Frequent rearrangement may explain the structural ...

Acta Veterinaria Hungarica 57 (3), pp. 453–461 (2009) DOI: 10.1556/AVet.57.2009.3.11

FREQUENT REARRANGEMENT MAY EXPLAIN THE STRUCTURAL HETEROGENEITY IN THE 11TH GENOME SEGMENT OF LAPINE ROTAVIRUSES – SHORT COMMUNICATION Krisztián BÁNYAI1,2*, Jelle MATTHIJNSSENS3, György SZÜCS2, Petra FORGÁCH4, Károly ERDÉLYI5, Marc van RANST3, Eleonora LORUSSO6, Nicola DECARO6, Gabriella ELIA6 and Vito MARTELLA6 1

Veterinary Medical Research Institute, Hungarian Academy of Sciences, Hungária krt. 21, H-1143 Budapest, Hungary; 2Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Pécs, Pécs, Hungary; 3Laboratory of Clinical and Epidemiological Virology, Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium; 4Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent István University, Budapest, Hungary; 5Department of Wildlife Diseases and Parasitology, Central Veterinary Institute, Budapest, Hungary; 6Department of Animal Health and Well-Being, University of Bari, Bari, Italy (Received 17 October 2008; accepted 11 December 2008) In rotaviruses, intragenic recombination or gene rearrangement occurs almost exclusively in the genome segments encoding for non-structural proteins. Rearranged RNA originates by mechanisms of partial sequence duplications and deletions or insertions of non-templated nucleotides. Of interest, epidemiological investigations have pointed out an unusual bias to rearrangements in genome segment 11, notably in rotavirus strains of lapine origin, as evidenced by the detection of numerous lapine strains with super-short genomic electropherotype. The sequence of the full-length genome segment 11 of two lapine strains with super-short electropherotype, LRV-4 and 3489/3, was determined and compared with rearranged and normal cognate genome segments of lapine rotaviruses. The rearranged genome segments contained head-to-tail partial duplications at the 3’ end of the main ORF encoding NSP5. Unlike the strains Alabama and B4106, intermingled stretches of non-templated sequences were not present in the accessory RNA of LRV-4 and 3489/3, while multiple deletions were mapped, suggesting the lack of functional constraints. Altogether, these findings suggest that independent rearrangement events have given origin to the various lapine strains that have super-short genome pattern. Key words: Gene duplication, deletion, recombination, evolution

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Corresponding author; E-mail: [email protected]; Phone: 0036 (1) 467-4082; Fax: 0036 (1) 467-4076 0236-6290/$ 20.00 © 2009 Akadémiai Kiadó, Budapest

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Group A rotaviruses, belonging to the family of Reoviridae, are considered one of the main causes of acute viral gastroenteritis of humans and animals. The rotavirus genome consists of 11 segments of double-stranded RNA (dsRNA) enclosed in a triple-layered capsid (Estes and Kapikian, 2007). The two outer capsid proteins, VP4 and VP7, are the main antigenic determinants. The inner capsid protein VP6 bears the subgroup (SG) specificities that allows the classification of group A rotaviruses into SGI, SGII, both SGI and II, or into SG nonInonII based on the reactivity with SG-specific monoclonal antibodies (MAbs). By visualisation of the migration pattern of the 11 dsRNA gene segments in polyacrylamide gel electrophoresis (PAGE), the majority of animal SGI and SGII, and human SGII rotavirus strains display a long electropherotype (E-type), while almost all SGI human rotavirus strains possess a short E-type, based on the mobility of genome segment 11 (Estes and Kapikian, 2007). Some human and animal strains possess super-short E-type, due to abnormal migration of genome segment 11 on polyacrylamide gel. Such super-short Etype has been observed rarely in bovine (VMRI), porcine (MC345, CC86, CC117, C60) simian (YK-1) and human (69M, B37, B38, AU19, Z10262) strains (Paul et al., 1988; González et al., 1989; Nuttall et al., 1989; Scott et al., 1989; Mattion et al., 1990; Ciarlet et al., 1997; Palombo et al., 1998; Nakagomi et al., 1999; Westerman et al., 2006). In addition, super-short RNA profile has been observed frequently in lapine rotaviruses (ALA, BAPwt, BAP-2, 3489/3, LRV-4; Thouless et al., 1986; Tanaka et al., 1988; Bányai et al., 2005; Martella et al., 2005) and in a lapine-like human strain, B4106, detected in Belgium (De Leener et al., 2004; Matthijnssens et al., 2006a). In addition, some tissue-culture adapted lapine rotavirus (LRV) strains has atypical long genome pattern (strain C-11) due to abnormal mobility of genome segment 10 or a short pattern (strain R-2) due to slower migration of genome segment 11 (Thouless et al., 1986). Altered mobility of rotavirus genome segments is generally accounted for by mechanisms of gene rearrangement (intragenic recombination) that is a result of partial head-to-tail duplication within a particular genome segment. Sequence duplications usually start after the open reading frame (ORF) and do not affect the ability of the RNAs to encode full-length proteins. Further, in rearranged dsRNA there may be deletions of RNA fragments and/or insertion of nontemplated nucleotides. In addition to genome segment 11, rearrangement has been also observed in genome segments 5, 6, 7, 8, and 10 (Estes and Kapikian, 2007). The presence of rearranged genome segments may be associated with changes in virus phenotypes. Hypotheses to explain intra-segmental recombination have implicated both the transcription (i.e. the plus-strand synthesis) and the replication step (i.e. the minus-strand synthesis), linking this phenomenon to the activity of viral RNA-dependent RNA-polymerase (RdRp) (Desselberger, 1996; Kojima et al., 1996; Matthijnssens et al., 2006b).

