Assortment and packaging of the segmented rotavirus genome

The rotavirus (RV) genome comprises 11 segments of double-stranded RNA (dsRNA) and is contained within a non-enveloped, icosahedral particle. During assembly, a highly coordinated selective packaging mechanism ensures that progeny RV virions contain one of each genome segment. Cis-acting signals thought to mediate assortment and packaging are associated with putative panhandle structures formed by base-pairing of the ends of RV plus-strand RNAs (+RNAs). Viral polymerases within assembling core particles convert the 11 distinct +RNAs to dsRNA genome segments. It remains unclear whether RV +RNAs are assorted before or during encapsidation, and the functions of viral proteins during these processes are not resolved. However, as reviewed here, recent insights gained from the study of RV and two other segmented RNA viruses, influenza A virus and bacteriophage Φ6, reveal potential mechanisms of RV assortment and packaging.     

Mutational analysis of cis-acting RNA signals in segment 7 of influenza A virus

The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.     

Genetic engineering of rotaviruses by reverse genetics

The rotavirus genome is composed of 11 gene segments of dsRNA. A recent breakthrough in the field of rotaviruses is the development of a reverse genetics system for generating recombinant rotaviruses possessing a gene segment derived from cloned cDNA. Although this approach is a helper virus‐driven system that is technically limited and gives low levels of recombinant viruses, it allows alteration of the rotavirus genome, thus contributing to our understanding of these medically important viruses. So far, this approach has successfully been applied to three of the 11 viral segments in our laboratory and others, and the efficiency of recovery of recombinant viruses has been improved. However, we are still waiting for the development of a helper virus‐free reverse genetics system for generating an infectious rotavirus entirely from cDNAs, as has been achieved for other members of the Reoviridae family.     

The Evolutionary Dynamics of Bluetongue Virus

Bluetongue virus (BTV) is a midge-borne member of the genus Orbivirus that causes an eponymous debilitating livestock disease of great agricultural impact and which has expanded into Europe in recent decades. Reassortment among the ten segments comprising the double-stranded (ds) RNA genome of BTV has played an important role in generating the epidemic strains of this virus in Europe. In this study, we investigated the dynamics of BTV genome segment evolution utilizing time-structured data sets of complete sequences from four segments, totalling 290 sequences largely sampled from ruminant hosts. Our analysis revealed that BTV genome segments generally evolve under strong purifying selection and at substitution rates that are generally lower (mean rates of ~0.5–7 × 10⁻⁴ nucleotide substitutions per site, per year) than vector-borne positive-sense viruses with single-strand (ss) RNA genomes. These also represent the most robust estimates of the nucleotide substitution rate in a dsRNA virus generated to date. Additionally, we determined that patterns of geographic structure and times to most recent common ancestor differ substantially between each segment, including a relatively recent origin for the diversity of segment 10 within the past millennium. Together, these findings demonstrate the effect of reassortment to decouple the evolutionary dynamics of BTV genome segments.     

cis effects in adeno-associated virus type 2 replication

We have utilized deletion mutants of adeno-associated virus (AAV) to investigate which elements of the AAV genome are required in cis for high yields of the wild-type virus in a plasmid transfection assay and in addition whether these elements affect primarily AAV DNA replication or encapsidation. All tested deletions from within the Rep region demonstrated a modest, approximately threefold, decrease in viral production. Deletions within the cap region resulted in markedly less virus. Previous observations suggested that in cells in which recombinant AAV (rAAV) was produced, as in our assay with the helper plasmid pDG, there is a substantial excess of empty capsids. Co-transfections of high- and low-yielding constructs demonstrated that under conditions where Cap is abundant, the constructs with cap deletions did not package efficiently. These observation suggest that the lower yields of rAAV cannot be entirely due to lack of capsids but that elements within the cap region of the wild-type genome are important for efficient encapsidation. The production of virus by the mutants we tested was, however, not consistent with the disruption of a cis-acting packaging signal. Apparently, when Cap is provided "in trans," encapsidation is inefficient. A second observation is that there were equivalent amounts of replicated but unencapsidated viral DNA in cells transfected with each of our constructs. We propose that, in accord with the previously proposed link between DNA replication and encapsidation, the total amount of AAV DNA replication can be limited by the efficiency of encapsidation.     

Cucumis sativus Cryptic Virus, a New Virus in Cucumber

Isometric virus-like particles (VLPs) have been purified from cucumber leaf tissue. Three dsRNA segments with estimated molecular weights of 1.8, 1.1 and 1.0 × 10⁶d have been isolated from VLPs occurring in CsCl density gradient fractions but were also readily detected in preparations from as little as 1 g of fresh leaf tissue. The VLPs resemble dsRNA containing cryptic viruses and have been named Cucumis sativus cryptic virus (CsCV).     

Occurrence of reoviruses in European cyprinid fishes (Tinea tinea Lin.; Leuciscus cephalus Lin.)

