Research Articles (Medical Bioscience)

Permanent URI for this collection

https://hdl.handle.net/10566/77

Browse

Now showing 1 - 20 of 257

Metronidazole in the Prophylaxis and treatment of Anaerobic Infection
(Health and Medical Publishing Group, 1978) Applebaum, P.C.; Moodley, J.; Chatterton, S.A.; Cowan, D.B.; Africa, Charlene W.J.
The influence of prophylactic metronidazole on vaginal carriage rates of anaerobes and the development of postoperative anaerobic infection was studied in 104 women who underwent abdominal hysterectomy. Metronidazole prophylaxis in 54 patients led to a decrease in the anaerobe vaginal carriage rate from 65% pre-operatively to 17% and 28% on the 3rd and 7th postoperative days respectively. In the control group (50 patients) no significant decrease in anaerobe yield was noted, corresponding percentages being 72%, 64%, and 74%. Postoperative infection occurred in 36 patients (28 controls; 8 on prophylactic metronidazole). Wound swabs from all 8 patients in the latter group yielded aerobes, and in 1 patient mixed infection (aerobes/anaerobes) occurred. In 7 of these patients (including the patient with mixed infection), the infection resolved spontaneously, while the 8th patient responded to therapy with metronidazole, kanamycin and ampicillin. In the control patients, 21 cases of postoperative wound infection and 4 of vault infection were seen; wound swabs from patients in the former group yielded aerobes in only 6 cases, and mixed growth of aerobes/ anaerobes in 10 cases. Postoperative wound/vault infections in control patients cleared spontaneously in 18 cases and responded to imidazole therapy, with or without ampicillin and kanamycin, in 7 cases.
The characterization and phylogenetic relationship of the trichoplusia ni single capsid nuclear polyhedrosis virus polyhedrin gene
(Kluwer Academic Publishers, 1999) Fielding, Burtram C.; Davison, Sean
The polyhedrin gene (polh) was identified from the Trichoplusia ni (Tni) single capsid nuclear polyhedrosis virus (SNPV). An EcoRI fragment containing the truncated polyhedrin gene was detected by hybridization with an AcMNPV expression vector probe; the remaining portion of the gene was amplified by reverse PCR. An open reading frame (ORF) of 741 nucleotides (nt), encoding a putative protein of 246 amino acids (a.a) with Mr 28,780 Da was identified. The 50-noncoding region contained the putative late (TAAG) transcription initiation motif. The 30 end, downstream of the translation stop codon, lacked an obvious putative poly (A) signal. Nucleotide and amino acid homology are greater than 80% to that of Mamestra brassicae polyhedrin sequences.Results suggest that T. niSNPV is a member of the group II nuclear polyhedrosis viruses.
Identification and characterization of the Trichoplusia ni Single Capsid Nuclear Polyhedrosis Virus p10 gene
(Springer Verlag, 2000) Fielding, Burtram C.; Davison, Sean
The p10 gene was identified and characterized from the Trichoplusia ni single capsid nuclear polyhedrosis virus (TniSNPV). The p10 open reading frame (ORF) sequence was identified following sequencing of the ends of the EcoRI-G clone. Subsequent sequencing of an EcoRI-SmaI subclone identified the entire p10 and a portion of a p26 homologue. The p10 ORF of 264 basepairs (bps), encoded a predicted protein of 88 amino acids (aas) with Mr 9527 Da. The putative late transcription initiation motif (TAAG) was found upstream of the translation initiation codon at position ÿ46. Downstream of the translation stop codon, a putative poly(A) signal was identified. The p10 amino acid sequence contained the three conserved domains reported for all other p10 genes. The p10 amino acid sequence was most homologous (85% similarity and 67% identity) to that of Buzura suppresaria NPV p10 sequence.
