key: cord-0984831-swxuix4n
authors: Oleksiewicz, M. B.; Bøtner, A.; Nielsen, J.; Storgaard, T.
title: Determination of 5′-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs
date: 2014-05-18
journal: Arch Virol
DOI: 10.1007/s007050050560
sha: 29bfdcefcceeb8d155f385ecd28a487c8b950514
doc_id: 984831
cord_uid: swxuix4n

We determined the untranslated 5′-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5′-leader from European- and American-type PRRSV differed in length (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication. These comparative data provide a priori knowledge for mutational identification of virulence determinants in the 5′ nontranslated part of the PRRSV genome.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently emerged pathogen, which belongs to the Arteriviridae family of enveloped, plussense RNA viruses. PRRSV was first isolated in Western Europe, and shortly thereafter in North America (for a recent review see [20] ). Despite the concurrent emergence of PRRSV-induced disease on the two continents, transatlantic PRRSV strains proved surprisingly divergent, with only 55-80% nucleotide homology in the structural genes [12] . In Denmark, the epidemic spread of indigenous, European-type PRRSV was attempted halted in 1996 by a largescale vaccination campaign, using a "classical" cell culture-attenuated live vaccine, based on the pathogenic North American VR2332 isolate. Unfortunately, this live vaccine strain proved unexpectedly adept at reverting to virulence and spreading to non-vaccinated herds [4, 10, 17, 21] . On a speculative note, many of the questions arising from the coexistence of European and American PRRSV types in Denmark may impinge on a single viral genetic element: the PRRSV untranslated 5 -leader. For the closely related coronaviruses, the 5 -leader regulates viral genomic RNA replication, transcription and mRNA translation [9, 22, 23] . Furthermore, the 5 -leaders appears to be a highly mobile genetic element, able of high-efficiency recombination between related viruses [11] , and 5 -leaders of other RNA viruses such as poliovirus and Venezuelan Equine Encephalitis virus contain attenuation/virulence determinants [5, 8] . In short, examination of the PRRSV 5 -leader sequence was expected to provide basic knowledge about the molecular basis of PRRSV replication/virulence/attenuation, as well as improve our means for understanding and monitoring any future exchange of genetic material between coexisting PRRSV types.

The Europeantype Lelystad virus is the only PRRSV strain for which the 5 -leader sequence has been published [15] . We determined the 5 -leader sequences for 111/92, a pathogenic European-type PRRSV isolate from 1992 [3, 10] , and VR2332, a pathogenic American-type isolate [2] , using the 5 RACE (rapid amplification of cDNA ends) kit from Life Technologies (Roskilde, Denmark). 5 RACE was carried out essentially according to the manufacturer's instructions. Cycle sequencing was done using fluorescent chain terminator chemistry (Perkin Elmer, Allerød, Denmark). A detailed description of the experimental protocol and primer sequences is available on request. The obtained 111/92 leader sequence was 96% similar to the Lelystad 5 -leader (Fig. 1) . The VR2332 5 -leader was 30 nt shorter, and had only approximately 50% nucleotide homology to the European-type leaders. The GC% also differed slightly between European (59-61%) and American (52%) viruses. The 111/92 and VR2332 5 -leader sequences shown in Fig. 1 have been submitted to GenBank, and given the accession numbers AF094476 and AF094475, respectively.

Identification of putative regulatory leader motifs: The 5 -leader is thought to act as a landing pad for viral and cellular proteins involved in PRRSV replication. Yet, identification of regulatory motifs in arteriviral leaders has so far proven elusive [6] . We observed that American and European-type-PRRSV were apparently evolutionary sufficiently distant, yet adequately close, for highly conserved motifs to become apparent in the 5 -leader sequences (Fig. 1) . A 12 nucleotide stretch containing a purine (A/G) and a pyrimidine (C/U) wobble position appeared to form a conserved "5 -leader start" motif ( Fig. 1, shaded) . Additionally, the 40 most 3 -terminal bases of the leader exhibited a high degree of conservation between European-and American-type PRRSV, and in this conserved area, a string of 8, 9 and 11 wholly invariable nucleotides appeared to be embedded ( Fig.  1 , shaded areas). Whereas these 5 -leader motifs are candidates for mediating the RNA-protein interactions thought important for regulating PRRSV replication, identification of protein partners, if any, will require further studies. Yet, we observed that the 9 nt motif contained a CACCC site, with another CACCC site occurring approximately 100 nt further upstream in American as well as Euro- [7, 18] , but their importance for non-retroviral RNA viruses is not known.

