virus preparations, it was revealed that (I) the virus particles of undiluted virus preparation had smaller polypeptides than normal ; (II) subgenomic RNA of large segment was found in virus particles of undiluted virus preparation. These two findings suggested that DI particles of HB-1 virus was a deletion mutant of standard virus.This is the first report indicating the polypeptides and RNA possibly related to IPN virus DI particles.