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Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric "crown" at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryoelectron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multidomain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a "proto-crown" precursor that assembles prior to RNA template recruitment, RNA synthesis, and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 Å resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding "legs." The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and nonsymmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility, and evolutionary plasticity and insights into (+)RNA virus replication and control.

Reactive oxygen species (ROS), including superoxide anion (O 2 - ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into stress adaptations in plants. A respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O 2 - production to inhibit pathogen infection during plant innate immunity. RBOH-derived O 2 - can be immediately converted into H 2 O 2 by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, hijacks the host's ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, what types of ROS are required for viral replication is currently unknown. Here, we found that RCNMV replication was sensitive to an O 2 - scavenger but insensitive to an H 2 O 2 scavenger. Interestingly, replication of another plant (+)RNA virus, brome mosaic virus, was sensitive to both types of scavengers. These results indicate a virus-specific pattern requirement of O 2 - and H 2 O 2 for (+)RNA virus replication and suggest a conserved nature of the roles of ROS in (+)RNA virus replication.

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

Viral capsid proteins (CPs) are characterized by their role in forming protective shells around viral genomes. However, CPs have additional and important roles in the virus infection cycles and in the cellular responses to infection. These activities involve CP binding to RNAs in both sequence-specific and nonspecific manners as well as association with other proteins. This review focuses on CPs of both plant and animal-infecting viruses with positive-strand RNA genomes. We summarize the structural features of CPs and describe their modulatory roles in viral translation, RNA-dependent RNA synthesis, and host defense responses., (© 2013 Elsevier Inc. All rights reserved.)

Recommendations released by the CDC in 2023 address the need to demonstrate that the RNA genome of positive-strand RNA viruses is inactivated in addition to viral particles. This recommendation is in response to the similarities between host mRNA and the viral genome that allow the viral RNA to be used as a template by host replication mechanisms to produce infectious viruses; therefore, there is concern that through artificial introduction into host cells, active positive-strand RNA genomes can be utilized to produce infectious viruses out of a containment facility. Utilizing 10% formalin for 7 days or 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M cacodylate buffer (glut/PFA) for 2 days to fix eastern equine encephalitis virus (EEEV)-infected non-human primate (NHP) brain tissue was found to effectively inactivate EEEV particles and genomic RNA. The methods assessed in this paper outline an effective means to validate both genomic RNA and viral particle inactivation. [ABSTRACT FROM AUTHOR]

New evidences are emerging to support the importance of viral replication complexes (VRCs) in not only viral replication, but also viral cell-to-cell movement. Currently, how VRCs grow in size and colocalize with viral movement proteins (MPs) remains unclear. Herein, we performed live-cell imaging of red clover necrotic mosaic virus (RCNMV) dsRNA by using reporter B2-GFP plants. Tiny granules of dsRNA were formed along the endoplasmic reticulum (ER) at an early stage of infection. Importantly, the colocalization of the dsRNA granules with the virus-encoded p27 replication protein showed that these structures are components of VRCs. These granules moved throughout the cytoplasm, driven by the acto–myosin system, and coalesced with each other to form larger aggregates; the MPs were not associated with these processes. Notably, the MPs colocalized preferentially with large dsRNA aggregates, rather than with tiny dsRNA granules, suggesting that the increase in the size of VRCs promotes their colocalization with MPs. • Early dsRNA granules of RCNMV coalesced each other to form larger aggregates. • Acto-myosin system drives the intracellular movement of dsRNA granules. • Tiny dsRNA granules contain viral replicase, while associate less with MP. • Maturation of VRCs could be an important process for viral movement. [ABSTRACT FROM AUTHOR]

Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo. Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5' untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or templateswitching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps. [ABSTRACT FROM AUTHOR]