The pathogenic North American VR2332 isolate underwent multiple cell culture passages to produce a live, attenuated PRRSV vaccine strain [10] . We determined the 5 -leader sequence of the PRRSV vaccine strain using 5 RACE. The vaccine 5 -leader sequence was completely identical to the VR2332 5 -leader sequence, except that the U preceding the 12 nt leader start motif in the VR2332 leader ( Fig. 1) was absent in the vaccine leader (the vaccine leader not included in Fig. 1 ). However, the 5 RACE results for 111/92 showed substantial variability in the first 1-2 nucleotides preceding the conserved 12 nt leader start motif (not shown). Therefore, the significance of a single nucleotide difference between VR2332 and the vaccine in this variable position vis-a-vis attenuation can unfortunately not be decided from our data. The 189/190 nucleotide identity between vaccine and VR2332 5 -leaders may indicate non-involvement of the 5 -leader in attenuation. Alternatively, the very high degree (99.7%, [10] ) of conservation between the vaccine and VR2332 in the structural genes may in turn imply that attenuation conditions were not sufficiently harsh to affect the 5 -leader sequence.

PCR amplification of subgenomic mRNAs: Free 5 -leader has been hypothesized to prime subgenomic viral mRNA transcription [1] . 5 -leader priming of subgenomic viral mRNA transcription is thought to require interaction between a 6 nt long junction sequence found at the 3 -end of the free leader, and partly homologous junction sites upstream of the various open reading frames in the viral genome. Thus, examining the junction sites in subgenomic viral mRNAs is integral to understanding the possible function of the 5 -leader in regulating of viral transcription. To determine 5 -leader-mRNA body junctions, total intracellular RNA from PRRSV-infected cultures was reverse transcribed using random hexamers and the "Ready-To-Go" reverse transcription system (Pharmacia, Allerød, Denmark). cDNA was PCR-amplified using standard protocols. The forward PCR primers were specific for the extreme 5 -end of the leader sequences determined in this study (Fig. 1) , and reverse PCR primers were situated 50-140 nt downstream of the start codon for each mRNA. Primer sequences are available on request. In virtually all cases, a single, predominant PCR product was observed, confirming previous reports that the PRRSV 5 -leader-mRNA junction mechanism is quite site-specific [16] . A highly notable exception was the ORF 7 (nucleocapsid) mRNA of VR2332, where the PCR band pattern indicated equal utilization of more than one junction site. Similar findings for VR2332 have recently been reported by others (see [20] and references therein). The sequence data are summarized in Table 1 . For all subgenomic mRNAs of 111/92, the junction sequences as well as the length of untranslated sequence between the 5 -leader and the start codon of the downstream open reading frame (Table  1) corresponded to the published values for Lelystad virus [16] . Thus, the 6 nt differences in the 5 -leaders (Fig. 1) , as well as the 7% nucleotide differences in the structural genes [10] , had no influence on the junction-choice of these two European-type viruses. Also, the published lengths of untranslated sequence between the 5 -leader and downstream ORF in mRNAs 3 and 4 of different North American PRRSV isolates [14] , and mRNAs 5, 6 and 7 of a Japanese PRRSV isolate [19] , were the same as the values determined for VR2332 in this study. Thus, for North American PRRSV types, the junction-choice also appears to be well conserved, despite relatively high (up to 11%) nucleotide differences in the structural genes [13, 14] . The VR2332 junctions determined in this study for mRNAs 5, 6 and 7 differed from those determined in another study for a Japanese, American-type PRRSV isolate [19] . However, the VR2332 junctions determined in this study for mRNAs 3 and 4 were identical to those determined previously for different North American isolates [14] . These findings may reflect genetic differences between Japanese and North American PRRSV type [19] . 