Two viruses have been isolated from tench (Tinea tinea) and from chub (Leuciscus cephalus). The agents replicated in EPC-and FHM cells at 20°C forming syncytia and lysis. Electron micrographs of the virion revealed icosahedral particles with a double capsid and a size of 70–75 nm. The genome of the isolated viruses consisted of 11 segments of dsRNA. SDS-PAGE migration patterns of the RNA revealed differences between the isolates and between the reoviruses of fishes. The isolates are serologically identical and they show serological relationships to the golden shiner virus (GSV) and to a lower extent to the chum salmon virus (CSV).     

Infectious Bursal Disease Virus: Ribonucleoprotein Complexes of a Double-Stranded RNA Virus

Genome-binding proteins with scaffolding and/or regulatory functions are common in living organisms and include histones in eukaryotic cells, histone-like proteins in some double-stranded DNA (dsDNA) viruses, and the nucleocapsid proteins of single-stranded RNA viruses. dsRNA viruses nevertheless lack these ribonucleoprotein (RNP) complexes and are characterized by sharing an icosahedral T = 2 core involved in the metabolism and insulation of the dsRNA genome. The birnaviruses, with a bipartite dsRNA genome, constitute a well-established exception and have a single-shelled T = 13 capsid only. Moreover, as in many negative single-stranded RNA viruses, the genomic dsRNA is bound to a nucleocapsid protein (VP3) and the RNA-dependent RNA polymerase (VPg). We used electron microscopy and functional analysis to characterize these RNP complexes of infectious bursal disease virus, the best characterized member of the Birnaviridae family. Mild disruption of viral particles revealed that VP3, the most abundant core protein, present at ∼ 450 copies per virion, is found in filamentous material tightly associated with the dsRNA. We developed a method to purify RNP and VPg-dsRNA complexes. Analysis of these complexes showed that they are linear molecules containing a constant amount of protein. Sensitivity assays to nucleases indicated that VP3 renders the genomic dsRNA less accessible for RNase III without introducing genome compaction. Additionally, we found that these RNP complexes are functionally competent for RNA synthesis in a capsid-independent manner, in contrast to most dsRNA viruses.     

Isolation, propagation and characterization of replication requirements of reiteration mutants of Simian Virus 40.

We have identified five reiteration mutants from serially-propagated, defective stocks of Simian Virus 40 and DAR virus (an SV40³ variant of human origin). The genomes of these mutants contain tandem repeats of specific segments of the SV40 genome. In order to propagate individual reiteration mutants, the monomer DNA segments from the mutant genomes are separated from wild-type SV40 DNA after cleavage by certain bacterial restriction endonucleases which produce short cohesive termini at their cleavage sites. These monomer segments, which are one-third, one-fourth, or one-fifth the size of wild-type SV40 DNA, are then circularized in vitro using bacteriophage T4 polynucleotide ligase and used to infect African green monkey kidney cells in the presence of wild-type or temperature-sensitive mutant DNAs as helpers. While wild-type SV40 and late temperature-sensitive mutants can serve as helpers in the replication and amplification of these minicircular DNAs, early temperature-sensitive mutant genomes are unable to do so at the nonpermissive temperature. The minicircular DNAs are amplified in vivo through an arithmetic series of oligomers. Encapsidation of reiterated molecules between 70 and 100% the size of wild-type SV40 DNA is observed, although reiterated viral DNA molecules much larger than unit size are formed in vivo.     

Two regions of an avian hepadnavirus RNA pregenome are required in cis for encapsidation.

We have constructed a series of deletion mutants spanning the genome of duck hepatitis B virus in order to determine which regions of the viral genome are required in cis for packaging of the pregenome into capsid particles. Deletion of sequences within either of two nonadjacent regions prevented replication of the mutant viral genomes expressed in a permissive avian hepatoma cell line in the presence of functionally active viral core and P proteins. Extraction of RNA from cells transfected with these replication-defective mutants showed that the mutants retained the capacity to be transcribed into a pregenomic-size viral RNA, but that these RNA species were not packaged into viral capsids. The two regions defined by these deletions are located 36 to 126 (region I) and 1046 to 1214 (region II) nucleotides downstream of the 5' end of the pregenome and contain sequences which are required in cis for encapsidation of the duck hepatitis B virus pregenome.     

Killer phenomenon in Ustilago maydis: Mapping viral functions

Summary Nonkiller progeny, lacking segments from the dsRNA genome of the virus associated with the P4 killer specifity, were recovered from a cross between a P4 killer strain and a sensitive strain. Three patterns of deletions were identified among the non-killers. In addition to the loss of killer activity these strains lost also the immunity and the ability to exclude the genomes of the virus associated with the P6 killer specifity but retained the essential information for viral coats. The patterns of deletions permitted the assignment of the killer function to 2 segments in the P4 genome, one in the medium group and the other in the lightest segment of the genome. Coat formation, as in the P6 virus, is associated with the heavy components of the dsRNA segmented genome but the information is organized somewhat differently from the organization of the virus associated with the P6 killer specifity. The loss of the exclusion function by the nonkillers enabled the reconstruction of hybrid viral genomes that restore specific killer activity. Thus, such hybrids indicate the position of the killer-related information in the P6 genome and suggest a role to the killer protein of P4 in the exclusion of specific dsRNA molecules.The study was supported in part by a Grant from the Branch of Basic Research of the Israel National Academy of Sciences     

