In order to suppress RNA silencing, many plant and some animal viruses encode RNA silencing suppressors to achieve infection. In this study, we report that B3 and B4, encoded by DNA3 and DNA4 of banana bunchy top virus (BBTV), exhibit RNA silencing suppression activity. B3 and B4 were able to increase the transient expression of green fluorescent protein (GFP) and dramatically enhanced the pathogenicity of potato virus X (PVX) in Nicotiana benthamiana. B4 was able to reverse established gene silencing on an inoculated leaf or on an upper leaf. B3, however, was only active during infection of an inoculated leaf. Furthermore, B4, but not B3, was able to enhance GFP expression in the transgenic N. benthamiana line 16c. In conclusion, B3 and B4 are the RNA silencing suppressors of BBTV, and they may act at different steps in the RNA silencing pathways.

An outbreak of a viral disease on chili pepper (Capsicum chinense Jacp. cv. Yellow Lantern) occurred in Hainan Province, China during 2003 and 2004. The disease was prevalent in five chili-producing counties surveyed. Leaves of infected plants initially displayed symptoms of dark green banding along veins and later became distorted with striking mosaic. Infected plants had reduced flower numbers and fruit set, resulting in a significant yield loss. The causative virus was characterized and identified as Chilli veinal mottle virus (ChiVMV) (3). An isolate of the virus was obtained via three single lesion passages through Chenopodium amaranticolor and was shown to reproduce the same symptoms on inoculated C. chinense cv. Yellow Lantern. Negative staining of crude extracts of the infected tissue and subsequent electron microscopy revealed flexuous rods of 12 to 13 × 750 nm, typical of a potyvirus. Pinwheel-like inclusion bodies were abundant in thin sections of infected leaves. Purified virus preparations contained one major protein of 32.8 kDa and one minor protein of 28 kDa when fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both of these protein bands were excised and subsequently analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Multiple peptide fragments from both proteins were identified as arising from ChiVMV capsid protein (CP) (1,2). Therefore, the 32.8-kDa protein is the full-length ChiVMV CP and the 28-kDa protein is presumably a degradation product of the CP. The combined biological and molecular data provided strong evidence that the viral disease on C. chinense was caused by ChiVMV. To our knowledge, this is the first report of ChiVMV infection on C. chinense in China and the first report of C. amaranticolor as an experimental host for ChiVMV. References: (1) P. Chiemsombat et al. Arch. Virol. 143:1855, 1998. (2). J. Joseph and H. S. Savithri. Arch. Virol. 144:1679, 1999. (3) P. Siriwong et al. Plant Pathol. 44:718, 1995.

Hibiscus latent Singapore virus (HLSV) mutants were constructed to study roles of its internal poly(A) tract (IPAT) in viral replication and coat protein (CP) expression. Shortening of the IPAT resulted in reduced HLSV RNA accumulation and its minimal length required for HLSV CP expression in plants was 24 nt. Disruption of a putative long range RNA-RNA interacting structure between 5' and 3' untranslated regions of HLSV-22A and -24A resulted in reduced viral RNA and undetectable CP accumulation in inoculated leaves. Replacement of the IPAT in HLSV with an upstream pseudoknot domain (UPD) of Tobacco mosaic virus (TMV) or insertion of the UPD to the immediate downstream of a 24 nt IPAT in HLSV resulted in drastically reduced viral RNA replication. Plants infected with a TMV mutant by replacement of the UPD with 43 nt IPAT exhibited milder mosaic symptoms without necrosis. We have proposed a model for HLSV replication.

Hibiscus latent Singapore virus (HLSV) is a member of Tobamovirus and its full-length cDNA clones were constructed. The in vitro transcripts from two HLSV full-length cDNA clones, which contain a hepta-adenosine stretch (pHLSV-7A) and an octo-adenosine stretch (pHLSV-8A), are both infectious. The replication level of HLSV-7A in Nicotiana benthamiana protoplasts was 5-fold lower, as compared to that of HLSV-8A. The replicase proteins of HLSV-7A were produced through programmed -1 ribosomal frameshift (-1 PRF) and the 7A stretch was a slippery sequence for -1 PRF. Mutations to the downstream pseudoknot of 7A stretch showed that the pseudoknot was not required for the frameshift in vitro. The stretch was found to be extended to 8A after subsequent replication cycles in vivo. It is envisaged that HLSV employs the monotonous runs of A and -1 PRF to convert its 7A to 8A to reach higher replication for its survival in plants.