key: cord-0004950-xh66h2yd
authors: Weiss, Susan R.; Zoltick, P. W.; Leibowitz, J. L.
title: The ns 4 gene of mouse hepatitis virus (MHV), strain A 59 contains two ORFs and thus differs from ns 4 of the JHM and S strains
date: 1993
journal: Arch Virol
DOI: 10.1007/bf01316905
sha: 59cfffac9635612c06e30bb8b5068dbbf49f92f0
doc_id: 4950
cord_uid: xh66h2yd

The sequence of the MHV-A 59 non-structural gene 4 (ns 4) reveals two open reading frames. The upstream ORF potentially encodes a 19 amino acid (2.2 kDa) polypeptide and the downstream ORF potentially encodes a 106 amino acid (11.7 kDa) polypeptide. This is in contrast to MHV-JHM gene 4 which expresses a 15 kDa protein. Cell free translation of a synthetic mRNA containing both ORFs of MHV-A 59 ns 4 results in the synthesis of a 2.2 kDa poylpeptide; the predicted 11.7 kDa product of the MHV-A 59 downstream ORF is not detected during cell free translation nor in infected cells. These results add to the recent data suggesting that expression of some of the ns proteins of MHV is not necessary for efficient growth in tissue culture.

Susan R. Weiss et al. approximately 22 kb gene encoding the putative viral polymerase(s) and several genes encoding small non-structural proteins. The polypeptides predicted to be encoded by these non-structural genes include the basic 30kDa polypeptide encoded by ORF 2a [2, 22] , the basic 13.9 and hydrophobic 9.6kDa potypeptides encoded by ORFs 5 a and 5 b respectively [4] . Both the 30 kDa and 9.6 kDa polypeptides have been detected in infected cells using specific antisera [2, 12, 22] . The 13.9kDa polypeptide is synthesized poorly during in vitro translation of a synthetic RNA containing ORF 5 a and it is not clear whether the 13.9 kDa polypeptide is in fact synthesized during infection [4] . While it is unclear what the functions of these ns proteins are, sequence analyses suggest that the 30kDa and 13.9 kDa polypeptides may play a role in RNA binding and the 9.6 kDa polypeptide may interact with membranes. Another non-structural polypeptide, 15 kDa in size, has been shown to be encoded by ORF 4 of MHV-JHM and has been detected, using an antibody, during infection with JHM [6] . While no function has been attributed to this 15 kDa JHM ns 4 gene product, it is predicted to be an integral membrane protein [17] . A 14kDa protein was reported in A 59 infected cells [ 13, 15] ; however, there are no data to link this protein to the ns 4 gene. We report here the sequence of ns 4 of MHV-A 59. We have found that the sequence of A 59 ns 4 differs from that of JHM such that deletions within the 15 kDa polypeptide coding region result in the presence of two ORFs instead of the one encoding the 15 kDa polypeptide. Figure 1 shows the sequence of the MHV-A 59 ns 4 gene including the intergenic consensus sequences preceeding the ns 4 and ns 5 genes. This was obtained by sequencing three A 59 cDNA clones. Clone 344, obtained by cloning of oligo (dT) primed cDNA transcribed from A 59 genome RNA [5] , was cleaved with Hae III and Hpa II and subcloned into the RF of M 13 bacteriophage. Subclones were sequenced using dideoxynucleotide sequencing technology and an M 13 universal sequencing primer [5] . Clones 613 and 332 were obtained from a cDNA library constructed from randomly primed cDNAs transcribed from A 59 genome RNA [14] ; the ns 4 region was sequenced using oligonucleotide primers on double stranded plasmid DNAs. The sequence up to nt 110 of A59 was confirmed by sequencing of genome RNA using an oligonucleotide primer complementary to nucteotides 123-150. This A 59 ns 4 sequence is similar to that of JHM but has single nucleotide deletions that result in frame shifts. This creates two ORFs in the A 59 sequence as opposed to one for the JHM sequence. The one base deletion just downstream of the AUG initiation codon of ORF 4 of JHM (nt 76 of the A 59 sequence) interrupts the 15kDa protein ORF and instead results in two ORFs in this region, one 19 amino acids or 2.2kDa and the other 106 amino acids or 11.7 kDa. (See Fig. 1 for nucleotide sequence and Fig. 2 for predicted amino acid sequence.) There is another one base deletion near the 3' end of JHM ORF 4 (nt 389 of the A 59 sequence) that results in a protein truncated at the carboxyterminus relative to the JHM protein. We are confident that this sequence (including the deletions) is not due to cloning artifacts as it was obtained from at least two cDNA clones 

A G -5 0 4 a~ A G G -C A G T C A -T A G C T A T G G C C G T G T T -G G T C C T A A G G C T A C A T T G G C T G - 97 A G G G CAG C A A G T A G T T A T G G C C CT C A T C G G T C C C A A G A C T A C T A T T G C T G -100 4~ 4a T E R M 4b--e~ C T G

* * * * * * * * * * * * * * * * * * * * , * * * * * * * * * * * * * * * * * * * * * * * * * * * 

A -1 5 0 A59 J H M

A T -2 0 0 A59 J H M A C G T G T G A C T G G C A A G C C A G C C A C T G T G T C T T A T A C T A C A A G T A C A C C A G -2 4 7 A C G C G T G A C T G G C A A G C C A G C C A C T G T G T C T T A T A C T A C A A G T A C A C C A G -2 5 0 A59 J H M T~C A C C G A G C G C G A C G A C G C T C

. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ******* The predicted 2.2 kDa protein encoded in ORF 4 a of A 59 would be quite hydrophobic (Fig. 3) and thus if expressed in vivo may contain a membrane spanning region. The predicted 11.7 kDa protein encoded in O R F 4 b is, for most of its length, similar to the JHM 15 kDa protein (Figs. 2 and 3 ). Both the A 59 and JHM proteins are predicted to be integral membrane proteins; however, because the A 59 protein is truncated at both ends there are some differences. The A 59 protein has two predicted membrane spanning domains (amino acids 1-17; 84-100) while the JHM has one (amino acid 10-26).

