Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavirus molecules

5' and 3' UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5' UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5' UTR and adjacent 289-nt sequences, and 3' UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3' UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5' and 3' replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members.     

Defective-interfering particles of murine coronavirus: mechanism of synthesis of defective viral RNAs

The mechanism of synthesis of the defective viral RNAs in cells infected with defective-interfering (DI) particles of mouse hepatitis virus was studied. Two DI-specific RNA species, DIssA of genomic size and DIssE of subgenomic size, were detected in DI-infected cells. Purified DI particles, however, were found to contain predominantly DIssA and only a trace amount of DIssE RNA. Despite its negligible amount, the DIssE RNA in virions appears to serve as the template for the synthesis of DIssE RNA in infected cells. This conclusion was supported by two studies. First, the uv target size for DIssE RNA synthesis is significantly smaller than that for DIssA. Second, when purified DIssE RNA was transfected into cells which had been infected with a helper virus, DIssE RNA could replicate itself and became a predominant RNA species in the infected cells. Thus, DIssE RNA was not synthesized from the genomic RNA of DI particles. By studying the relationship between virus dilution and the amount of intracellular viral RNA synthesis, we have further shown that DIssE RNA synthesis requires a helper function, but it does not utilize the leader sequence of the helper virus. In contrast, DIssA synthesis appears to be helper-independent and can replicate itself. Thus DIssA codes for a functional RNA polymerase.     

The Taiwanese hepatitis C virus genome: sequence determination and mapping the 5' termini of viral genomic and antigenomic RNA.

The complete nucleotide sequence of hepatitis C virus (HCV) cloned from the liver tissue of a Taiwanese patient with post-transfusion type C hepatitis was determined. The 5' end of HCV genomic RNA was located 341 nucleotides upstream from the initiation codon for the viral polyprotein open reading frame. The 5' end of the viral antigenomic RNA was shown to have 13 consecutive As. Thus the 3' terminus of the viral genome is a stretch of U which ends about 50 nucleotides downstream from the stop codon of the large open reading frame. The nucleotide sequence homology between this HCV strain and two Japanese isolates was 90.5 and 90.7%, respectively. Homology with the United States strain, however, was only 77.8%. Accordingly, the indigenous Taiwanese HCV strain is of the same subtype as the Japanese isolates. Novel features of the viral genome termini are possibly relevant to HCV genome replication.     

Identification of the 5' end of the rubella virus subgenomic RNA

The 5' end of the subgenomic RNA of rubella virus was determined by primer extension. The Maxam-Gilbert sequence ladder of the primer extension product contained a determinable sequence which was colinear with the complement of the sequence of the genomic RNA through nucleotide 3325 from the 3' end of the genomic RNA and a pair of bands in every lane above the determinable sequence. These results indicated that synthesis of the subgenomic RNA is initiated internally on the minus-polarity genome-length RNA template and that the length of subgenomic RNA is 3327 nucleotides excluding the poly(A) tail. There are thus 77 nucleotides between the 5' end of the subgenomic RNA and the first AUG, which is the initiation codon for the structural protein open reading frame. The initiation site of rubella virus subgenomic RNA synthesis is 20 nucleotides downstream from a block of 28 nucleotides which shares homology with the nucleotide sequence which is conserved at the subgenomic RNA initiation site in the Alphaviruses, the other genus of Togaviruses.     

Complete nucleotide sequence of the genomic RNA of Sindbis virus

The entire nucleotide sequence of the genomic RNA of the type virus of the alphavirus genus, Sindbis virus, has been determined. The genome is 11,703 nucleotides in length, exclusive of the 5' cap and the 3'-terminal poly(A) tract. After the 5'-terminal cap there are 59 nucleotides of 5' nontranslated nucleic acid followed by a reading frame of 7539 nucleotides that encodes the nonstructural polypeptides and which is open except for a single opal termination codon. Following 48 untranslated bases located in the junction region which separates the nonstructural and structural protein coding sequences, there is an open reading frame 3735 nucleotides long that encodes the structural proteins. Finally, the 3' untranslated region is 322 nucleotides long. The nonstructural proteins are translated from the genomic RNA as two polyprotein precursors. The first is 1896 amino acids in length and terminates at an opal codon at position 1897. This polyprotein is processed to produce three polypeptides called nsP1, nsP2, and nsP3. Sites of post-translational cleavage to produce these three proteins have been tentatively located using available N-terminal amino acid sequence data. In both cases cleavage probably occurs between the two alanine residues in the sequence Gly-Ala-Ala. The fourth nonstructural protein, nsP4, is produced when readthrough of the opal codon produces a second polyprotein precursor of length 2513 amino acids, which is also cleaved posttranslationally. The structural proteins are translated from a subgenomic message which begins at nucleotide 7598, is 4106 nucleotides in length (exclusive of the poly(A) tract), and is coterminal with the 3' end of the genomic RNA. The structural proteins are also translated as a polyprotein precursor which is cleaved to produce a nucleocapsid protein and two integral membrane glycoproteins as well as two small peptides not present in the mature virion. A replication strategy for Sindbis virus based upon the complete nucleotide sequence, as well as prior data, is presented.     