Identification, sequence analysis, and phylogeny of the immediate early gene 1 of the trichoplusia ni single nucleocapsid polyhedrosis virus
(Springer Verlag, 2001) Wang, Weizhou; Leat, Neil; Fielding, Burtram C.; Davison, Sean
Substantial research has been conducted on the immediate early 1 (ie-1) genes from the prototype baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). In both cases ie-1 gene products have been implicated in transcriptional activation and repression. In this study an ie-1 homolog was identified from Trichoplusia ni single nucleocapsid polyhedrosis virus (TniSNPV). Nucleotide sequence analysis indicated that the TniSNPV ie-1 gene consists of a 2217 nucleotide open reading frame (ORF), encoding a protein with a molecular mass of 84.464 kDa. This represents the largest baculovirus ie-1 gene characterised to date. Of the seven ie-1 homologs identified to date, the TniSNPV ie-1 shared most sequence similarity with the ie-1 gene of Spodoptera exigua MNPV (SeMNPV) (41%). At the nucleotide level, expected TATA and CAGT motifs were found to precede each ie-1 ORF. At the protein level, it was confirmed that the N-termini are poorly conserved, but share the characteristic of having a high proportion of acidic amino acids. In addition it was found that N-terminal regions significantly matched the SET domain in the Swiss-Prot prosite database. The C-terminal regions of the deduced IE-1 sequences were found to be substantially more conserved than the N-termini. Several conserved motifs were identified in the C-terminal sequences. A phylogenetic tree of nine baculovirus IE-1 proteins was constructed using maximum parsimony analysis. The phylogenetic estimation of the ie-1 genes shows that TniSNPV is a member of the previously described lepidopteran NPV group II and it is most closely related to SeMNPV.
The genetic organisation of a 2,966 basepair DNA fragment of a Single Capsid Nucleopolyhedrovirus isolated from Trichoplusia ni
(Springer Verlag, 2002) Fielding, Burtram C.; Khan, Sehaam; Wang, Weizhou; Kruger, Courtney; Abrahams, Rayaana; Davison, Sean
In order to investiagte the genomic organization of the Trichoplusia ni Single Capsid Nucleoplyhedrovirus (TnSNPV) , a 2,966 base pairs (bp) genomic fragment was sequenced. The fragment was found to contain five open reading frames (ORF's) homologous to baculovirus genes, including p26, fibrillin (p10), AcMNPV ORF-29, late expression facor 6 (lef 6) and the C-terminal portion of p74, on either stand of DNA. Predicted amini acid sequences for the ORFs were compared and identity values of between 12% and 54% were observed. Clustering and arrangement of the TnSNPV genes were similar to the clustering reported for SeMNPV, confirming TnSNPV was a Group II NPV.
Development of molecular tools for honeybee virus research: the South African contribution
(Academic Journals, 2003) Davison, Sean; Leat, Neil; Benjeddou, Mongi
Increasing knowledge of the association of honeybee viruses with other honeybee parasites, primarily the ectoparasitic mite Varroa destructor, and their implication in the mass mortality of honeybee colonies has resulted in increasing awareness and interest in honeybee viruses. In addition the identification, monitoring and prevention of spread of bee viruses is of considerable importance, particularly when considering the lack of information on the natural incidence of virus infections in honeybee populations worldwide. A total of eighteen honeybee viruses have been identified and physically characterized. Most of them have physical features resembling picornaviruses, and are referred to as picorna-like viruses. The complete genome sequences of four picorna-like honeybee viruses, namely Acute Bee Paralysis Virus (ABPV), Black Queen Cell Virus (BQCV), Sacbrood Virus (SBV) and Deformed Wing Virus (DWV) have been determined. The availability of this sequence data has lead to great advances in the studies on honeybee viruses. In particular, the development of a reverse genetics system for BQCV, will open new opportunities for studies directed at understanding the molecular biology, persistence, pathogenesis, and interaction of these bee viruses with other parasites. This review focuses on the contribution of the Honeybee Virus Research Group (HBVRG), from the University of the Western Cape of South Africa, in the development of molecular tools for the study of molecular biology and pathology of these viruses.
