Characterization of the Tupaia rhabdovirus genome reveals a long open reading frame overlapping with P and a novel gene encoding a small hydrophobic protein

Abstract

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.

FIG. 1.

TRV genome organization and transcription/replication control sequences. (A) Map of the TRV genome. The black bar represents the 11,440-nucleotides single-stranded TRV antigenome, the seven open arrows indicate the position of the open reading frames of the N, P, C, M, SH, G, and L proteins. Relevant cDNA clones are indicated by small bars labeled A, B, and CH-2-2-2, respectively. (B) Sequences of the gene junctions. The gene start and end sequences (right and left columns, respectively) and the intergenic nucleotides (middle column) of the six TRV genes are indicated. A consensus sequence is indicated below the gene sequences (shown in bold type, nucleotide base code of the Nomenclature Committee of the International Union of Biochemistry). This sequence is compared to the sequences of other rhabdoviruses (bottom). (C) The TRV leader and trailer sequences. The leader sequence includes the first ten nucleotides of the N gene, the trailer sequence the ten last nucleotides of the L gene (underlined and in bold type). (D) Alignment of the TRV leader with the leaders of other rhabdoviruses. The first three and the tenth nucleotide are perfectly conserved. (E) Complementarity of the 3′ and 5′ end of the TRV genome.

FIG. 2.

Predicted sequence of the proteins encoded in the TRV P gene mRNA (A) and comparison of the TRV C protein to other rhabdovirus C proteins (B). In (A) the start and stop codons of the P and C proteins are underlined and in bold type, and the deduced amino acid sequences (one-letter code) are presented above the nucleotide sequence. The alignment in B was created with the ClustalX program (59) using the default settings except for a gap penalty of 25 instead of 10. The basic amino acids arginine, lysine, and histidine are shown in bold type. TRV, tupaia rhabdovirus; VSNJV, vesicular stomatitis virus New Jersey; VSVIV, vesicular stomatitis Indiana; PIRY, Piry virus; CHAPV, Chandipura virus. The accession numbers of the sequences are given in Fig. 5.

FIG. 3.

Nucleotide sequence of the TRV SH gene mRNA (A), Northern blot analysis of TRV infected cells (B) and hydropathicity plot of the SH protein (C). In (A) the SH protein start and stop codons are underlined and in bold type, and the deduced amino acid sequences (one-letter code) is presented above the nucleotide sequence. In the Northern blot in B two probes against the TRV SH and M genes have been used as indicated above the lanes. Lanes containing mRNA from infected cells or uninfected cells are labeled with a plus or minus symbol, respectively, and the lane containing the molecular weight marker is marked with an “M”. The sizes of the markers are given on the right, and the positions of the different TRV mRNAs are indicated on the left. The hydropathicity plot (C) was created with the program ProtScale on the proteomics server of the Swiss Institute of Bioinformatics according to the method of Kyte and Doolittle (40) with a window size of nine.

FIG. 4.

Comparison of the TRV genome structure with the genome structure of representatives of the different rhabdovirus genera. The reading frames for the conserved rhabdovirus genes N, P, M, G, and L are depicted as open arrows, additional genes are shown in grey. The size of the genomes and the rhabdovirus genera are indicated. The abbreviations of virus names are the same as in Fig. 5.

FIG. 5.

Phylogenetic trees for the L proteins (conserved regions A to D) of selected members of the Mononegavirales (A) and N proteins of selected rhabdoviruses (B). The details of the construction of the phylogenetic trees are described in the Materials and Methods section. Family names are bolded, underlined, and italicized. Genera names within the Rhabdoviridae are bolded and italicized. The bar indicates 0.1 expected substitutions per site. In B, bootstrap values for the major branches from 1000 resampled data sets are indicated in percent. ABLV, Australian bat lyssavirus (accession number NC_003243); ARV, Adelaide river virus (AAC54627); BEFV, bovine ephemeral fever virus (NC_002526); BDV, Borna disease virus (NC_001607); CDV, canine distemper virus (NC_001921); CHPV, Chandipura virus (P11211); EBOV, Ebola virus (NC_002549); FDLV, fer-de-lance virus (NC_005084); FLAV, Flanders virus (AH012179); HeV, Hendra virus (NC_001906); HIPV1, human parainfluenza virus 1 (NC_003461); HIPV3, human parainfluenza virus 3 (NC_001796); HIRRV, Hirame rhabdovirus (NC_005093); HMPV, human metapneumovirus (NC_004148); HRSV, human respiratory syncytial virus (NC_001781); IHNV, infectious hematopoietic necrosis virus (NC_001652); LBV, Lagos bat virus (AAR03475); MARV, Marburg virus (NC_001608); MFSV, maize fine streak virus (NC_005974); MMV, maize mosaic virus (NC_005975); MOKV, Mokola virus (AAR03476); MuV, Mumps virus, (NC_002200); MV, measles virus (NC_001498); NCMV,northern cereal mosaic virus (NC_002251); NDV, Newcastle disease virus (NC_002617); NiV, Nipah virus (NC_002728); OITAV, Oita virus(BAD13431); PIRYV, Piry virus (VHVNPV); RABV, rabies virus (NC_001542); RYSV, rice yellow stunt virus (NC_003746); SHRV, snakehead rhabdovirus (NC_000903); SIGMAV, Sigma virus (CAA54327); SV5, simian virus 5 (Q88434); SVCV, spring viremia of carp virus (NC_002803); SYNV, Sonchus yellow net virus (NC_001615); TPMV, tupaia paramyxovirus (NC_002199); TROURV, trout rhabdovirus 903/87 (AAL35756); TRV, tupaia rhabdovirus; VHSV, viral hemorrhagic septicemia virus (NC_000855); VSIV, vesicular stomatitis Indiana virus (NC_001560); VSNJV,; Vesicular stomatitis New Jersey virus (P04881); WCBV, west Caucasian bat virus (AAR03481).