Insertion of a bovine SMT3B gene in NS4B and duplication of NS3 in a bovine viral diarrhea virus genome correlate with the cytopathogenicity of the virus
Introduction
Bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus (BDV) of sheep, comprise the genus Pestivirus, which represents a group of important animal pathogenes (Franchi et al., 1991). BVDV has a single strand RNA genome of 12.5 kb with positive polarity (Collett et al., 1988a, Meyers et al., 1989, Deng and Brock, 1992). Two biotypes of BVDV, cytopathic (cp) and non-cytopathic (ncp), can be distinguished by their effect on cell culture (Gillespie et al., 1960, Gillespie et al., 1962). Both biotypes are required for the development of a fatal syndrome, mucosal disease (MD), in cattle (Brownlie et al., 1984, Bolin et al., 1985). The only reported biochemical difference between these two biotypes is the production of an 80-kDa protein (NS3) only by cp viruses (Donis and Dubovi, 1987, Howard et al., 1987, Pocock et al., 1987, Corapi et al., 1988). Biochemical and immunological analyses showed that NS3 (formerly P80, Collett et al., 1988b) is co-linear with the C-terminal portion of the non-structural protein, NS2–3 (P125, Tautz et al., 1993, and references therein).
Previous studies by our group and others suggested that, in some cases, RNA recombination events in the NS2–3 region of a ncp viral genome lead to the generation of NS3 and conversion of a ncp to a cp virus (Meyers et al., 1991, Meyers et al., 1992, Qi et al., 1992, Tautz et al., 1994). For all cpBVDV analyzed so far, RNA recombinations result in either rearrangements of the viral genome or insertion of a cellular sequence, which is sometimes accompanied by a large duplication of the viral genome (Collett et al., 1988a, Meyers et al., 1990, Meyers et al., 1991, Meyers et al., 1992, Qi et al., 1992, Tautz et al., 1994, Tautz et al., 1996). Similar cellular gene insertions, genomic duplications and rearrangements also exist in cpCSFV and cpBDV (Meyers and Thiel, 1995, Becher et al., 1996). In all these cases, it appears that a signal is created by the RNA recombination events for the cleavage of the NS2–3 protein. In this communication, we report the finding of a novel cellular gene insertion in a cpBVDV genome. Characterization of this insert lends further support to the hypothesis that RNA recombinations in a ncpBVDV result in the generation of a protein processing signal, which then leads to the formation of NS3 and, consequently, the conversion of a ncpBVDV to a cpBVDV.
Section snippets
Viruses
The cpBVDV strain SNC was isolated from a spontaneous MD case at the National Animal Disease Center, Ames, IA. Isolation and propagation of the virus have been described previously (Wilhelmsen et al., 1991).
RNA preparation, reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing
RNA purification from BVDV-infected MDBK cells, first strand cDNA synthesis, polymerase chain reaction and DNA sequencing were performed as previously described (Qi et al., 1992). The three primers used in RT-PCR were designed based on the sequence of the prototype BVDV NADL genome; the
Cloning and sequencing of part of the NS2–3 region
Our previous studies showed that 75% of the analyzed cpBVDV isolates have ubiquitin gene insertions accompanied by NS3 gene duplications in their genomes (Qi et al., 1992). To further assess the frequency of cellular gene insertions or viral gene duplications in the cpBVDV genomes, 16 cpBVDV field isolates were screened by the RT-PCR technique described in Materials and methods. PCR reactions with primers 11 and 10 produced a fragment about 900 bp in 14 of the cpBVDV isolates and a fragment of
Discussion
Cellular gene insertions, gene duplications or rearrangement in a cpBVDV genome play an important role in the processing of the non-structural protein NS2–3 to generate NS3, although other examples exist in which no insertions or gene rearrangement have been found (Qi et al., 1992, Greiser-Wilke et al., 1993, Kummerer et al., 1998). Bovine ubiquitin genes are the most frequently reported cellular gene insertions in cpBVDV genomes. A second bovine cellular gene called cINS was found in the
Acknowledgements
We thank Dr Todd Hansen for the gift of bovine endometrium cDNA library, and Dr Yuzhi Zheng for some technical assistance. The sequences presented in this paper have been submitted to GenBank with accession numbers U89437, U89438 and U89439. This work was supported by USDA grant #93-37204-9212 to E.S. Berry and F. Qi.
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2019, VirologyCitation Excerpt :Noncytopathic viruses can establish persistent, non-apparent infections, both in vitro and in vivo. Cytopathic viruses arise from noncytopathic viruses either by incorporation of cellular RNA sequences into the NS2/NS3 coding region or by duplication of the NS3 region (Baroth et al., 2000; Meyers et al., 1991a, 1991b; Muller et al., 2003; Qi et al., 1998, 1992; Rinck et al., 2001; Tautz et al., 1993; Vilcek et al., 2000). Point mutations causing cleavage of the NS2/NS3 are known (Kummerer et al., 1998; Pellerin et al., 1995).
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2015, Virus ResearchCitation Excerpt :One of the most common insertions is ubiquitin mRNA coding sequence (Meyers et al., 1991; Qi et al., 1992; Tautz et al., 1993; Becher et al., 1999). Studies of BVDV type I revealed cellular insertions including sequences coding for S27a-ubiquitin fusion ribosomal protein (Becher et al., 1998; Becher et al., 2001) and ubiquitin-like proteins (SMT3B, NEDD8)(Qi et al., 1998; Baroth et al., 2000) and proteins with ubiquitin-like folding domains including light chain 3 (LC3) (Fricke et al., 2004), GABA(A)-RAP and GATE-16 (Becher et al., 2002). Another common cellular insertion is J-domain protein, a member of the DnaJ-chaperone family.
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2015, Advances in Virus ResearchCitation Excerpt :Further studies identified other cellular insertions upstream of glycine 1590, the conserved N-terminus of NS3. They include sequences coding for a ribosomal protein S27a-ubiquitin fusion protein (Becher, Orlich, & Thiel, 1998b, 2001), ubiquitin-like proteins SMT3B and NEDD8 (Baroth, Orlich, Thiel, & Becher, 2000; Qi, Ridpath, & Berry, 1998), and a group of proteins with ubiquitin-like fold like LC3 (Fricke, Gunn, & Meyers, 2001; Fricke, Voss, Thumm, & Meyers, 2004), GABA(A)-RAP, and GATE-16 (Becher, Thiel, Collins, Brownlie, & Orlich, 2002). All these insertions are (I) substrates for cellular proteases residing in the cytoplasm of the cell (Becher et al., 2002; Fricke et al., 2004; Hemelaar, Lelyveld, Kessler, & Ploegh, 2003; Nishida, Kaneko, Kitagawa, & Yasuda, 2001; Wu et al., 2003) and (II) are located right upstream of NS3.