The RNA-dependent RNA polymerases (RdRPs) encoded by the RNA viruses are a unique class of nucleic acid polymerases. Each viral RdRP contains a 500–600 residue catalytic module with palm, fingers, and thumb domains forming an encircled human right hand architecture. Seven polymerase catalytic motifs are located in the RdRP palm and fingers domains, comprising the most conserved parts of the RdRP and are responsible for the RNA-only specificity in catalysis. Functional regions are often found fused to the RdRP catalytic module, resulting in a high level of diversity in RdRP global structure and regulatory mechanism. In this review, we surveyed all 46 RdRP-sequence available virus families of the positive-strand RNA viruses listed in the 2018b collection of the International Committee on Virus Taxonomy (ICTV) and chose a total of 49 RdRPs as representatives. By locating hallmark residues in RdRP catalytic motifs and by referencing structural and functional information in the literature, we were able to estimate the N- and C-terminal boundaries of the catalytic module in these RdRPs, which in turn serve as reference points to predict additional functional regions beyond the catalytic module. Interestingly, a large number of virus families may have additional regions fused to the RdRP N-terminus, while only a few of them have such regions on the C-terminal side of the RdRP. The current knowledge on these additional regions, either in three-dimensional (3D) structure or in function, is quite limited. In the five RdRP-structure available virus families in the positive-strand RNA viruses, only the Flaviviridae family has the 3D structural information resolved for such regions. Hence, future efforts to solve full-length RdRP structures containing these regions and to dissect the functional contribution of them are necessary to improve the overall understanding of the RdRP proteins as an evolutionarily integrated group, and our analyses here may serve as a guideline for selecting representative RdRP systems in these studies. [ABSTRACT FROM AUTHOR]

Membrane association is a hallmark of the genome replication of positive-strand RNA viruses [(+)RNA viruses]. All well-studied (+)RNA viruses remodel host membranes and lipid metabolism through orchestrated virus-host interactions to create a suitable microenvironment to survive and thrive in host cells. Recent research has shown that host lipids, as major components of cellular membranes, play key roles in the replication of multiple (+)RNA viruses. This review focuses on how (+)RNA viruses manipulate host lipid synthesis and metabolism to facilitate their genomic RNA replication, and how interference with the cellular lipid metabolism affects viral replication. [ABSTRACT FROM AUTHOR]

The genome of red clover necrotic mosaic virus is divided into two positive-stranded RNA molecules of RNA1 and RNA2, which have no 5′ cap structure and no 3′ poly(A) tail. Previously, we showed that any mutations in the cis -acting RNA replication elements of RNA2 abolished its cap-independent translational activity, suggesting a strong link between RNA replication and translation. Here, we investigated the functions of the 5′ untranslated region (UTR) of RNA2 and revealed that the basal stem-structure (5′BS) predicted in the 5′ UTR is essential for robust RNA replication. Interestingly, RNA2 mutants with substitution or deletion in the right side of the 5′BS showed strong translational activity, despite their impaired replication competency. Furthermore, nucleotide sequences other than the 5′BS of the 5′ UTR were essential to facilitate the replication-associated translation. Overall, these cis -acting RNA elements seem to coordinately regulate the balance between RNA replication and replication-associated translation. [ABSTRACT FROM AUTHOR]

Melandrium yellow fleck virus belongs to the genus Bromovirus, which is a group of tripartite plant RNA viruses. This virus has an approximately 200-nucleotide direct repeat sequence in the 5' untranslated region (UTR) of RNA3 that encodes the 3a movement protein. In the present study, protoplast assays suggested that the duplicated region contains amplification-enhancing elements. Deletion analyses of the 5' UTR of RNA3 showed that mutations in the short base-paired region, which is located dozens of bases upstream of the initiation codon of the 3a gene, greatly reduced the accumulation of RNA3. Disruption and restoration of the base-paired structure caused the accumulation of RNA3 to be decreased and restored, respectively. In vitro translation/replication assays demonstrated that the base-paired structure is important for the efficient amplification of negative-stand RNA3. A similar base-paired structure in RNA3 of another bromovirus, brome mosaic virus (BMV), also facilitated the efficient amplification of BMV RNA3, but only in combination with melandrium yellow fleck virus (MYFV) replicase and not with BMV replicase, thereby suggesting specific interactions between base-paired structures and MYFV replicase. [ABSTRACT FROM AUTHOR]

Replication of positive-strand RNA [(+)RNA] viruses proceeds through well-orchestrated actions of both viral and host factors. Remarkable features of eukaryotic (+)RNA virus replication include hijacking of host factors by viral components and remodeling of intracellular membranes to establish the viral replication factory, where viral RNA is synthesized. Here we review recent progress in our understanding of how (+)RNA plant viruses use host factors to create favorable environments for viral RNA replication. [ABSTRACT FROM AUTHOR]

