| Tomato spotted wilt virus(TSWV),the type member of the genus Tospovir?s in the family Bunyaviridae,consists of three single-stranded RNA segments(L,M and S).RNA L is a negative-stranded RNA,whose complementary strand encodes RNA-dependent RNA polymerase(RdRp).RNA M and S are both ambisense RNA encoding two ORFs by viral and complementary strand rspectively.RNA M encodes a nonstruct?ral protein(NSm)responsible for cell-to-cell movement and its complementary strand encodes a Gn-Gc glycoprotein.RNA S encodes a nonstruct?ral protein(NSs)responsible for cell-to-cell movement and its complementary strand encodes coat protein(N).All four proteins encoded by ambisense RNA M or S are expressed from corresponding subgenomic RNA.However,the accurate 3' terminal location of subgenomic RNAs is not reported.The complete nucleotide sequences of three new isolates from different hosts(tobacco,red pepper and green pepper)in Yunnan province,China were determined by RT-PCR and subsequent sequencing.Based on the Genbank date including 29 RNA L,62 RNA M and 66 RNA S of TSWV,following characteristics were summarized: 1.The length variation of RNA M and RNA S is bigger than RNA L.2.The length of all ORFs regions is stable.3.The length variation of RNA M and S are mainly due to the length varization of internal gene region(IGR).Molecular variation analysis using DNAMAN and phylogenetic analysis using MEGA5.0 show that the three new isolates in China have the closest relationship with tomato isolate in china and have further relationship with lettuce isolate in china.It is also suggested the inherent and frequent reassortment among TSWV isolates,which often occurred in multi-segmented RNA virus.Recombination assay performed using the RDP4 program showed a large amount of recombination events among TSWV,while previous reports on negative strand RNA viruses only detected a few recombination events.In addition,recombination events among TSWV showed remarkable preference of location,i.e,almost all of recombination events in TSWV RNA M and RNA S occurring within or around the 5' first ORF of viral RNAs.In order to analyze the translational characteristics of subgenomic RNAs for TSWV RNA M(KM65718)and RNA S(KM657115),the 3' terminal nucleotides of these subgenomic RNAs were determined by 3' RACE since previous report only mapped them roughly.The 3' terminal nucleotide of sgRNA-NSm having 5' co-terminal with RNA M was A1080 in RNA M,while 3' terminal nucleotide of sgRNA-(Gn-Gc)having 5' co-terminal with complementary strand of RNA M was complementary to U1200 in RNA M.The 3' terminal nucleotide of sgRNA-NSs having 5' co-terminal with RNA S was C1702 in RNA S,while 3'terminal nucleotide of sgRNA-N having 5' co-terminal with complementary strand of RNA S was complementary to A1865 in RNA S.The length of sgRNA-NSm,sgRNA-(Gn-Gc),sgRNA-NSs and sgRNA-N was 1080 nt,3573 nt,1702 nt and 1106 nt respectively.Although the length and sequences of IGR in different TSWV RNA M or RNA S was variable,the 3' terminal nucleotide of subgenomic RNAs and its flanking sequences was conserved.The 3'UTRs of subgenomic RNAs only contain partial stem-loop located within the viral or complementary strand of RNA M and RNA S,which bring about single-stranded A-rich region at the 3' terminal of subgenomic RNAs.The 5'UTR and 3'UTR of subgenomic RNAs from RNA S were fused upstream and downstream of firefly luciferase(Fluc)to evaluate their effect on translation.5'UTR of sgRNA-NSs and sgRNA-N upregulated the translation of Fluc,further the 3'UTR enhanced the upregulation of 5'UTR synergistically.Deletion mutagenesis suggested the essential function of 5' terminal 1-15 nt on translation.Moreover,preliminary result of trans-competition assay implied that 5'UTR may bind some translation initiation factors or ribosomal subunits to facilitate translation.Deletion mutagenesis also suggested that A-rich region within 3'UTR played essential role in enhancing translation and it may act as poly(A). |
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