Not applicable UCCACCCCK * UAAACC * UUGUCA genomic sequence> AGGGAGUGG * UAAACC * UUGUCA

The 5 leader-mRNA junction sequence of the complete set of 6 subgenomic mRNAs (2-7) was determined for both PRRSV strains, as described in the text. The mRNA sequence immediately surrounding the junction between 5 -leader and mRNA body is shown aligned to the published genomic sequence. The 6 nt junction is sandwiched between * , and the junction in the mRNA sequence is also italicized. The part of the mRNA sequence identical to the genomic sequence is underlined. The distance from the last nt of the junction to the first nt of the start codon is indicated following the > above each mRNA sequence. The nucleotides shown in bold lettering at the 5 -end of the mRNA sequence are part of the 9 nt conserved motif in the 5 -leader, see Fig 1. Y in mRNA 6 of 111/92 and K in mRNA 7 of VR2332 indicate C/U and U/G ambiguities, respectively, due to clonal sequence variation in the mRNA population [16] . The genomic site 5 for 111/92 (EMBL accession number AJ223078) and genomic site 2 for VR2332 (GenBank accession number U87392) were re-sequenced for the present study. For 111/92, no sequence information exists for genomic site 2, and published sequence from Lelystad virus (GenBank accession M96262) was substituted. mRNA 7 is a prevalent variant of VR2332 mRNA 7

In summary, we determined the 5 -leader sequences for three PRRSV isolates. This data provided the first insights into the degree of variability in this important, regulatory part of the PRRSV genome. Additionally, the new sequences allowed, for the first time, prediction of putative regulatory elements in the PRRSV 5leader, which may be an aid in developing second-generation, genetically engineered PRRSV vaccines. Finally, scrutiny of subgenomic mRNAs identified intriguing differences in the junction-choices for the ORF 7 (nucleocapsid) mRNA of European-and American-type PRRSV, which are currently under further investigation.

An in vitro system for the leader-primed transcription of coronavirus mRNAs

Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332)

Isolation of porcine reproductive and respiratory syndrome (PRRS) virus in a Danish swine herd and experimental infection of pregnant gilts with the virus

Appearance of acute PRRS-like symptoms in sow herds after vaccination with a modified live PRRS vaccine

Increased neurovirulence associated with a single nucleotide change in a noncoding region of the Sabin type 3 poliovaccine genome

A 68-nucleotide sequence, within the 3 noncoding region of simian hemorrhagic fever Virus negative-strand RNA binds to four MA 104 cell proteins

Transactivation of the human keratin 4 and Epstein-Barr virus ED-L2 promoters by gut-enriched Krüppel-like factor

Attenuation of Venezuelan equine encephalitis virus strain TC -83 is encoded by the 5 -noncoding region and the E2 envelope glycoprotein

Requirement of the 5 -end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription

Detection of porcine reproductive and respiratory syndrome virus of the American type in Danish swine herds

High-frequency leader sequence switching during coronavirus defective interfering RNA replication

Phylogenetic analyses of the putative M (ORF 6) and N (ORF 7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe

Sequence comparison of open reading frames 2 to 5 of low and high virulence United States isolates of porcine reproductive and respiratory syndrome virus

A nested set of six or seven subgenomic mRNAs is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus

Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus

Subgenomic RNAs of Lelystad virus contain a conserved leader-body junction sequence

Experimental inoculation of late-term pregnant sows with a field isolate of PRRS vaccine-like virus

Sp1 family proteins recognize the U5 repressive element of the long terminal repeat of human T cell leukemia virus type I through binding to the CACCC core motif

Characteristics of major structural protein coding gene and leader-body sequence in subgenomic mRNA of porcine reproductive and respiratory syndrome virus isolated in Japan

The molecular biology of arteriviruses

Blocking ELISA's for the distinction between antibodies against European and American strains of porcine reproductive and respiratory syndrome (PRRS) virus

Coronavirus translational regulation: leader affects mRNA efficiency

Coronavirus leader RNA regulates and initiates subgenomic mRNA transcription both in trans and in cis

P. Normann and F. Lihme are thanked for technical assistance. S. Alexandersen is thanked for helpful discussions. This work was partly supported by a grant from the Norma and Frode Jacobsen foundation, and grant 9600926 to TS from the Danish Agricultural and Veterinary Research council.

Since the submission of this manuscript, a study containing a partial 5 -leader sequence of VR2332 has appeared in print [Nelsen CJ et al. (1999) 

Authors' address: Dr. T. Stargaard, Ministry of Food, Agriculture, and Fisheries, Danish Veterinary Institute for Virus Research, Lindholm, DK-4771 Kalvehave, Denmark.Received October 12, 1998