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Short Communication |
1 Department of Entomology, The Pennsylvania State University, 501 ASI Building, University Park, PA 16802, USA
2 Department of Microbiology, 32 Pearson Hall, Miami University, Oxford, OH 45056, USA
3 Proteomics and Mass Spectrometry Core Facility, Huck Institutes of the Life Sciences, The Pennsylvania State University, 104 Chemistry Building, University Park, PA 16802, USA
4 Department of Biochemistry, Virginia Polytechnic Institute and State University, 111 Engel Hall, Blacksburg, VA 24061, USA
Correspondence
Liwang Cui
luc2{at}psu.edu
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ABSTRACT |
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). The name of this virus family is derived from the unique cellular pathology that they cause, i.e. the formation of large, virion-containing vesicles of 5–10 µm diameter in the haemolymph of infected insects. Another distinguishing feature of this virus family is the unusual mode of viral transmission: whilst ascoviruses (AVs) are poorly infectious through the oral route, they may rely on parasitoid wasps for mechanical transmission (Govindarajan & Federici, 1990). Although these viruses were discovered more than 20 years ago, it was not until recently that three AV genomes [Trichoplusia ni ascovirus 2c (TnAV-2c), Spodoptera frugiperda ascovirus 1a (SfAV-1a) and Heliothis virescens ascovirus 3e (HvAV-3e)] were sequenced (Asgari et al., 2007; Bideshi et al., 2006; Wang et al., 2006). Both the genome organization and phylogenetic analysis of the DNA polymerase and major capsid protein (MCP) genes suggest that AVs are related closely to viruses in the family Iridoviridae (Cheng et al., 2005; Stasiak et al., 2003). Compared with other groups of large DNA viruses, AVs have been relatively poorly studied and the functions of most of the predicted open reading frames (ORFs) are unknown (Asgari et al., 2007; Bideshi et al., 2006; Wang et al., 2006).
Virions of AVs are enveloped and contain more than 12 polypeptides ranging from 10 to 200 kDa (Federici et al., 1990). With the deciphering of three AV genomes, we sought to identify the virion structural proteins of TnAV-2c by using a high-accuracy mass-spectrometry approach. TnAV-2c, originally isolated from a Helicoverpa zea larva (Cheng et al., 2005), was propagated in H. zea larvae of a laboratory colony. Viral vesicles and particles were purified on sucrose gradients by using a previously described method (Federici & Govindarajan, 1990). The virion particles were denatured and analysed by SDS-PAGE (12 % gel). Virion structural proteins were visualized by staining with Coomassie brilliant blue R-250. Eight protein bands that were clearly visible were excised individually from the gel and digested with trypsin by using an in-gel digestion procedure (http://www.pharma.ethz.ch/institute_groups/biomacromolecules/protocols/ingelding). Peptide sequencing was performed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). After digestion, the resulting peptides were delivered to a reverse-phase trap column by an autosampler for desalting and then fractionated in a nanoflow reverse-phase column (75 µmx150 mm), using acetonitrile gradient elution (5–90 % acetonitrile in 0.1 % formic acid). Eluted peptides were ionized by electrospray and transferred directly into a hybrid quadrupole/time of flight mass spectrometer (Water Company). The spectral data were recorded and searched against the non-redundant protein databases (EST, MSDB, NCBInr and SwissProt) by using the Mascot search engine for protein identification.
