Effect of mutations in Barley yellow dwarf virus genomic RNA on the 5' termini of subgenomic RNAs.

To investigate the role in transcription initiation of sequences upstream of the promoter for subgenomic RNA2 (sgRNA2) of Barley yellow dwarf virus PAV, deletion and insertion clones were constructed and the 5' termini of all three sgRNAs were determined. Deletion and insertion of sequences upstream of the transcription start site of sgRNA2 altered the position and nucleotide used to initiate transcription of sgRNA2, but not of sgRNAs 1 or 3. Since the promoter for sgRNA2 is downstream of the mutations, these results show that sequences outside of the core promoter can influence transcription start site selection from the sgRNA2 promoter.

Sequence requirements for translation initiation of Rhopalosiphum padi virus ORF2.

Rhopalosiphum padi virus (RhPV) is an aphid-infecting virus with a 10-kb ssRNA genome that contains two large open reading frames (ORFs). ORF1 and ORF2 encode the nonstructural and structural polyproteins, respectively. Both ORFs are preceded by noncoding regions of 500 nt that could function as internal ribosome entry segments (IRESes). The sequence for ORF2 lacks an obvious initiation codon, but an out-of-frame AUG codon is present that could translate ORF2 through a +1 frameshift. To investigate the mechanisms of translation initiation of ORF2, a series of point and deletion mutations were constructed and transcribed and translated in vitro. A bicistronic plasmid containing two copies of the RhPV intergenic region translated both ORFs efficiently, indicating that the region functioned as an IRES in vitro. Deletion analysis showed that the region required for activity of the IRES extended from 178 nt upstream and 6 nt downstream of the 5' border of ORF2. Changes in the out-of-frame AUG codon had little effect on translation initiation, but mutations that included the first and second codons of ORF2 or that disrupted a putative pseudoknot structure near the 3' end of the IRES failed to initiate protein synthesis. Sequence analysis of the in vitro synthesized proteins showed that the first amino acid of the polyprotein corresponded to the second codon of ORF2. These results show that in vitro the noncoding region upstream of RhPV ORF2 functions as an IRES that contains a pseudoknot that directs translation initiation at a non-AUG codon. If the in vitro synthesized proteins have not been processed by an aminopeptidase, translation is initiated at the second (GCA) codon of ORF2.

Solanaceous Weeds as Possible Sources of Cucumber mosaic virus in Southern Illinois for Aphid Transmission to Pepper.

Over 5,000 individual plants representing approximately 55 species from an area in southern Illinois where Cucumber mosaic virus (CMV) has been a major problem in pepper (Capsicum annuum) were tested for the presence of CMV by enzyme-linked immunosorbent assay (ELISA). Representative ELISA-positive samples were checked by western blot tests to confirm virus-specific reactions. Nearly all of the infected plants detected were either Solanum ptycanthum (eastern black nightshade) or Physalis spp. (principally P. heterophylla, groundcherry). Over 1,000 pepper transplants and approximately 500 tomato transplants, collected prior to planting, were negative for CMV by ELISA. In aphid transmission (arena) experiments, all five aphid species tested were capable of transmitting CMV from nightshade to pepper: Aphis fabae subsp. solanella, Aphis gossypii, Myzus persicae, Rhopalosiphum padi, and Sitobion avenae. Aphis fabae subsp. solanella, A. gossypii, and A. nerii were able to transmit CMV from P. heterophylla to pepper. Aphis fabae subsp. solanella was commonly found colonizing nightshade from May through October in southern Illinois.

Nucleotide sequence analysis shows that Rhopalosiphum padi virus is a member of a novel group of insect-infecting RNA viruses.

Rhopalosiphum padi virus (RhPV) is an aphid virus that has been considered a member of the Picornaviridae based on physicochemical properties. The 10,011-nt polyadenylated RNA genome of RhPV was completely sequenced. Analysis of the sequence revealed the presence of two open reading frames (ORFs). The predicted amino acid sequence of ORF1, representing the first 6600 nt of the RhPV genome, showed significant similarity to the nonstructural proteins of several plant and animal RNA viruses. Direct sequence analysis of the RhPV capsid proteins showed that ORF2, which represents the last 2900 nt, encodes the three structural proteins (28, 29, and 30 kDa). The predicted amino acid sequence of ORF2 is very similar to the corresponding regions of Drosophila C virus, Plautia stali intestine virus, and to a partial sequence from the 3' end of the cricket paralysis virus genome. The site of initiation of protein synthesis for ORF2 could not be determined from the amino acid and nucleotide sequences. ORF1 is preceded by 579 nt of noncoding RNA and the two ORFs are separated by more than 500 nt of noncoding RNA. Like picornaviruses, these regions may function to facilitate the cap-independent initiation of translation of the two ORFs. These data suggest that RhPV, Drosophila C virus, Plautia stali intestine virus, and probably cricket paralysis virus are members of a unique group of small RNA viruses that infect primarily insects.