Nucleotide sequence of RNA2 and polyprotein processing sites of a Chinese isolate of broad bean wilt virus 2

RNA2 of broad bean wilt virus 2 (BBWV2) isolate B935 is composed of 3601 nucleotide (nt) residues, exclusive of the polyadenylate at the 3' end. Only one of the six possible reading frames has a long open reading frame, which extends from nt 231 to nt 3428 in the polarity of encapsidated RNA, and encodes a polyprotein of 119 kD. The N-terminus of the large coat protein (LCP) is located at 599 nt and the small coat protein (SCP) at 197 nl from the C-terminus of the 119 kD protein, which suggests that the coat proteins are released from the polyprotein by cleavages of a glulamine-glycine (Q-G) and a glutamine-alanine (Q-A) bond respectively. The sequence comparison of B935 with fabaviruses shows that B935 has very high sequence homology with other BBWV2 isolates and with patchoul' mild mosaic virus, but has lower homology with BBWV1 isolates. B935 has a similar genomic organization, but a low sequence homology to RNA2 molecules of comoviruses and nepoviruses.     

Cloning, sequencing and expression of the coat protein gene of Cocksfoot mottle virus

The coat protein (CP) gene of Cocksfoot mottle virus (CfMV) was amplified by RT-PCR and inserted into expression vector pGEX-4T-1, and the resulting plasmid was designated as pGEXCfMV-JANCP. The fusion protein GST-CP was expressed in BL21 (DE3) pLysS after IPTG induction. The results of SDS-PAGE and Western blot analysis showed that the CfMV-CP gene was efficiently expressed in E.coli BL21 (DE3) pLysS through IPTG induction and the 56.0 kD protein was obtained.     

Genome sequencing and characterization analysis of a Beijing isolate of chicken coronavirus infectious bronchitis virus

Avian infectious bronchitis virus (AIBV) is classified as a member of the genus coronavirus in the family coronaviridae. The enveloped virus has a positive-sense, single-stranded RNA genome of approximately 28 kilo-bases,which has a 5‘ cap structure and 3‘ polyadenylation tract.The complete genome sequence of infectious bronchitis virus (IBV), Beijing isolate, was determined by cloning sequencing and primer walking. The whole genome is 27733 nucleotides in length, has ten open reading frames:5′-orfla-orflab-s-3a-3b-e-m- 6a-6b-n-3′. Alignments of the genome sequence of IBV Beijing isolate with those of two AIBV strains and one SARS coronavirus were performed respectively. The genome sequence of IBV Beijing isolate compared with that of the IBV strain LX4 (uncompleted, 19440 bp in size) was 91.2% similarity. However, the full-length genome sequence of IBV Beijing isolate was 85.2% identity to that of IBV Strain Beaudette, and was only 50.8% homology to that of SARS coronavirus. The results showed that the genome of IBV has remarkable variation. And IBV Beijing isolate is not closely related to SARS coronavirus. Phylogenetic analyses based on the whole genome sequence, S protein, M protein and N protein, also showed that AIBV Bering isolate is lone virus in group Ⅲ and is distant from SARS coronavirus. In conclusion, this study will contribute to the studies of diagnosis and diseases control on IBV in China.     

Function of A-Rich region in DNAβ associated with Tomato yellow leaf curl China virus

A novel type of circular single-stranded satellite DNA, known as DNAβ, was recently characterized and demonstrated to be associated with monopartite begomoviruses. DNAβ was essential for induction of characteristic symptoms in plants. DNAβ has three structural features: an 115 bp highly conserved region, tiC/gene and A-Rich region. The in-frame ATG mutation of βC1 gene of Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-TY10) DNAβ demonstrated that βC1 gene is required for leaf curl symptom. Here, the function of A-Rich region in TYLCCNV-Y10 DNAβ was identified. When A-Rich region was deleted, the A-Rich deleted mutant was still capable of replication and systemic infection in plant, indicating that A-Rich region is not required for trans-replication of DNAβ. The immunotrapping-PCR demonstrated that A-Rich deleted mutant could be encapsidated in the coat protein encoded by TYLCCNV-Y10 DNA-A, suggesting that A-Rich region is not related with DNAβ encapsidation. However, the A-Rich region deleted mutant caused milder symptom.     