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Sequence analysis of additional rearranged genome segments is necessary to confute or validate the models that have been proposed to understand the mechanism by which rearrangement occurs. During the investigation of local strain diversity in rabbit herds, two unusual rotavirus strains, LRV-4 and 3489/3, were identified by the analysis of their RNA profiles. Both strains displayed a super-short E-type of RNA migration by PAGE (Bányai et al., 2005; Martella et al., 2005). LRV-4, a P[14],G3 rotavirus, was identified in an Italian rabbitry in Foggia, Italy, in 2003 (Martella et al., 2005). Strain 3489/3 is a lapine P[22],G3 rotavirus, identified from the intestinal content of a dead rabbit in 2004 in Hungary (Bányai et al., 2005). The sequence of genome segment 11 of these two lapine strains was determined and analysed along with rearranged and normal cognate genome segments of other rotavirus strains of lapine origin. Two additional lapine strains, Alabama with super-short E-type (isolated in the USA in 1984; Thouless et al., 1986) and 30/96 with long E-type (isolated in Italy in 1996; Martella et al., 2003), and the human strain, B4106 (De Leener et al., 2004), representing a zoonotic, lapine-derived P[14],G3 rotavirus (Matthijnssens et al., 2006a), were also included in this study for comparison. The nucleotide sequence of genome segment 11 of the two strains was determined by direct sequencing after amplification of the dsRNA by reverse transcription-PCR (Bányai et al., 2005; Martella et al., 2005). The alignment of genome segments 11 was prepared with the Multalin (Corpet, 1988) server and then manually adjusted using the GeneDoc software to obtain the best matches (Nicholas et al., 1997). Further comparison between LRV-4, 3489/3 and 30/96 was performed with the pairwiseFLAG software (http://bioinformatics.itri.org.tw/ prflag/prflag.php), and manually edited in Adobe Illustrator. ORFs were searched with the ORF Finder at the NCBI’s web site (http//www.ncbi.nlm.nih.gov/gorf/ orfig.cgi). Phylogenetic analysis was carried out using the p-distance algorithm and the neighbour-joining method supported by bootstrap analysis in the MEGA2 software (Kumar et al., 2001). The full-length genome segments 11 of the Italian strain LRV-4 and the Hungarian strain 3489/3 were aligned with rearranged genome segment 11 of two lapine strains, B4106 and Alabama. Significantly matching regions included only the 618 nt at the 5’ ends and the 49 nt at the 3’ ends (not shown), that are actually the regions constituting the entire length of normal genome segment 11. The primary structure of the rearranged regions spanning these matching regions was found less conserved. To understand this heterogeneity, both LRV-4 and 3489/3 strains were compared with the reference sequence of 30/96. Strain 30/96 was used as hypothetical ancestor because it is currently the single known lapine rotavirus strain that has a normal structure for genome segment 11. Data shown in Fig. 1 suggest that a hypothetical intermediate, formed after rearrangement, occurred in the ancestors of LRV-4 and 3489/3. These contained secondary deletions in the 3’ non-coding duplicated regions. It is striking that LRV-4 and 3489/3 have both their Acta Veterinaria Hungarica 57, 2009

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rearrangement break points in common (nucleotide positions 139 and 619). Nucleotide 619 is the position adjacent to the stop codon of the ORF encoding the NSP5 protein. Interestingly, both Alabama and B4106 share the break point at nucleotide 619, but they have different second break points (Alabama, nt 168; B4106, nt 257; Matthijnssens et al., 2006a). The differences in length of the partially duplicated regions (3489/3, 399 nt; LRV-4, 339 nt; Alabama, 369 nt; B4106, 376 nt) and the differences in subsequent deletions and/or insertions, results in slight differences in the total lengths of genome segment 11 of these strains.

