No abstract

A virus isolate causing mosaic disease of commercial sugarcane was purified to homogeneity. Electron microscopy revealed flexuous filamentous virus particles of ca 890 x 15 nm. The virus isolate reacted positively with heterologous antiserum to narcissus latent virus form UK, but failed to react with potyvirus group specific antiserum. N-terminal sequencing of the intact coat protein (CP) and the tryptic peptides indicated that the virus was probably a potyvirus but distinct from several reported potyviruses. Comparison of the 3'-terminal 1084 nucleotide sequence of the RNA genome of this virus revealed 93.6% sequence identity in the coat protein coding region with the recently described sugarcane streak mosaic virus (Pakistani isolate). The molecular weight of the coat protein (40 kDa) was higher than that deduced from the amino acid sequence (34 kDa). The apparent increase in size was shown to be due to glycosylation of the coat protein which has not been reported thus far in the family, Potyviridae. This is the first report on the molecular characterization of a virus causing mosaic disease of sugarcane in India and the results demonstrate that the virus is a strain of sugarcane streak mosaic virus, a member of the Tritimovirus genus of the Potyviridae. We have named it sugarcane streak mosaic virus--Andhra Pradesh isolate (SCSMV-AP).

Bromoviral templates for plus-strand RNA synthesis are rich in A or U nucleotides in comparison to templates for minus-strand RNA synthesis. Previous studies demonstrated that plus-strand RNA synthesis by the brome mosaic virus (BMV) RNA replicase is more efficient if the template contains an A/U-rich template sequence near the initiation site (K. Sivakumaran and C. C. Kao, J. Virol. 73:6415-6423, 1999). These observations led us to examine the effects of nucleotide changes near the template's initiation site on the accumulation of BMV RNA3 genomic minus-strand, genomic plus-strand, and subgenomic RNAs in barley protoplasts transfected with wild-type and mutant BMV transcripts. Mutations in the template for minusstrand synthesis had only modest effects on BMV replication in barley protoplasts. Mutants with changes to the ؉3, ؉5, and ؉7 template nucleotides accumulated minus-strand RNA at levels similar to the the wild-type level. However, mutations at positions adjacent to the initiation cytidylate in the templates for genomic and subgenomic plus-strand RNA synthesis significantly decreased RNA accumulation. For example, changes at the third template nucleotide for plus-strand RNA3 synthesis resulted in RNA accumulation at between 18 and 24% of the wild-type level, and mutations in the third template nucleotide for subgenomic RNA4 resulted in accumulations at between 7 and 14% of the wild-type level. The effects of the mutations generally decreased as the mutations occurred further from the initiation nucleotide. These findings demonstrate that there are different requirements of the template sequence near the initiation nucleotide for BMV RNA accumulation in plant cells.Efficient viral RNA synthesis requires specific recognition and proper interaction between the template RNA and the membrane-bound viral replicase (7,12,40,41). This recognition process is likely to be quite complex because an RNA virus expresses different classes of RNAs at regulated levels and times (for review of viral RNA replication, see references 8 and 30). We study viral RNA replication with Brome mosaic virus (BMV) as a model plus-strand RNA virus.BMV is a single-stranded, positive-sense RNA virus that belongs to the Bromoviridae family of plant viruses in the alphavirus-like superfamily (18, 23). The tripartite BMV genome is composed of RNAs designated RNA1 (3.2 kb), RNA2 (2.8 kb), and RNA3 (2.1 kb). Viral replication is dependent on BMV genomic RNAs 1 and 2, which encode the replicationassociated proteins 1a and 2a, respectively. The dicistronic RNA3 encodes the 3a movement protein and coat protein, both of which are required for systemic infection in plants (5, 34, 38). Minus-strand RNA3 can also be transcribed to produce a subgenomic RNA4 (0.9 kb) that directs translation of the viral capsid (3).Complete replication of the BMV genome requires three classes of RNA promoters that direct the BMV replicase to synthesize genomic minus-strand, genomic plus-strand, and subgenomic RNAs (Fig. 1A) (22, 23). A tRNA-like structure at the 3Ј end...