In the TnAV-2c virion protein gel, at least eight protein bands were clearly visible and were analysed by LC/MS/MS (Fig. 1). Except for the MCP, the functions of other proteins have not been characterized. The two most abundant proteins had molecular masses of approximately 72 and 54 kDa. The two proteins had significant peptide coverage from the MS/MS analysis (Table 1). The 54 kDa band corresponded to the MCP (ORF153), which was identified by 13 peptides. This protein is highly conserved among AVs and iridoviruses and has been used in phylogenetic analysis of these viruses (Cheng et al., 2005; Stasiak et al., 2003). The 72 kDa protein was identified by 10 peptides corresponding to ORF141. This protein, weakly conserved in SfAV-1a, HvAV-3e and iridoviruses, also showed similarity to periphilin, a protein expressed in the nuclear matrix (Kurita et al., 2004). Interestingly, this protein is highly enriched in Arg and Ser, which constitute 41 % of all residues. The largest structural protein was identified by seven peptides, encoded by ORF147. Intriguingly, it has similarity to Smc (structural maintenance of chromosomes) domain-containing proteins and also has weak similarity to myosin. Smc proteins are conserved evolutionarily from bacterial and archaeal species to eukaryotes and are involved in regulating the structural and functional organization of chromosomes (Hirano, 2006). The four less abundant proteins identified correspond to ORFs 142, 43, 2 and 115 (Fig. 1; Table 1). ORF142 has no homologues in the SfAV-1a and HvAV-3e genomes, but contains a DNA-binding motif with similarity to that present in the forkhead transcription factor. ORF43 has 32 % amino acid identity to the predicted dynein-like 
-chain protein in SfAV-1a (ORF84) and HvAV-3e (ORF146), a protein also conserved in iridoviruses and miniviruses. ORF2 encodes a predicted protein with no homologues in other organisms, and its N terminus shows similarity to ORF6. Its high content of basic residues (Arg and Lys) and the presence of an RNA-binding motif at the C terminus suggest that it may be involved in nucleic acid binding. ORF115 has significant similarity to predicted proteins in HvAV-3e (ORF77), SfAV-1a (ORF64) and Chilo iridovirus (CIV) (ORF209R). These proteins may function as Ser/Thr kinases, as they contain a conserved Ser/Thr kinase motif.
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REFERENCES |
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Bideshi, D. K., Demattei, M. V., Rouleux-Bonnin, F., Stasiak, K., Tan, Y., Bigot, S., Bigot, Y. & Federici, B. A. (2006). Genomic sequence of Spodoptera frugiperda ascovirus 1a, an enveloped, double-stranded DNA insect virus that manipulates apoptosis for viral reproduction. J Virol 80, 11791–11805.
Cheng, X. W., Wang, L., Carner, G. R. & Arif, B. M. (2005). Characterization of three ascovirus isolates from cotton insects. J Invertebr Pathol 89, 193–202.[CrossRef][Medline]
Federici, B. A. & Govindarajan, R. (1990). Comparative histopathology of three ascovirus isolates in larval noctuids. J Invertebr Pathol 56, 300–311.[CrossRef][Medline]
Federici, B. A., Vlak, J. M. & Hamm, J. J. (1990). Comparative study of virion structure, protein composition and genomic DNA of three ascovirus isolates. J Gen Virol 71, 1661–1668.
Federici, B. A., Bigot, Y., Granados, R. R., Hamm, J. J., Miller, L. K., Newton, I., Stasiak, K. & Vlak, J. M. (2005). Family Ascoviridae. In Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 269–274. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. San Diego: Elsevier Academic Press.
Govindarajan, R. & Federici, B. A. (1990). Ascovirus infectivity and effects of infection on the growth and development of noctuid larvae. J Invertebr Pathol 56, 291–299.[CrossRef][Medline]
Hirano, T. (2006). At the heart of the chromosome: SMC proteins in action. Nat Rev Mol Cell Biol 7, 311–322.[CrossRef][Medline]
Kurita, M., Suzuki, H., Masai, H., Mizumoto, K., Ogata, E., Nishimoto, I., Aiso, S. & Matsuoka, M. (2004). Overexpression of CR/periphilin downregulates Cdc7 expression and induces S-phase arrest. Biochem Biophys Res Commun 324, 554–561.[CrossRef][Medline]
Stasiak, K., Renault, S., Demattei, M. V., Bigot, Y. & Federici, B. A. (2003). Evidence for the evolution of ascoviruses from iridoviruses. J Gen Virol 84, 2999–3009.
Wang, L., Xue, J., Seaborn, C. P., Arif, B. M. & Cheng, X. W. (2006). Sequence and organization of the Trichoplusia ni ascovirus 2c (Ascoviridae) genome. Virology 354, 167–177.[CrossRef][Medline]
Received 26 February 2007;
accepted 25 April 2007.
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