Fig. 1. Analysis of the rearranged NSP5 gene of lapine strains LRV-4 and 3489/3. Schematic representation of the duplication, the hypothetical intermediate, and the subsequent deletion events. Nucleotide sequences are freely available from the corresponding author upon request

The NSP5 gene of 3489/3 and LRV-4 shared a relatively high genetic relatedness (97.5% amino acid identities), raising the possibility of their common origin (data not shown). However, inspection of their rearranged gene structure revealed that both strains had secondary deletions in various, partly overlapping regions, suggesting that the final gene structure we observed was generated through independent evolutionary pathways. Acta Veterinaria Hungarica 57, 2009

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Fig. 2. Computational prediction of coding capacity of the rearranged genome segment 11 of lapine rotaviruses. Boxes show the ORFs encoding NSP5 and NSP6, while arrow indicate the putative ORFs originated as a result of intragenic recombination. Strain names are indicated

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As a rule, group A rotaviruses encode two ORFs in their genome segment 11; ORF1 (nt 22–618) encodes the NSP5 protein, while ORF2 (nt 80–370) encodes the NSP6 protein. However, exceptions to this structure have been published, demonstrating the existence of either slightly shorter or longer ORFs, or alternatively, point mutations in the start codon, or non-sense mutations in the NSP6 coding region (Gorziglia et al., 1989; Kojima et al., 1996; Wu et al., 1998). In addition to the NSP5 and NSP6, some potential ORFs were identified in the LRV-4 and 3489/3 strains in the duplicated sequence regions. The putative ORF3 and ORF4 of strain LRV-4 are derived from ORF2 and ORF1, respectively, and encode potential NSP6- and NSP5-related proteins, both truncated at their N-terminal ends. In case of 3489/3, the ORF3 encodes a putative 90 amino acid (aa) long polypeptide with a mosaic structure that had acquired its Nterminal end (aa 1–20) from the NSP6 (corresponding to region aa 65–84) and its C-terminal end (aa 21–90) from the coding region for NSP5 (corresponding to region aa 129–198; Fig. 2). The putative ORF4 did not show significant matches with protein sequences deposited in public databases and possessed no known amino acid sequence motifs; however, it displayed similarities with the possible protein of ORF3 of B4106, likely due to serendipitous parallel RNA structure reorganisations (data not shown). From the viewpoint of rotavirus epidemiology it is important to note that we see very few strains with rearranged genome segments, and even if they are identified, their subsequent spread in the population usually cannot be tracked. Therefore, detection of different rearranged genome segment 11 structures in lapine (-derived) strains seems to be of interest and raises the possibility that this phenomenon occurs more frequently in certain animals (e.g. in rabbits) than in other species. If this reflects well the true occurrence of rotavirus strains with rearranged genome segments in nature, it seems an intriguing question why gene rearrangement is so frequent in these host species. One explanation is that rotavirus might determine a prolonged persistence in rabbits kept in commercial herds, thus increasing the odds for genomic rearrangements. This ecological background seems to parallel with that seen in human rotaviruses with a rearranged genome, which have been isolated most frequently from chronically infected immunocompromised patients (Pedley et al., 1984; Hundley et al., 1987; Palombo et al., 1998; Gault et al., 2001; Mori et al., 2002). Immune selection and force to effectively adapt to new host species drives the molecular evolution of rotaviruses. Accumulation of point mutations, frequent gene reassortment and intermolecular recombination between cognate genes may facilitate both escaping the immune response and introducing heterologous genes in the rotavirus population and adaptation to heterologous hosts, thus opening new ecological niches for a particular strain or, at least, for parts of its genetic material. The evolutionary benefits of gene rearrangement, resulting in partial duplications and deletions within a particular gene segment is, howActa Veterinaria Hungarica 57, 2009


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ever, much more controversial and has not yet been formally demonstrated. There is experimental evidence that genome segments longer than their counterparts of normal length are transcribed more efficiently, resulting in higher number of transcripts in the early step of viral life cycle (Patton et al., 1999). Hence, rotaviruses with a rearranged genome may overgrow the virus population with standard genome and become predominant within the host during a single infection course. However, the ability of strains with rearranged genome segment 11 to overgrow other viral strains in vitro has been related to point mutations affecting the phosphorylation pattern of the NSP5, rather than to the length of the rearranged genome segment (Chnaiderman et al., 1998). In addition, a more recent survey on the incidence of rearrangement among viral progenies in immunocompetent children found no evidence for the abilty of strains with rearranged genome segment 11 to overgrow strains with normal genome during acute rotavirus infection (Schnepf et al., 2008). These data, together with the relative rarity of rearranged genome segments in viable wild type rotavirus strains in a variety of host species, indicate that field rotavirus strains, in general, do not systematically exploit this possibility, raising questions on the real evolutionary significance of rearrangement. Thus, additional features such as serendipitious increase in capacity to encode new viral proteins useful in the rotavirus life cycle might be taken into account as a possible contribution to the relative evolutionary fitness of strains with rearranged genome in rabbits, although the question whether such secondarily acquired ORFs are indeed expressed is open and needs further scrutiny (Gorziglia et al., 1989). In conclusion, data presented in this study revealed common breakpoint sites and considerable structural heterogeneity in genome segment 11 of lapine rotaviruses, suggesting (i) a non-random initiation site for RNA duplication and (ii) a lack of functional/structural constraints in the subsequent evolution of duplicated gene fragments, respectively. Reverse genetics (Komoto et al., 2006) might help address unanswered questions related to rearrangement and understand the implications of such genetic alterations for rotavirus fitness and evolution. Acknowledgements Financial support was provided by the EVENT programme (grant no. SP22-CT2004-502571). K. B. is a recipient of Bolyai János Fellowship from the Hungarian Academy of Sciences. J. M. was supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen).


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