Translational Cross-Activation of the Encapsidated RNA of Potexviruses

We had shown the genomic RNA of potexviruses potato virus X and the alternanthera mosaic virus to be inaccessible in vitro to ribosomes while in intact virion form, but the RNAs can be translationally activated following the binding of movement protein 1 (MP1) to virus particles. Here, we present the results of the follow-up study targeting two more potexvirus species - the Narcissus mosaic virus and the Potato aucuba mosaic virus. We found encapsidated potexviral RNA to share common translational features in vitro and the MP1 to be potent over homological virions of its own species and over heterological virions of other species, as well exhibiting selective specificity. Reciprocal cross-activation is observed among viral species phylogenetically either close or distant. There is direct evidence that MP1 binding to the end of the virion is necessary, but not sufficient, for translational activation of encapsidated RNA.

Flexible filamentous virus structures from fiber diffraction

Fiber diffraction data have been obtained from Narcissus mosaic virus, a potexvirus from the family Flexiviridae, and soybean mosaic virus (SMV), a potyvirus from the family Potyviridae. Analysis of the data in conjunction with cryo-electron microscopy data allowed us to determine the symmetry of the viruses and to make reconstructions of SMV at 19 Å resolution and of another potexvirus, papaya mosaic virus, at 18 Å resolution. These data include the first well-ordered data ever obtained for the potyviruses and the best-ordered data from the potexviruses, and offer the promise of eventual high resolution structure determinations.

First Report of Narcissus mosaic virus Infecting Crocus spp. Cultivars in the Netherlands

A survey to identify virus diseases affecting Crocus spp. in the Netherlands was conducted during April 2004. Crocus spp. (cvs. Flavus, Pick-wick, Remembrance, and Grand Maitre) with symptoms suggestive of virus infection (stunting, yellowing, necrosis, and flower color breaking) were collected from several fields in the Breezand and Lisse districts in northern and southern Netherlands, respectively. All samples were tested for the presence of six known crocus-infecting viruses (1,2) using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. The ELISA assay was performed with the following polyclonal and monoclonal antibodies: Iris severe mosaic virus (ISMV); Tobacco rattle virus (TRV) isolates F, Y, and J obtained from the Applied Plant Research Institute, Lisse, Netherlands; Arabis mosaic virus; Cucumber mosaic virus from the Plant Research International Institute, Wageningen, Netherlands; Iris yellow spot virus (IYSV) from the Virology Department at Wageningen University, Netherlands; and the potyvirus group-specific monoclonal antiserum from the DSMZ, Braunschweig, Germany. All samples that tested positive with a potyvirus antiserum were further tested for the presence of Bean yellow mosaic virus (BYMV) using a BYMV-specific antiserum. Serological results obtained indicated that BYMV, detected with the potyvirus antiserum and BYMV-specific antiserum, and ISMV were the most commonly encountered viruses. Tobacco necrosis virus (TNV) and TRV were only found occasionally, whereas IYSV, was not detected in any of the samples tested. To study the presence of viruses not yet reported, total RNA was extracted and tested with a RT-PCR assay with carlavirus, potexvirus, necrovirus (R. Miglino, unpublished), and potyvirus (3) genus-specific oligonucleotides. In accordance with the ELISA results, PCR amplicons were obtained with the potyvirus, TNV, and TRV primer sets. Furthermore, a 280-bp amplicon corresponding to the expected size was amplified in a RT-PCR assay performed on total RNA with a potexvirus genus-specific primer set. The reverse primer (5′-AGC ATG GCG CCA TCT TGT GAC TG-3′) was located upstream in the conserved viral replicaseencoding region at position 4254-4231 of Narcissus mosaic virus (NMV) RNA genome (Genbank Accession No. D13747) and the forward primer (5′-CTG AAG TCA CAA TGG GTG AAG AA-3′) was located downstream at position 3969–3992. Sequence homology using BLAST analysis of the cloned and sequenced PCR product showed 98% identity with NMV. Although the virus has a very narrow host range, the results of this study may have a significant impact on the crocus industry in the Netherlands. To our knowledge, this is the first report of NMV infecting crocus. References: (1) M. G. Bellardi and A. Pisi. Inf. Fitopatol. 37:33, 1987. (2) A. F. L. M. Derks. Crocus spp. Pages 260–264 in: Virus and Virus-like Diseases of Bulbs and Flower Crops. G. Loebenstein et al., eds. Wiley publishers, West Sussex, UK, 1995. (3) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991.

Solution structures of potato virus X and narcissus mosaic virus from Raman optical activity

Potato virus X (PVX) and narcissus mosaic virus (NMV) were studied using vibrational Raman optical activity (ROA) in order to obtain new information on the structures of their coat protein subunits. The ROA spectra of the two intact virions are very similar to each other and similar to that of tobacco mosaic virus (TMV) studied previously, being dominated by signals characteristic of proteins with helix bundle folds. In particular, PVX and NMV show strong positive ROA bands at ∼1340 cm−1 assigned to hydrated α-helix and perhaps originating in surface exposed helical residues, together with less strong positive ROA intensity in the range ∼1297–1312 cm−1 assigned to α-helix in a more hydrophobic environment and perhaps originating in residues at helix–helix interfaces. The positive ∼1340 cm−1 ROA band of TMV is less intense than those of PVX and NMV, suggesting that TMV contains less hydrated α-helix. Small differences in other spectral regions reflect differences in some loop, turn and side-chain compositions and conformations among the three viruses. A pattern recognition program based on principal component analysis of ROA spectra indicates that the coat protein subunit folds of PVX and NMV may be very similar to each other and similar to that of TMV. These results suggest that PVX and NMV may have coat protein subunit structures based on folds similar to the TMV helix bundle and hence that the helical architecture of the PVX and NMV particles may be similar to that of TMV but with different structural parameters.