|Scheets, K - OKLAHOMA STATE UNIVERSITY|
Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 7, 2006
Publication Date: June 13, 2006
Citation: Scheets, K., Redinbaugh, M.G. 2006. Infectious cDNA transcripts of maize necrotic streak virus: infectivity and translational characteristics. Virology. 350:171-183. Interpretive Summary: Characterization of the genomes of emerging viruses is important for developing specific and sensitive diagnostics and for understanding how the virus infects its crop host. To continue characterization of maize necrotic streak virus (MNeSV), an emerging maize virus isolated from field-grown corn in Arizona, we completed the sequence of the virus' genome, analyzed the genome sequence for regulatory elements, characterized the expression of genome sequences in plants and in vitro, and constructed clones of the virus that produce infectious transcripts. Our results indicated that the regulatory elements contained in MNeSV and genome expression, show a high degree of conservation with those found in Tomato bushy stunt virus (TBSV). These results indicate that overall virus replication and gene expression strategies are likely to be conserved between the two viruses. However, the genome sequence and some biological characteristics of TBSV are distinct from those of TBSV. These results suggest MNeSV should be taxonomically aligned with the genus Tombusvirus, of which TBSV is the primary example, or MNeSV should be assigned to a new genus related to that containing TBSV. These results are of interest to researchers developing an understanding of virus replication in plants, and to those interested in developing virus-based vectors for gene silencing in cereals. This research will impact scientific peers and will influence funding opportunities. Maize necrotic streak virus has a relatively small genome, and the virus replicates to high levels in plants. These characteristics make it a potential target for development of virus-based vectors for gene silencing in maize and other cereals. Such vectors are urgently needed so that researchers can characterize the enormous volume of genomic information that is rapidly accumulating for maize, rice and other cereals. The work performed in this study is part of the necessary background information required for development of successful vectors.
Technical Abstract: The completion of the sequence of Maize necrotic streak virus (MNeSV) indicated a genome of 4094 nt. Northern blotting and primer extension mapping identified two sgRNAs, 1607 nt and 781 nt long. Comparison of the MNeSV and Tomato bushy stunt virus genome sequences indicated that tombusvirus regulatory sequences and RNA secondary structures required for virus replication were highly conserved in MNeSV, despite lower sequence similarity in untranslated regions (UTRs). RNA ligase mediated-rapid analysis of cDNA ends of vRNA indicated MNeSV is not capped. Transcripts from six variant cDNAs were all infectious in maize (Zea mays L.) protoplasts and plants. In protoplasts, uncapped transcripts starting with GG had extremely low infectivity, which could be ameliorated by capping. Translation of genomic and subgenomic RNA transcripts in wheat germ extracts produced five proteins with sizes corresponding to the expected gene products, and showed that MNeSV has a 3' cap-independent translational enhancer located within the 3' 156 nt that functions in vitro [kms1].