Characterization of a unique group-specific protein (U122) of the severe acute respiratory syndrome coronavirus
(American Society for Microbiology, 2004) Fielding, Burtram C.; Tan, Yee-Joo; Shen, Shuo; Tan, Timothy H.P.; Ooi, Eng-Eong; Lim, Seng Gee; Hong, Wanjin; Goh, Phuay-Yee
A novel coronavirus (CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV genome encodes the characteristic essential CoV replication and structural proteins. Additionally, the genome contains six group-specific open reading frames (ORFs) larger than 50 amino acids, with no known homologues. As with the group-specific genes of the other CoVs, little is known about the SARS-CoV group-specific genes. SARS-CoV ORF7a encodes a putative unique 122-amino-acid protein, designated U122 in this study. The deduced sequence contains a probable cleaved signal sequence and a C-terminal transmembrane helix, indicating that U122 is likely to be a type I membrane protein. The C-terminal tail also contains a typical endoplasmic reticulum (ER) retrieval motif, KRKTE. U122 was expressed in SARS-CoVinfected Vero E6 cells, as it could be detected by Western blot and immunofluorescence analyses. U122 is localized to the perinuclear region of both SARS-CoV-infected and transfected cells and colocalized with ER and intermediate compartment markers. Mutational analyses showed that both the signal peptide sequence and ER retrieval motif were functional.
Overexpression of 7a, a Protein Specifically Encoded by the Severe Acute Respiratory Syndrome Coronavirus, Induces Apoptosis via a Caspase-Dependent Pathway
(American Society for Microbiology, 2004) Tan, Yee-Joo; Fielding, Burtram C.; Goh, Phuay-Yee; Shen, Shuo; Tan, Timothy H.P.; Lim, Seng Gee; Hong, Wanjin
Besides genes that are homologous to proteins found in other coronaviruses, the severe acute respiratory syndrome coronavirus genome also contains nine other potential open reading frames. Previously, we have characterized the expression and cellular localization of two of these “accessory” viral proteins, 3a (previously termed U274) and 7a (previously termed U122). In this study, we further examined whether they can induce apoptosis, which has been observed clinically. We showed that the overexpression of 7a, but not of 3a or the viral structural proteins, nucleocapsid, membrane, and envelope, induces apoptosis. 7a induces apoptosis via a caspase-dependent pathway and in cell lines derived from different organs, including lung, kidney, and liver.
Profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers
(American Society for Microbiology, 2004) Tan, Yee-Joo; Goh, Phuay-Yee; Fielding, Burtram C.; Shen, Shuo; Chou, Chih-Fong; Fu, Jianlin; Leong, Hoe Nam; Leo, Yee Sin; Ooi, Eng-Eong; Ling, Ai Ee; Lim, Seng Gee; Hong, Wanjin
A Novel Severe Acute Respiratory Syndrome Coronavirus Protein, U274, is transported to the Cell Surface and undergoes Endocytosis
(American Society for Microbiology, 2004) Tan, Yee-Joo; Teng, Eileen; Shen, Shuo; Tan, Timothy H.P.; Goh, Phuay-Yee; Fielding, Burtram C.; Ooi, Eng-Eong; Tan, Hwee-Cheng; Lim, Seng Gee; Hong, Wanjin
The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains open reading frames (ORFs) that encode for several genes that are homologous to proteins found in all known coronaviruses. These are the replicase gene 1a/1b and the four structural proteins, nucleocapsid (N), spike (S), membrane (M), and envelope (E), and these proteins are expected to be essential for the replication of the virus. In addition, this genome also contains nine other potential ORFs varying in length from 39 to 274 amino acids. The largest among these is the first ORF of the second longest subgenomic RNA, and this protein (termed U274 in the present study) consists of 274 amino acids and contains three putative transmembrane domains. Using antibody specific for the C terminus of U274, we show U274 to be expressed in SARS-CoV-infected Vero E6 cells and, in addition to the full-length protein, two other processed forms were also detected. By indirect immunofluorescence, U274 was localized to the perinuclear region, as well as to the plasma membrane, in both transfected and infected cells. Using an N terminus myc-tagged U274, the topology of U274 and its expression on the cell surface were confirmed. Deletion of a cytoplasmic domain of U274, which contains Yxx and diacidic motifs, abolished its transport to the cell surface. In addition, U274 expressed on the cell surface can internalize antibodies from the culture medium into the cells. Coimmunoprecipitation experiments also showed that U274 could interact specifically with the M, E, and S structural proteins, as well as with U122, another protein that is unique to SARS-CoV.