Two virus-encoded RNA silencing suppressors, P14 of Beet necrotic yellow vein virus and S6 of Rice black streak dwarf virus

Functional analysis for gene silencing suppressor of P14 gene of Beet necrotic yellow vein virus and S6 gene of Rice black streak dwarf virus was carried out by agro- infiltration with recombinant vectors of Potato virus X. The phenotype observation of green fluorescent protein (GFP)expression and Northern blot showed that the gene silencing of gfp transgenic Nicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the $6. In the suppressed plants, the gfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially the gfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.     

Functional analysis of DNA 4 coding region from a Chinese Zhangzhou isolate of banana bunchy top virus

The DNA 4 coding region of banana bunchy top virus from a Chinese Zhangzhou isolate (BBTV-ZZ) is cloned by PCR. The sequencing analysis shows that it is 351 nucleotides long and it putatively encodes a protein of 116 amino acids. On the basis of a plant binary vector pBin438, the plant expression vector pBBTV-4B harboring the BBTV-ZZ DNA 4 coding region has been constructed and then transferred to tobacco (Nicotiana tobacum cv. Xanthi nc) by a Agrobacterium-mediated procedure. Under insect-free condition, movement-defective mutant of CMV-Fny strain (CMV-Fny-△MP) is mechanically inoculated on the lower leaves of transgenic plants. Systemic symptoms with different degrees of severity are developed in the upper uninoculated leaves of transgenic plants at 12 days postinoculation (dpi), while no symptoms can be seen in the uninoculated leaves of untransformed plants at any time. Accumulation of CMV-Fny is detected on the upper uninoculated leaves of transgenic plants, but is not on that of untransformed plants by indirect double antibody sandwich enzyme-link immunosorbent assay (DAS-ELISA). The results reveal that transgenic plants have acquired the property of cell-to-cell movement and systemic spread of CMV-Fny-△MP. This suggests that the protein encoded by BBTV-ZZ DNA 4 might have function of viral movement protein.     

Identification of a bi-directional promoter from Tomato yellow leaf curl China virus

A bi-directional promoter of Tomato yellow leaf curl China virus (TYLCCNV) was obtained with the total DNA from TYLCCNV isolate Y10 infected tobacco leaves as a template. Plant expression vectors were constructed by fusing the amplified DNA fragment with the gus gene and nopaline terminator in different orientations. The vectors containing promoter fragments were transferred into leaf cells and plant stems of Nicotiana benthamiana by Agrobacterium-mediated method. Transient expression results showed that both the complementary and virion-sense promoters could drive the gus gene to express, and the GUS activity of the complementary-sense promoter was stronger than that of the virion-sense. Co-expression of the vector containing βC1 gene of TYLCCNV DNAβ with the vector containing a bi-directional promoter revealed that the βC1 protein has no impact on expression of either the virion- or the complementary-sense promoter.     

Delayed resistance to Cucumber mosaic virus mediated by 3′ UTR-derived hairpin RNA

RNA silencing has been shown to function in the plant antivirus defense response,leading to viral RNA degradation induced by vsiRNA-containing RISC cleavage activity.Cucumber mosaic virus(CMV) 3′UTR sequences share a high conservation of nucleotide sequence and secondary structures that are important for CMV replication.Here,in an attempt to simultaneously target the multiple genomic and subgenomic RNAs of CMV for degradation,CMV 3′UTR were used to design hairpin RNA(hpRNA) to transform tobacco(Xanthi.nc) so as to constitutively produce viral siRNAs.Most of the transgenic plants expressing CMV Q strain(Q-CMV,subgroup Ⅱ strain) RNA3 3′UTR-derived hpRNA showed de-layed resistance to Q-CMV infection and exhibited recovery phenotypes.Compared with Q-CMV-in-oculated leaves,the upper leaves showed weak or no disease symptoms and a reduced accumulation level of viral RNAs.Together with transient assays,our results indicate that the 3′UTR-derived siRNAs were biologically active in targeting viral RNA for degradation.Recovery resistance in transgenic plants was also observed against subgroup IB strain SD-CMV infection,indicating a broad-spectrum anti-CMV effect of the 3′UTR-based antiviral silencing.Northern blot assays indicated that there was no strong correlation between the degree of resistance and the accumulation level of 3′UTR-derived siRNAs,suggesting that to target a highly structured RNA,such as the CMV 3′UTR,the quantity of siRNAs may not be the only determinant of silencing efficiency.Target RNA secondary structures may also affect target accessibility,siRNA-containing RISC-target recognition and the consequent antiviral effect.     