Abstract: Movement protein (MP) of Red clover necrotic mosaic virus (RCNMV) forms punctate structures on the cortical endoplasmic reticulum (ER) of Nicotiana benthamiana cells, which are associated with viral RNA1 replication (Kaido et al., Virology 395, 232–242. 2009). We investigated the significance of ER-targeting by MP during virus movement from cell to cell, by analyzing the function of a series of MPs with varying length deletions at their C-terminus, either fused or not fused with green fluorescent protein (GFP). The C-terminal 70 amino acids were crucial to ER-localization of MP-GFP and cell-to-cell movement of the recombinant virus encoding it. However, C-terminal deletion did not affect MP functions, such as increasing the size exclusion limit of plasmodesmata, single-stranded RNA binding in vitro, and MP interacting in vivo. We discuss the possible role of this MP region in virus movement from cell to cell. [Copyright &y& Elsevier]

Abstract: The specific recognition of genomic RNAs by viral replicase proteins is a key regulatory step during the early replication process in positive-strand RNA viruses. In this study, we characterized the RNA-binding activity of the auxiliary replicase protein p27 of Red clover necrotic mosaic virus (RCNMV), which has a bipartite genome consisting of RNA1 and RNA2. Aptamer pull-down assays identified the amino acid residues of p27 involved in its specific interaction with RNA2. The RNA-binding activity of p27 correlated with its activity in recruiting RNA2 to membranes. We also identified the amino acids required for the formation of the 480-kDa replicase complex, a key player of RCNMV RNA replication. These amino acids are not involved in the functions of p27 that bind viral RNA or replicase proteins, suggesting an additional role for p27 in the assembly of the replicase complex. Our results demonstrate that p27 has multiple functions in RCNMV replication. [Copyright &y& Elsevier]

Abstract: Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA virus with a bipartite genome. The movement protein (MP) encoded by RNA2 is essential for viral movement. To obtain further insights into the viral movement mechanism, subcellular localizations of RCNMV MP fused with green fluorescent protein (MP:GFP) were examined in Nicotiana benthamiana epidermal cells and protoplasts. The MP:GFP expressed from the recombinant virus first appeared in the cell wall and subsequently was observed on the cortical endoplasmic reticulum (ER) as punctate spots. In contrast, the MP:GFP expressed transiently in the absence of other viral components was localized exclusively in the cell wall. Transient expression of the MP:GFP with a variety of RCNMV components revealed that the ER localization of the MP:GFP was associated with RNA1 replication, or its negative-strand RNA synthesis, but not those of RNA2 or replicase proteins per se. A model of RCNMV cell-to-cell movement is discussed. [Copyright &y& Elsevier]

Abstract: Multiplex reverse transcription and polymerase chain reaction (PCR) methods were developed to detect Solenopsis invicta viruses -1, -2, and -3 simultaneously in their host, the red imported fire ant, S. invicta. cDNA synthesis was conducted in a single reaction containing an oligonucleotide primer specific for each virus. Multiplex PCR was subsequently conducted with oligonucleotide primer pairs specific for each virus. The method was specific and sensitive, capable of detecting as few as 500 copies of the viral genomes consistently. Specificity was verified by PCR and amplicon sequencing. The method was evaluated against field-collected samples of ant workers from colonies in Argentina (n =135 ant colonies) and the United States (n =172 ant colonies). The prevalence of each virus in fire ant colonies varied considerably from site to site. A number of colonies exhibited multiple virus infections. However, the multiple SINV infection rate was lower than for single infections. Comparison of viral infection prevalence between S. invicta colonies in Argentina and the U.S. showed no statistical differences, regardless of infection category. This method is anticipated to facilitate epidemiological and related studies concerning the S. invicta viruses in fire ants. [Copyright &y& Elsevier]