Cellular Characterization of SARS Coronavirus Nucleocapsid
(Leibniz Research Centre for Working Environment and Human Factors, 2004) Goh, Phuay-Yee; Choi, Yook-Wah; Shen, Shuo; Tan, Yee-Joo; Fielding, Burtram C.; Tan, Timothy H.P.; O, Eng-Eong; Lim, Seng Gee; Hong, Wanjin
The Severe and Acute Respiratory Syndrome coronavirus (SARS CoV) is a newly-emerged virus that caused an outbreak of atypical pneumonia in the winter of 2002-2003. Polyclonal antibodies raised against the nucleocapsid (N) of the SARS CoV showed the localization of N to the cytoplasm and the nucleolus in virus-infected and N-expressing Vero E6 cells. Like other coronavirus N proteins, the SARS N is probably a phosphoprotein. N protein expressed in mammalian cells is apparently able to “spread” to neighboring cells. For N to spread to neighboring cells, it must be exported out of the expressing cells. This is shown by the immunoprecipitation of N from the culture medium of a stable cell line expressing myc-N. Deletion studies showed that the 27 kD C-terminal domain of N (C1/2) is the minimal region of N that can spread to other cells. The nucleolar localization and spreading of N are artefacts of fixation, reminiscent of other protein-transduction domain (PTD)-containing proteins
Amino acids 1055 to 1192 in the S2 Region of severe acute respiratory syndrome Coronavirus S Protein induce neutralizing antibodies: Implications for the development of vaccines and antiviral agents
(American Society for Microbiology, 2005) Keng, Choong-Tat; Zhang, Aihua; Shen, Shuo; Lip, Kuo-Ming; Fielding, Burtram C.; Tan, Timothy H.P.; Chou, Chih-Fong; Loh, Chay Boon; Wang, Sifang; Fu, Jianlin; Yang, Xiaoming; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-Joo
The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-S 10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.
Genetic lesions within the 3a gene of SARS-CoV
(BioMed Central, 2005) Tan, Timothy H.P.; Barkham, Timothy; Fielding, Burtram C.; Chou, Chih-Fong; Shen, Shuo; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-Joo
A series of frameshift mutations within the 3a gene has been observed in culture-derived severe acute respiratory syndrome coronavirus (SARS-CoV). We report here that viral RNA from clinical samples obtained from SARS-CoV infected patients also contains a heterogeneous population of wild-type and mutant 3a transcripts.