The cis-acting elements for Brome mosaic virus (BMV) RNA synthesis have been characterized primarily for RNA3. To identify additional replicase-binding elements, nested fragments of all three of the BMV RNAs, both plus-and minus-sense fragments, were constructed and tested for binding enriched BMV replicase in a template competition assay. Ten RNA fragments containing replicase-binding sites were identified; eight were characterized further because they were more effective competitors. All eight mapped to noncoding regions of BMV RNAs, and the positions of seven localized to sequences containing previously characterized core promoter elements ( The replication of positive-strand RNA viruses is essential for pathogenesis. The responsible enzyme, the viral replicase, is a complex that includes the virus-encoded RNA-dependent RNA polymerase, additional viral replicase proteins, and host factors (6, 32). In addition, specific interactions between the viral genomic RNAs and viral replicase are required for replication and/or transcription of the viral RNAs (1, 6). The interaction process is likely to be quite complex because an RNA virus expresses different classes of RNAs at programmed levels and times.Brome mosaic virus (BMV) belongs to the alphavirus-like superfamily of plant and animal positive-strand RNA viruses. The BMV genome is divided into three capped RNAs, designated RNA1, RNA2, and RNA3 (3, 26). RNA1 and RNA2 encode replication-associated proteins, while RNA3 encodes two proteins required for systemic movement of the virus in plants and encapsidation of viral RNAs. Due to the dicistronic nature of RNA3, the second cistron encoding the capsid is translated from subgenomic RNA that is transcribed from the subgenomic promoter. In all, three classes of RNAs must be produced during successful BMV replication: genomic minusstrand, genomic plus-strand, and subgenomic RNAs.cis-acting elements for efficient genomic plus-strand, minusstrand, and subgenomic BMV RNA synthesis have been characterized by a combination of approaches, including RNA synthesis by the BMV replicase in vitro (37), transfection of BMV RNAs into protoplasts (34), analysis in plants (7), and replication in Saccharomyces cerevisiae, which is permissive for BMV replication and transcription (23). Each study generally focused on one cis-acting element and on RNA3, which does not contain functions directly required for replication. Nonetheless, a number of required elements have been identified (Fig. 1).The 3Ј noncoding regions (NCR) of BMV genomic plusstrand RNAs form a tRNA-like structure that directs the initiation of minus-strand RNA synthesis in vitro (10, 15) and in vivo (38, 46). Stem-loop C (SLC) within the tRNA-like structure of RNA3 binds the BMV replicase through an RNA structure called a clamped adenine motif (29). Given the highly similar structures predicted for RNA1 and RNA2, it is likely that the same structures are required to direct their minusstrand replication (Fig. 1). A mutation in SLC in RNA2 was shown to affect RNA replicat...

The 3 portions of plus-strand brome mosaic virus (BMV) RNAs mimic cellular tRNAs. Nucleotide substitutions or deletions in the 3 CCA of the tRNA-like sequence (TLS) affect minus-strand initiation unless repaired. We observed that 2-nucleotide deletions involving the CCA 3 sequence in one or all BMV RNAs still allowed RNA accumulation in barley protoplasts at significant levels. Alterations of CCA to GGA in only BMV RNA3 also allowed RNA accumulation at wild-type levels. However, substitutions in all three BMV RNAs severely reduced RNA accumulation, demonstrating that substitutions have different repair requirements than do small deletions. Furthermore, wild-type BMV RNA1 was required for the repair and replication of RNAs with nucleotide substitutions. Results from sequencing of progeny viral RNA from mutant input RNAs demonstrated that RNA1 did not contribute its sequence to the mutant RNAs. Instead, the repaired ends were heterogeneous, with one-third having a restored CCA and others having sequences with the only commonality being the restoration of one cytidylate. The role of BMV RNA1 in increased repair was examined.The 3Ј ends of viral RNAs are required for the proper translation, stability, and replication of the RNAs. For RNA replication, the end of the RNA must be unwound and recognized by the viral replication machinery (7). Consistent with this need, several RNA-dependent RNA polymerases (RdRps) have a narrow template channel that can only accommodate single-stranded RNA or have mechanisms that discriminate against the use of double-stranded templates for de novo initiation (10,43,53). A potential cost of having a single-stranded 3Ј sequence is increased susceptibility to cellular nucleases. It is therefore to be expected that RNA viruses have mechanisms to protect the ends from degradation and/or to repair the end sequences.Strategies that could prevent degradation include the formation of base paired structures that can be opened through alternative base pairing, as in carmovirus (51), the binding of cellular proteins, as in phage Q␤ (5), or covalent linkage of viral proteins to the ends of viral genomes, as in picornaviruses and some DNA viruses (60). Several viral RNA repair processes have also been identified. The 3Ј ends of viral RNA genomes and their associated satellite (sat) RNAs can be repaired either by viral polymerase, by RNA recombination, or by a host-terminal transferase, including the poly(A) polymerase complex (9,11,12,19,20,28,31,47,48,61). Minus-strand viruses such as Hantaan virus (29) and Respiratory Syncytial virus (41) have ends with short repeats that apparently allow initiation of RNA synthesis within an internal repeat and then realign the nascent RNA at the end of the genome, thereby regenerating the ends of the RNAs. Other strategies may include the use of abortive initiation products or the synthesis of initiation products from mutated initiation sequence to prime the synthesis of the turnip crinkle virus (TCV) satellite RNA (10,47).A number of plant-infecting RNA viruses...