Abstract: Viral capsid proteins (CPs) can regulate gene expression and encapsulate viral RNAs. Low-level expression of the brome mosaic virus (BMV) CP was found to stimulate viral RNA accumulation, while higher levels inhibited translation and BMV RNA replication. Regulation of translation acts through an RNA element named the B box, which is also critical for the replicase assembly. The BMV CP has also been shown to preferentially bind to an RNA element named SLC that contains the core promoter for genomic minus-strand RNA synthesis. To further elucidate CP interaction with RNA, we used a reversible cross-linking–peptide fingerprinting assay to identify peptides in the capsid that contact the SLC, the B-box RNA, and the encapsidated RNA. Transient expression of three mutations made in residues within or close by the cross-linked peptides partially released the normal inhibition of viral RNA accumulation in agroinfiltrated Nicotiana benthamiana. Interestingly, two of the mutants, R142A and D148A, were found to retain the ability to down-regulate reporter RNA translation. These two mutants formed viral particles in inoculated leaves, but only R142A was able to move systemically in the inoculated plant. The R142A CP was found to have higher affinities for SLC and the B box compared with those of wild-type CP and to alter contacts to the RNA in the virion. These results better define how the BMV CP can interact with RNA and regulate different viral processes. [Copyright &y& Elsevier]

Flock House virus (FHV) is a nonenveloped, icosahedral insect virus whose genome consists of two molecules of single-stranded, positive-sense RNA. FHV is a highly tractable system for studies on a variety of basic aspects of RNA virology. In this review, recent studies on the replication of FHV genomic and subgenomic RNA are discussed, including a landmark study on the ultrastructure and molecular organization of FHV replication complexes. In addition, we show how research on FHV B2, a potent suppressor of RNA silencing, resulted in significant insights into antiviral immunity in insects. We also explain how the specific packaging of the bipartite genome of this virus is not only controlled by specific RNA-protein interactions but also by coupling between RNA replication and genome recognition. Finally, applications for FHV as an epitopepresenting system are described with particular reference to its recent use for the development of a novel anthrax antitoxin and vaccine. [ABSTRACT FROM AUTHOR]

Abstract: Feline calicivirus (FCV) is a common cause of upper respiratory and oral disease in cats. Highly virulent, systemic strains of FCV (vs FCV) have been recently described. These vs FCV isolates cause edema, cutaneous ulcers and high mortality in affected cats. This study reports a disease model with such a vs FCV isolate (FCV-33585). It also describes a full-length capsid gene sequence of this vs FCV isolate and the capsid sequence comparison of this strain with 35 other virulent and non-virulent FCV strains. In addition, sequence comparison of this strain with other 114 known sequences in the hyper-variable region of capsid gene was analyzed. Two amino acids were identified within the hyper-variable region as potentially unique signature for this vs FCV strain. This study also describes the attenuation of FCV-33585 by two methods: serial passaging at low temperature, and the generation of a temperature sensitive (ts) mutant by UV irridiation. Moreover, the potential use of attenuated vs FCV as vaccine was also explored. Monoclonal antibodies were also identified which could differentiate commonly used FCV vaccine strain from this vs strain (FCV-33585). And two monoclonal antibodies were found to react specifically the wild-type, not the attenuated FCV-33585. [Copyright &y& Elsevier]

Positive-strand RNA viruses constitute the largest group of plant viruses and have an important impact on world agriculture. These viruses have small genomes that encode a limited number of proteins and depend on their hosts to complete the various steps of their replication cycle. In this review, the contact points between positive-strand RNA plant viruses and their hosts, which are necessary for the translation and replication of the viral genomes, are discussed. Special emphasis is placed on the description of viral replication complexes that are associated with specific membranous compartments derived from plant intracellular membranes and contain viral RNAs and proteins as well as a variety of host proteins. These complexes are assembled via an intricate network of protein–protein, protein–membrane, and protein–RNA interactions. The role of host factors in regulating the assembly, stability, and activity of viral replication complexes are also discussed. [ABSTRACT FROM AUTHOR]

In the family Bromoviridae, a mixture of the three genomic RNAs of bromo-, cucumo-, and oleaviruses is infectious as such, whereas the RNAs of alfamo-and ilarviruses require binding of a few molecules of coat protein (CP) to the 3′ end to initiate infection. Most studies on the early function of CP have been done on the alfamovirus Alfalfa mosaic virus (AMV). The 3′ 112 nucleotides of AMV RNAs can adopt two different conformations. One conformer consists of a tRNA-like structure that, together with an upstream hairpin, is required for minus-strand promoter activity. The other conformer consists of four hairpins interspersed by AUGC-sequences and represents a strong binding site for CP. Binding of CP to this conformer enhances the translational efficiency of viral RNAs in vivo 40-fold and blocks viral minus-strand RNA synthesis in vitro. AMV CP is proposed to initiate infection by mimicking the function of the poly(A)-binding protein. [ABSTRACT FROM AUTHOR]