Structure of the L (polymerase) protein gene of sonchus yellow net virus.

The complete nucleotide sequence of the L protein gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined by dideoxynucleotide sequencing of cloned cDNAs derived from the negative-strand genomic RNA. The L protein gene is composed of 6401 nucleotides (nt) located between positions 7158 and 13558 relative to the 3' end of the genomic RNA. Sequence analysis suggests that the complementary mRNA contains a 44 nt untranslated 5' leader sequence preceding an open reading frame of 6348 nucleotides that is capable of encoding a polypeptide of 2116 amino acids with a deduced molecular weight of 241,569 Da. The L protein is positively charged, has a high proportion of the amino acids Leu and Ile, and contains putative polymerase and RNA binding domains. Extended alignment of the SYNV L protein amino acid sequence with those of other nonsegmented negative-strand RNA virus polymerases reveals conservation of sequences within 12 blocks that appear sequentially along the protein. A cluster dendrogram derived from the L protein alignments indicates that SYNV is more closely related to animal rhabdoviruses than to the paramyxoviruses and that the animal rhabdoviruses have diverged less from each other than from SYNV.     

Comparison of the full-length genome sequence of avian metapneumovirus subtype C with other paramyxoviruses

We determined the nucleotide (nt) sequence of the small hydrophobic (SH), attachment glycoprotein (G), and RNA polymerase (L) genes, plus the leader and trailer regions of the Colorado strain of Avian metapneumovirus subtype C (aMPV/C) in order to complete the genome sequencing. The complete genome comprised of 13,134 nucleotides, with a 40 nt leader at its 3' end and a 45 nt trailer at its 5' end. The aMPV/C L gene was the largest with 6173 nt and consisting of a single open reading frame encoding a 2005 amino acids (aa) protein. Comparison of the aMPV/C SH, G, and L nt and predicted aa sequences with those of Human metapneumoviruses (hMPV) revealed higher nt and aa sequence identities than the sequence identities between the aMPV subtypes A, B, C, and D, supporting earlier finding that aMPV/C was closer evolutionary to hMPV than the other aMPV subtypes.     

The complete genome sequence of Perina nuda picorna-like virus,an insect-infecting RNA virus with a genome organization similar to that of the mammalian picornaviruses

Perina nuda picorna-like virus (PnPV) is an insect-infecting RNA virus with morphological and physicochemical characters similar to the Picornaviridae. In this article, we determine the complete genome sequence and analyze the gene organization of PnPV. The genome of PnPV consists of 9476 nucleotides (nts) excluding the poly(A) tail and contains a single large open reading frame (ORF) of 8958 nts (2986 codons) flanked by 473 and 45 nt noncoding regions on the 5' and 3' ends, respectively. Northern blotting did not detect the presence of any subgenomic RNA. The PnPV genome codes for four structural proteins (CP1-4), and determination of their N-terminal sequences by Edman degradation, showed that all four are located in the 5' region of the genome. The 3' part of the PnPV genome contains the consensus sequence motifs for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) in that order from the 5' to the 3' end. In all of these characters, the genome organization of PnPV resembles the mammalian picornaviruses and two other insect picorna-like viruses, infectious flacherie virus (IFV) of the silkworm and Sacbrood virus (SBV) of the honeybee. In a phylogenetic tree based on the eight conserved domains in the RdRp sequence, PnPV formed a separate cluster with IFV and SBV, which suggests that these three insect picorna-like viruses might constitute a novel group of insect-infecting RNA viruses.     