A novel cell-based binding assay system reconstituting interaction between SARS-CoV S protein and its cellular receptor
(Elsevier, 2005) Chou, Chih-Fong; Shen, Shuo; Tan, Yee-Joo; Fielding, Burtram C.; Tan, Timothy H.P.; Fu, Jianlin; Xu, Qiurong; Lim, Seng Gee; Hong, Wanjin
Severe acute respiratory syndrome (SARS), a life-threatening disease, is caused by the newly identified virus SARS coronavirus (SARSCoV). In order to study the spike (S) protein of this highly contagious virus, we established a clonal cell-line, CHO-SG, from the Chinese hamster ovary cells that stably expresses C-terminally EGFP-tagged SARS-CoV S protein (S-EGFP). The ectodomain of the S glycoprotein is localized on the surface of CHO-SG cells with N-acetyl-glucosamine-terminated carbohydrate structure. CHO-SG cells associated tightly with Vero E6 cells, a SARS-CoV receptor (ACE2) expressing cell-line, and the interaction remained stable under highly stringent condition (1MNaCl). This interaction could be blocked by either the serum from a SARS convalescent patient or a goat anti-ACE2 antibody, indicating that the interaction is specific. A binding epitope with lesser degree of glycosylation and native conformation was localized by using rabbit anti-sera raised against five denatured recombinant S protein fragments expressed in Escherichia coli. One of the sera obtained from the fragment encompassing amino acids 48-358 significantly blocked the interaction between CHO-SG and Vero E6 cells. The region is useful for studying neutralizing antibodies in future vaccine development. This paper describes an easy and safe cell-based assay suitable for studying the binding between SARS-CoV S protein and its receptor.
The Severe Acute Respiratory Syndrome Coronavirus 3a Protein Up-Regulates Expression of Fibrinogen in Lung Epithelial Cells
(American Society for Microbiology, 2005) Tan, Yee-Joo; Tham, Puay-Yoke; Chan, Daphne Z. L.; Chou, Chih-Fong; Shen, Shuo; Fielding, Burtram C.; Tan, Timothy H.P.; Lim, Seng Gee; Hong, Wanjin
Here we analyzed the gene expression profile of cells that stably express the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein to determine its effects on host functions. A lung epithelial cell-line, A549, was chosen for this study because the lung is the primary organ infected by SARS-CoV and fatalities resulted mainly from pulmonary complications. Our results showed that the expression of 3a up-regulates the mRNA levels of all three subunits, A , B , and , of fibrinogen. Consequently, the intracellular levels as well as the secretion of fibrinogen were increased. We also observed increased fibrinogen levels in SARS-CoV-infected Vero E6 cells.
Severe acute respiratory syndrome coronavirus protein 7a interacts with hSGT
(Elsevier, 2006) Fielding, Burtram C.; Gunalan, Vithiagaran; Tan, Timothy H.P.; Chou, Chih-Fong; Shen, Shuo; Khan, Sehaam; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-Joo
Severe acute respiratory syndrome coronavirus (SARS-CoV) 7a is an accessory protein with no known homologues. In this study, we report the interaction of a SARS-CoV 7a and small glutamine-rich tetratricopeptide repeat-containing protein (SGT). SARS-CoV 7a and human SGT interaction was identified using a two-hybrid system screen and confirmed with interaction screens in cell culture and cellular co-localization studies. The SGT domain of interaction was mapped by deletion mutant analysis and results indicated that tetratricopeptide repeat 2 (aa 125-158) was essential for interaction. We also showed that 7a interacted with SARS-CoV structural proteins M (membrane) and E (envelope), which have been shown to be essential for virus-like particle formation. Taken together, our results coupled with data from studies of the interaction between SGT and HIV-1 vpu indicated that SGT could be involved in the life-cycle, possibly assembly of SARS-CoV.