The genomic RNA of Turnip yellow mosaic virus (TYMV) has an 82-nucleotide-long tRNA-like structure at its 3′-end that can be valylated and then form a stable complex with translation elongation factor eEF1A·GTP. Transcription of this RNA by TYMV RNA-dependent RNA polymerase (RdRp) to yield minus strands has previously been shown to initiate within the 3′-CCA sequence. We have now demonstrated that minus strand synthesis is strongly repressed upon the binding of eEF1A·GTP to the valylated viral RNA. eEF1A·GTP had no effect on RNA synthesis templated by non-aminoacylated RNA. Higher eEF1A·GTP levels were needed to repress minus strand synthesis templated by valyl-EMV TLS RNA, which binds eEF1A·GTP with lower affinity than does valyl-TYMV RNA. Repression by eEF1A·GTP was also observed with a methionylated variant of TYMV RNA and with aminoacylated tRNAHis, tRNAAla, and tRNAPhe transcripts. It is proposed that minus strand repression by eEF1A·GTP binding occurs early during infection to help coordinate the competing translation and replication functions of the genomic RNA. [Copyright &y& Elsevier]

Positive-strand RNA viruses such as hepatitis C virus (HCV), flaviviruses, and coronaviruses are medically important. Assembly of replicase on host membranes is a conserved replication strategy and an attractive antiviral target. The mechanisms of replicase assembly are largely unknown, due to the technical difficulties in purifying the replicase and carrying out structural studies. Here, with an HCV replicase assembly surrogate system, we employed a bioorthogonal system to introduce the photolabile unnatural amino into each residue in the cytosolic regions of NS4B and the amphipathic helix (AH) of NS5A. Photocrosslinking enabled visualization of NS4B oligomerization and NS5A dimerization at pinpointed interacting residues and identifying contacting sites among the replicase components. Characterization of the interacting sites revealed hub elements in replicase assembly by docking replicase components to prompt protein-protein interactions. The results provide information about the molecular architecture of the replicase, advancing understanding of the mechanism of replicase assembly. [Display omitted] • N-terminal AH1 and C-terminal residue L237 mainly mediate NS4B oligomerization • NS4B C-terminal residues S227, D228, A229, and R232 are the docking sites of NS3 • NS5A AH plays a role in NS5A-NS4B interaction and NS5A dimerization • NS4B C-terminal H2 and NS5A AH are hub elements in HCV replicase assembly Zhang et al. use a polyprotein expression model to introduce a photoactivatable unnatural amino acids at individual residues to characterize the interaction between HCV replicase components NS4B, NS5A, and NS3. The detailed landscape of protein-protein interactions provides a model for replicase assembly. [ABSTRACT FROM AUTHOR]

Abstract: Purification of Solenopsis invicta virus 1 (SINV-1) from its host, S. invicta, and subsequent examination by electron microscopy revealed a homogeneous fraction of spherical particles with a diameter of 30–35nm. Quantitative PCR with SINV-1-specific oligonucleotide primers verified that this fraction contained high copy numbers of the SINV-1 genome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the SINV-1 purified fraction revealed three major and one minor protein bands. The protein bands were labeled VP1 (40.8±1.4kDa), VP2 (35.7±2.8kDa), VP3 (25.2±1.8kDa), and VP4 (22.2±2.5kDa) based on mass. N-terminal sequence was acquired successfully for VP1, VP2, and VP3, but not VP4, and delineated each capsid protein within the 3′-proximal open reading frame of SINV-1. Positional organization of the viral proteins within the SINV-1 structural polyprotein was consistent with dicistroviruses (when based on sequence similarity). Blastp analysis of SINV-1 VP1, VP2, and VP3 revealed significant identity with corresponding structural capsid proteins of positive-strand RNA viruses, particularly acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV). Amino acid residues about the scissile bonds for VP1 and VP3 were consistent with dicistroviruses and insect-infecting picorna-like viruses. N-terminal sequencing of VP2 also established that translation initiation of the SINV-1 structural polyprotein was mediated by an internal ribosomal entry site and is AUG-independent. [Copyright &y& Elsevier]