Molecular cloning and sequence analysis of the mumps virus gene encoding the L protein and the trailer sequence.

We have cloned and determined the nucleotide sequences of the seventh gene of the Miyahara strain of mumps virus (MuV) encoding the L protein. The L gene is 6925 nucleotides in length and contains a single long open reading frame which is capable of coding for a protein of 2261 amino acids with a calculated molecular weight of 256,571 Da. The deduced amino acid sequence of the L protein of MuV showed significant homology with those of six other paramyxoviruses, human parainfluenza type 2 virus, Newcastle disease virus, Sendai virus, measles virus, human parainfluenza type 3 virus, and human respiratory syncytial virus. The predicted MuV L protein contained distinct elements thought to be essential for RNA polymerase activity. A noncoding sequence of 24 nucleotides downstream of the presumed polyadenylation site of the L gene showed significant complementarity with the leader sequence composed of 55 nucleotide at the 3' end of the genomic RNA.     

Structure of the glycoprotein gene of sonchus yellow net virus, a plant rhabdovirus

The nucleotide sequence of the glycoprotein (G) gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined from viral genomic and mRNA cDNA clones. The G cistron is 2045 nucleotides (nt) long and the G protein mRNA open reading frame (ORF), as determined from the cDNA sequence, contains 1896 nt and encodes a protein of 632 amino acids. Immunoblots with antibodies elecited against the purified glycoprotein from virus particles reacted with a fusion protein produced in Escherichia coli, indicating that the cloned ORF encodes the G protein. The 5' end of the G protein mRNA corresponds to nt 5111, relative to the 3' end of the viral (minus sense) genome, as determined by primer extension from mRNA isolated from infected plants, and extends to nt position 7155 on the genomic RNA. A 34-nt untranslated 5' leader sequence and a 115-nt untranslated 3' end flank the ORF on the mRNA. The gene junctions on either side of the G gene on the genomic RNA are identical to those previously described for other SYNV genes and are similar to sequences separating genes of animal rhabdoviruses. The predicted molecular weight of the G protein is 70,215 Da, a value less than the 77,000 Da estimated for the glycosylated G protein from virus particles. The deduced amino acid sequence of the SYNV G protein shares little direct relatedness with the G proteins of other rhabdoviruses, but appears to contain a similar signal sequence, a transmembrane anchor domain, and glycosylation signals. In addition, the SYNV G protein contains a putative nuclear targeting site near the carboxy terminus, which may be involved in transit to the nuclear membrane prior to morphogenesis.     

Determination of the complete genomic sequence and analysis of the gene products of the virus of Spring Viremia of Carp, a fish rhabdovirus

The complete genome of spring viremia of carp virus (SVCV) was cloned and the sequence of 11019 nucleotides was determined. It contains five open reading frames (ORF's) encoding for the nucleoprotein N; phosphoprotein P; matrix protein M; glycoprotein G; and the viral RNA dependent RNA polymerase L. Genes are organised in the order typical for rhabdoviruses: 3'-N-P-M-G-L-5'. The short leader and trailer regions of SVCV exhibit inverse complementarity and are similar to the respective 3' and 5' ends of the genome of vesicular stomatitis virus. To verify the predicted open reading frames proteins were expressed in bacteria and analysed with a polyclonal anti-SVCV serum. Furthermore, monospecific antisera against the distinct viral proteins were generated. Comparison of genome and protein confirm the assignment of SVCV to the genus Vesiculovirus.     

Sequence and organization of southern bean mosaic virus genomic RNA

The genomic RNA sequence of the cowpea strain of southern bean mosaic virus (SBMV-C) has been determined. The genome is 4194 nucleotides in length and has four open reading frames. A 5' proximal open frame, from base 49 to base 603, corresponds to the length of the P4 proteins translated in cell-free extracts from full-length and smaller virion RNA. The largest open frame extends from base 570 to base 3437 and encodes the two largest proteins translated in cell-free extracts from full-length virion RNA. Segments of this open reading frame's predicted amino acid sequence resemble those of known viral RNA polymerases, ATP-binding proteins, and viral genome-linked proteins. A third open frame extends from base 1895 to base 2380 and has not been correlated with an in vitro translation product. The fourth open reading frame is located in the 3' terminal region of the genome extending from base 3217 to base 4053. This frame encodes the SBMV capsid protein which is translated from subgenomic, virion RNA.