Monoclonal Antibodies targeting the HR2 Domain and the region immediately upstream of the HR2 of the S Protein neutralize in Vitro Infection of Severe Acute Respiratory Syndrome Coronavirus
(American Society for Microbiology, 2006) Lip, Kuo-Ming; Shen, Shuo; Yang, Xiaoming; Keng, Choong-Tat; Zhang, Aihua; Oh, Hsueh-Ling Janice; Li, Zhi-Hong; Hwang, Le-Ann; Chou, Chih-Fong; Fielding, Burtram C.; Tan, Timothy H.P.; Mayrhofer, Josef; Falkner, Falko G.; Fu, Jianlin; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-Joo
We have previously shown that an Escherichia coli-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. T. Keng, A. Zhang, S. Shen, K. M. Lip, B. C. Fielding, T. H. Tan, C. F. Chou, C. B. Loh, S. Wang, J. Fu, X. Yang, S. G. Lim, W. Hong, and Y. J. Tan, J. Virol. 79:3289–3296, 2005). In this study, monoclonal antibodies (MAbs) were raised against this fragment to identify the linear neutralizing epitopes in the functional domain and to investigate the mechanisms involved in neutralization. Eighteen hybridomas secreting the S protein-specific MAbs were obtained. Binding sites of these MAbs were mapped to four linear epitopes. Two of them were located within the HR2 region and two immediately upstream of the HR2 domain. MAbs targeting these epitopes showed in vitro neutralizing activities and were able to inhibit cell-cell membrane fusion. These results provide evidence of novel neutralizing epitopes that are located in the HR2 domain and the spacer region immediately upstream of the HR2 of the S protein.
Over-expression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in Vero E6 cells
(Elsevier, 2006) Khan, Sehaam; Fielding, Burtram C.; Tan, Timothy H.P.; Chou, Chih-Fong; Shou, Shen; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-Joo
The genome of the severe acute respiratory syndrome coronavirus encodes for eight accessory viral proteins with no known homologues in other coronaviruses. One of these is the 3b protein, which is encoded by the second open reading frame in subgenomic RNA 3 and contains 154 amino acids. Here, a detailed time-course study was performed to compare the apoptosis and necrosis profiles induced by full-length 3b, a 3b mutant that was deleted by 30 amino acids from the C terminus (3b 124-154) and the classical apoptosis inducer, Bax. Our results showed that Vero E6 cells transfected with a construct for expressing 3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time-points. At all the time-points analysed, the apoptosis induced by the expression of 3b was less than the level induced by Bax but the level of necrosis was comparable. The 3b 124-154 mutant behaves in a similar manner indicating that the localization of the 3b protein does not seems to be important for the cell-death pathways since full-length 3b is localized predominantly to the nucleolus, while the mutant is found to be concentrated in the peri-nuclear regions. To our knowledge, this is the first report of the induction of necrosis by a SARS-CoV protein.
The scientific merit of Carpobrotus mellei L. based on antimicrobial activity and chemical profiling
(Academic Journals, 2006) Springfield, E. P.; Weitz, Frans
Species of the genus Carpobrotus are similar in appearance and have been used for medicinal purposes over many generations. Carpobrotus mellei is endemic to the south-Western Cape of South Africa, and also used for various ailments. To date no scientific validation and information has been reported on C. mellei. This study investigated the antimicrobial potential of C. mellei against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Mycobacterium smegmatis, and determined the minimum inhibitory concentrations (MIC) by two-fold serial dilution. C. mellei showed antimicrobial activity against S. aureus and M. smegmatis in the disc diffusion method. Eight chemical compounds showed clear zones of inhibition in the bioautograms, seven against S. aureus and three compounds were active against M. smegmatis. The ethyl acetate extracts have MIC values of 7.5 mg/ml and 15 mg/ml against S. aureus and M. smegmatis, respectively. Phytochemical tests indicated the presence of flavonoids, hydrolysable tannins, phytosterols and aromatic acids. High performance liquid chromatography (HPLC) showed a species-specific spectrum at a wavelength of 280 nm. The results confirm that C. mellei has scientific merit, and can substitute one of the other Carpobrotus species for antimicrobial usage within the south-Western Cape of South Africa.
The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein
(Elsevier, 2007) Kumar, Purnima; Gunalan, Vithiagaran; Liu, Boping; Chow, Vincent T.K.; Druce, Julian; Birch, Chris; Catton, Mike; Fielding, Burtram C.; Tan, Yee-Joo; Lal, Sunil K.
Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein–protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8– ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication

Browse

Browsing by Issue Date

Results Per Page

Sort Options