Delayed resistance to Cucumber mosaic virus mediated by 3’UTR-derived hairpin RNA

RNA silencing has been shown to function in the plant antivirus defense response, leading to viral RNA degradation induced by vsiRNA-containing RISC cleavage activity. Cucumber mosaic virus （CMV） 3′UTR sequences share a high conservation of nucleotide sequence and secondary structures that are important for CMV replication. Here, in an attempt to simultaneously target the multiple genomic and subgenomic RNAs of CMV for degradation, CMV 3′UTR were used to design hairpin RNA （hpRNA） to transform tobacco （Xanthi. nc） so as to constitutively produce viral siRNAs. Most of the transgenic plants expressing CMV Q strain （Q-CMV, subgroup Ⅱ strain） RNA3 3′UTR-derived hpRNA showed delayed resistance to Q-CMV infection and exhibited recovery phenotypes. Compared with Q-CMV-inoculated leaves, the upper leaves showed weak or no disease symptoms and a reduced accumulation level of viral RNAs. Together with transient assays, our results indicate that the 3′UTR-derived siRNAs were biologically active in targeting viral RNA for degradation. Recovery resistance in transgenic plants was also observed against subgroup IB strain SD-CMV infection, indicating a broad-spectrum anti-CMV effect of the 3′UTR-based antiviral silencing. Northern blot assays indicated that there was no strong correlation between the degree of resistance and the accumulation level of 3′UTR-derived siRNAs, suggesting that to target a highly structured RNA, such as the CMV 3′UTR, the quantity of siRNAs may not be the only determinant of silencing efficiency. Target RNA secondary structures may also affect target accessibility, siRNA-containing RISC-target recognition and the consequent antiviral effect.     

A complete sequence and comparative analysis of a SARS-associated virus （Isolate BJO1）

The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-assoclated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs.We report the complete genome sequence and comparative analysis of an isolate (B J01) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 20RFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARS-associated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymous mutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host.Two amino acid changes have been detected in the M protein,which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s).     

Cloning, sequencing and expression of the coat protein gene of Cocksfoot mottle virus

The coat protein (CP) gene of Cocksfoot mottle virus (CfMV) was amplified by RT-PCR and inserted into expression vector pGEX-4T-1, and the resulting plasmid was designated as pGEXCfMV-JANCP. The fusion protein GST-CP was expressed in BL21 (DE3) pLysS after IPTG induction. The results of SDS-PAGE and Western blot analysis showed that the CfMV-CP gene was efficiently expressed in E. coli BL21 (DE3) pLysS through IPTG induction and the 56.0 kD protein was obtained.     

Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.     

AC2 and AC4 proteins of <Emphasis Type="Italic">Tomato yellow leaf curl China virus</Emphasis> and <Emphasis Type="Italic">Tobacco curly shoot virus</Emphasis> mediate suppression of RNA silencing

Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) alone could systemically infect host plants such as Nicotiana benthamiana without symptoms. In contrast, Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) alone induces leaf curl symptoms in N. benthamiana. When inoculated into transgenic N. benthamiana plants expressing GFP gene (line 16c), TYLCCNV-Y10 neither reverses the established GFP silencing nor blocks the onset of GFP silencing. In contrast, TbCSV-Y35 can partially reverse the established GFP silencing and block the onset of GFP silencing in new leaves. In the patch co-infiltration assays, the AC2 and AC4 proteins of TYLCCNV-Y10 and TbCSV-Y35 could suppress local GFP silencing and delay systemic GFP silencing, suggesting that they are suppressors of RNA silencing. Comparison of the accumulation levels of GFP mRNA in the co-infiltration patches showed that Y10 AC2 and Y35 AC2 proteins had similar efficiency for suppression of RNA silencing. However, Y35 AC4 protein functioned as a stronger suppressor of RNA silencing than Y10 AC4 protein. There-fore, the pathogenicity difference between TbCSV-Y35 and TYLCCNV-Y10 may be related to the functional difference in their AC4 proteins.     

Identification of a bi-directional promoter from Tomato yellow leaf curl China virus

A bi-directional promoter of Tomato yellow leaf curl China virus (TYLCCNV) was obtained with the total DNA from TYLCCNV isolate Y10 infected tobacco leaves as a template. Plant expression vectors were constructed by fusing the amplified DNA fragment with the gus gene and nopaline terminator in different orientations. The vectors containing promoter fragments were transferred into leaf cells and plant stems of Nicotiana benthamiana by Agrobacterium-mediated method. Transient expression results showed that both the complementary and virion-sense promoters could drive the gus gene to express, and the GUS activity of the complementary-sense promoter was stronger than that of the virion-sense. Co-expression of the vector containing βC1 gene of TYLCCNV DNAβ with the vector containing a bi-directional promoter revealed that the βC1 protein has no impact on expression of either the virion- or the complementary-sense promoter.     

Interaction of hepatitis B virus with tumor suppressor gene p53: its significance and biological function

The mechanism of the interaction of hepatitis B virus (HBV) with tumor suppressor p53 and its role in the hepatocar-cinogenesis have been studied by PCR-directed sequencing, gel shift assays and in situ ultraviolet cross-linking assay. The biological function of the interaction of HBV with p53 gene was investigated by co-transfection of chloramphenicol acetyltransferase ( CAT) reporter gene. p53 and HBV DNA. and quantitative PCR. Among the 16 primary hepatocellular carcinoma (PHC) samples. 13 were HBV-DNA positive. 10 HBxAg positive and 9 p53 protein positive. The p53 gene point mutation was found in 5 samples, one of which had a G to T substitution located at codon 249. After analyzing the HBV genome by a computer program, a p53 response element binding sequence was found in HBV genome at upstream of enhancer I. from 1047 to 1059 nucleotides. This sequence could specifically bind to p53 protein, increase p53 protein accumulation in the PHC cells and stimulate the transactivating activity of p53 and HBV replication . The results also revealed that HBxAg could combine with p53 protein to form a complex in the cells and enhance CAT expression. Immunocytochemical staining showed that p53 protein complex was located in the cytoplasm and the process of p53 entry to nuclei was. in part, blocked. From our results, we conclude that the mutation of p53 gene at codon 249 is infrequent in HBV-associated PHC. the DNA-protein binding between HBV and p53. and the protein-protein binding between HBxAg and p53 might lead to the reduction or inactivation of p53 protein, which in turn resulting in HBV-associated hepatocarcinogenesis.     

Identification of a bi-directional promoter from Tomato yellow leaf curl China virus

A bi-directional promoter of Tomato yellow leaf curl China virus (TYLCCNV) was obtained with the total DNA from TYLCCNV isolate Y10 infected tobacco leaves as a template. Plant expression vectors were constructed by fusing the amplified DNA fragment with the gus gene and nopaline terminator in different orientations. The vectors containing promoter fragments were transferred into leaf cells and plant stems of Nicotiana benthamiana by Agrobacterium-mediated method. Transient expression results showed that both the complementary and virion-sense promoters could drive the gus gene to express, and the GUS activity of the complementary-sense promoter was stronger than that of the virion-sense. Co-expression of the vector containing βC1 gene of TYLCCNV DNAβ with the vector containing a bi-directional promoter revealed that the βC1 protein has no impact on expression of either the virion- or the complementary-sense promoter.