Like MHV-A 59, the antigenically related bovine coronavirus (BCV) genome contains two ORFs (potentially encoding 4.9 and 4.8 kDa polypeptides). Abrahams etal. [1] suggested that these ORFs could have arisen from a one-nucleotide deletion in an ORF similar to ns 4 of JHM or the 4 b polypeptide encoded by A 59 described here.

Thus, ns 4 of A 59 resembles in structure the ns 5 gene in which two ORFs follow one intergenic consensus region and are present on one mRNA. In the case of ns 5 the downstream ORF is the more frequently expressed by cell free translation and also has been the only one detected in infected cells [12] . Thus we wanted to determine which is the more likely to be translated with ns 4. The region encoding ns 4 (containing ORF 4 a and ORF 4 b) was excised from A 59 cDNA clone 613 and ligated into pIBI 31 downstream of the T 7 bacteriophage RNA polymerase promoter (pIBI 31.A 594 ab). Briefly pIBI 31.A594 ab was constructed as follows. Clone 613 was cleaved with Ssp I (about 60 nts upstream of the gene 3/4 intragenic sequence) and Dra I (about 150 nts downstream of the O R F 4 b terminator). This fragment was then treated with Bal31. The resulting 650 nt fragment was ligated into Hinc II cut pIB131 downstream of the T 7 RNA polymerase promoter. This plasmid was cleaved with HindIII and Pst I, blunt-ended with T 4 DNA polymerase and religated. This last step removed a potential ATG initiation site for protein synthesis in the multiple II site of pIBI 31 downstream of the  T 7 promoter. The viral sequences in this clone were sequenced and the gene 4 insert found to have the identical sequence to that reported previously [17] . After linearization of plasmids pIBI 131.JHM 4 and pIBI 31.A 594 ab with Eco RI, capped R N A transcripts were synthesized using T 7 R N A polymerase in reactions containing 300 ng of template D N A as described by Krieg and Melton [8] . R N A was transcribed in vitro using T 7 R N A polymerase [8] and translated in a wheat germ lysate. The results of these translations are shown in Fig. 4 . The major product of translation of the A 59 R N A is the predicted in the polypeptide encoded in the upstream ORF. This is not surprising as the A U G initiation codon for ORF 4 a is not only near the 5' end of the RNA but also is in a better context for translation. ORF 4 a is initiated with the sequence GCU AUG G while ORF 4 b contains U G U AUG C. The consensus sequence for protein synthesis initiation by ribosome scanning is A(G)CC A UG GG with the -3 and + 4 positions (the A of the A U G is position 1) being the most important for efficency of initiation of translation [7] . Given the sequence of ORF 4 a, b of MHV-A 59 and the cell-free translation results described above, it seemed unlikely that the 15 kDa protein observed in JHM infected cells would be present in A 59 infected cells. However, to confirm this, and to determine if the predicted 11.7 kDa protein was expressed from MHV-A 59 ORF 4 b, we used an antiserum directed against the JHM 15 kDa ns 4 gene product to detect products of ORF 4 in cells infected with MHV-A 59 or MHV-JHM. Since this antiserum (obtained from Dr. Stuart Siddell, Wiirzburg, Federal Republic of Germany) is directed against a peptide containing the 71 carboxyterminal amino acids of the JHM 15 kDa ns 4 gene product [6] , it should detect an A 59 polypeptide encoded in A 59 ORF 4 b (see Fig. 2 ). As shown in Fig. 5 , using a Western blot assay, this antiserum detected the JHM 15 kDa ORF 4 product, but did not detect a polypeptide in MHV-A 59 infected cells. Thus it is unlikely that the predicted A 59 ORF 4 b product is synthesized in infected cells. This antiserum would not be expected to detect the predicted 2.2 kDa product of MHV-A 59 ORF 4 a.

Recent data have suggested that some of the MHV small ns proteins may not be necessary for efficient replication in cell culture. MHV strains lacking expression of ORF 2 a [16] in one case and ORF 4 and 5 a [21] in another have been identified. In both these cases the corresponding mRNAs are not synthesized due to mutations in the intergenic region or deletions in the coding regions (Table 1 ). In the case ofns 4 of A 59, it is clear that the 15 kDa protein synthesized by JHM is not synthesized and thus is not necessary for A 59 replication in cell culture. It is also unlikely that an alternative polyprotein is expressed from The data for J H M were taken from Skinner and Siddell [17] and the data for MHV-S were taken from Yokomori and Lai [21] . The proteins predicted from the ORFs were deduced from the nucleotide sequences of the ns 4 genes of the three strains, mRNA 4 of A 59 has been shown to be expressed in many publications (for example [5, 20] 

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