Particle and naked RNA mycoviruses in industrially cultivated mushroom Pleurotus ostreatus in China

Many cultivated mushroom strains, such as Pleurotus ostreatus TD300, displayed symptoms of degeneration. A spherical virus POSV and four dsRNA segments were extracted from mycelium of P. ostreatus TD300. POSV had a diameter of 23 nm and encapsidated a 2.5kb dsRNA segment with coat proteins whose molecular weights were 39 kDa and 30 kDa. Four dsRNA segments were 8.2 kb, 2.5 kb, 2.0 kb, and 1.1 kb in size, respectively. The 1.1 kb dsRNA segment often escaped detection. The cDNA and the amino acid sequences of the 8.2 kb dsRNA were homologous to those of RNA-dependent RNA polymerases (RDRP) of ssRNA oyster mushroom spherical virus (OMSV), and contained conserved motifs A to D which were almost identical to those in RDRP of OMSV. The cDNA and amino acid sequences of the 2.5 kb and 2.0 kb dsRNA segments were homologous to that of RDRP and capsid protein of dsRNA virus P. ostreatus virus 1 (PoV1), respectively. In particular, the amino acid sequence of 2.5 kb dsRNA segment had high identity with the conserved motifs A to C in RDRP of PoV1, a Partiviridae virus. After eliminating the viruses in P. ostreatus TD300, the symptoms of degeneration completely disappeared. The results reveal that P. ostreatus TD300 was at least infected by a particle virus POSV, and two naked viruses, one was a dsRNA virus with a 2.0 kb dsRNA segment, the other was an ssRNA virus whose replicating form of genome was an 8.2 kb dsRNA segment. Mycoviruses infection is a causative agent of mushroom strain degeneration.     

Mycocin production in <Emphasis Type="Italic">Cryptococcus aquaticus</Emphasis>

Double-stranded RNA viruses of about 35 nm in diameter were isolated from a mycocin-secreting strain of Cryptococcus aquaticus. A derivative of this strain, lacking small dsRNA, was non-mycocinogenic and sensitive to its own toxin. The killing pattern of this mycocin was restricted to some species of the Cystofilobasidiales clade. Despite the differences in genome size of dsRNA viruses in mycocinogenic strains of Cryptococcus aquaticus, Cystofilobasidium sp. CBS 6569, Cystofilobasidium bisporidii, Cystofilobasidium infirmominiatum, Trichosporon pullulans and Xanthophyllomyces dendrorhous and killing patterns of their mycocins, the viral genomes showed homology in hybridisation experiments.     

Effects of the Number of Genome Segments on Primary and Systemic Infections with a Multipartite Plant RNA Virus

Multipartite plant viruses were discovered because of discrepancies between the observed dose response and predictions of the independent-action hypothesis (IAH) model. Theory suggests that the number of genome segments predicts the shape of the dose-response curve, but a rigorous test of this hypothesis has not been reported. Here, Alfalfa mosaic virus (AMV), a tripartite Alfamovirus, and transgenic Nicotianatabacum plants expressing no (wild type), one (P2), or two (P12) viral genome segments were used to test whether the number of genome segments necessary for infection predicts the dose response. The dose-response curve of wild-type plants was steep and congruent with the predicted kinetics of a multipartite virus, confirming previous results. Moreover, for P12 plants, the data support the IAH model, showing that the expression of virus genome segments by the host plant can modulate the infection kinetics of a tripartite virus to those of a monopartite virus. However, the different types of virus particles occurred at different frequencies, with a ratio of 116:45:1 (RNA1 to RNA2 to RNA3), which will affect infection kinetics and required analysis with a more comprehensive infection model. This analysis showed that each type of virus particle has a different probability of invading the host plant, at both the primary- and systemic-infection levels. While the number of genome segments affects the dose response, taking into consideration differences in the infection kinetics of the three types of AMV particles results in a better understanding of the infection process.     

Cucumber mosaic virus genome is encapsidated in alfalfa mosaic virus coat protein expressed in transgenic tobacco plants

The expression of viral coat protein (CP) in transgenic plants has been shown to be very effective in virus plant protection. However, the introduction of CP genes into plants presents the potential risk of the encapsidation of a superinfecting viral genome in the transgenic protein, an event which could change the epidemiology of the disease. To detect the potential heterologous encapsidation of the cucumber mosaic virus (CMV) genome by alfalfa mosaic virus (AIMV) CP expressed in transgenic tobacco plants, a system of immunocapture (IC) and amplification by polymerase chain reaction (PCR) was optimized. This provided high sensitivity and reliable selection of the heterologously encapsidated CMV genome in the presence of natural CMV particles. As little as 2 pg of virus could be detected by immunocapture/polymerase chain reaction (IC/PCR) technique. Evidence for heterologous encapsidation of the CMV genome was found in 11 of the 33 transgenic plants tested two weeks after CMV inoculation. This demonstrates a significant rate of heterologous encapsidation events between two unrelated viruses in transgenic plants. Since CP is involved in the interactions of the virus particle with its vector, the release in the field of such transgenic plants could alter the transmission properties of some important viruses.