SELF-INTERACTION OF COAT PROTEIN OF CARNATION MOTTLE VIRUS

(Funded by the Third World Academy of Sciences, Italy)

Self-interaction of the coat protein (CP) of Carnation mottle virus (CarMV) infecting carnations was investigated. The CP gene was characterized at the sequence level by RT-PCR amplification using indigenously designed primers (AJ566191 and AJ566192). The gene was cloned, sequenced and found to be 1047bp in length. The gene and protein showed >95 % similarity to already known sequences in the database. From the deduced sequence, primers were designed to amplify various regions of the CP gene for expression in Saccharomyces cerevisae strain AH109. Ten pairs of primers were designed to amplify the complete, N-terminal, C-terminal and middle portion of the protein. These amplified products were cloned in plasmids pGADT7 and pGBKT7 inframe to the activation and binding domain (AD & BD). The cloned fragments in AD and BD were co-transformed into AH109 and analyzed for interaction by determining the requirement of His by the transformed cells for their growth so as to assess the tendency of the proteins to interact or not. Only clones showing positive interaction would grow in the media not supplemented with His. Thirty different combinations were transformed for self-interaction and interaction with different portions of the protein. The complete CP gene did not interact with itself in the yeast two-hybrid system. This could be due to the requirement of viral RNA for interaction or even if the proteins were interacting, the AD and BD were not closer enough to activate His expression. Further, in most of the cases (except 180-349 and N-terminal), the complete CP gene did not interact with other portions of the protein. Similarly, analysis of the other portions indicated the following: N-terminal of the protein (1-180AA) did not self-interact while showing interaction with the protein of 100-349 aminoacids; N-terminal of the protein showed interaction with 100-200AA and 180-349AA; 100-200AA showed interaction with 180-349 and 100AA from the N-terminal; 180-349AA residues selfinteracted and complete coat protein showed interaction with 180-349AA.

The figure showing the requirement of Histidine for cotransformants with ADA/BDA (CP-CP) and ADA/BDB (CP 180-349/CP). As can be seen from the photograph, the complete coat protein does not self-interact. On the other hand if N-terminal 180 amino acids are deleted, the protein does shown interaction withcomplete CP. On the left colonies were plated onto media lacking histidine while media supplemented with His is on the right

The figure showing the requirement of Histidine for cotransformants with ADB/BDD (CP180-349AA/CP1-180AA), and ADC/BDD (CP100-349AA/CP1- 180AA). As can be seen from the photograph, 180 AA residues from the N terminal interact with complete coat protein where 180 AA residues have been deleted from the N-terminal (ADB/BDD). However, N-terminal 180 residues show weaker interaction with 100-349AA residues. On the right colonies were plated onto media containing histidine while media lacking His is on the left

Based on the above results, it could be concluded that N-N interaction was not important while other portions of the protein seemed to be important for assembly. Further, prior interaction with viral RNA might be a necessity for initiation of capsid assembly.

Carnation 

Continuing development of diagnostics for Carnation etched ring virus (CERV), polyclonal antiserum was raised against in vitro expressed and purified coat protein (CP). In view of its efficacy and efficiency, it was purified, tagged and formulated as a diagnostic kit

Investigation on self-interaction of the CP of CERV infecting carnations was initiated. The CP gene was characterized at the sequence level by PCR amplification using indigenously designed primers. The gene was cloned and sequenced (1485 bp). Both gene and protein showed >96% similarity to reported sequences in database. From the deduced sequence, primers were designed to amplify complete, N & C-terminals and middle portions of the CP gene for expression in Saccharomyces cerevisae strain AH109. Cloning of the amplified products inframe to the activation and binding domain (AD and BD) is being studied.         

Chrysanthemum

Studies on Tomato aspermy virus coat protein gene variability.

Thirty three chrysanthemum samples were collected during 2002-2005 from different locations of India to screen for Tomato aspermy virus (TAV). ELISA and hybridization assays detected infection in 18 samples (54.54%). RT-PCR with designed primers amplified the entire CP gene (657 bp) of TAV from all positive samples. The CP gene sequences exhibited an overall nucleotide homology of 87- 100%, while deduced amino acid homology ranged between 80-100%. Phylogenetic analysis revealed two major groups of TAV, one minor group of two isolates (B and P) and an Indian isolate As with an extra origin. Recombination was also observed at various regions of CP gene of different TAV isolates. The Indian isolates Ut, Del, AP and Har contained unique sequences with no recombination event.

MOLECULAR CHARACTERIZATION OF A CARLAVIRUS INFECTING CHRYSANTHEMUMS IN INDIA

(Funded by Department of Biotechnology, New Delhi)

CPs of 31 CVB isolates were cloned and sequenced. The sequences were analyzed by multalin program for similarity and variability at nucleotide and protein level. A high level of homology (> 90%) was observed at protein level. The isolates in general formed five groups. Group I  composed of isolates from Uttarkashi (UK), Orissa (Or) and Chamba (HP2), group II  from Leh (JK3), Jammu (JK1), Arunachal Pradesh (ArP), Tamilnadu (TN), Sikkim (SK) and Maharashtra (MH), group III  from Dehradun (UA1), Lucknow (UP), Srinagar (JK2), Delhi (DL) and Calcutta (WB1), group IV from Rajasthan (RJ), Bihar (BR) and Palampur (HP1) and group V from Jharkhand (JH), Siligudi (WB2), Haryana (HR), Guwahati (AS), Gwalior (MP), Hyderabad (AP), Ludhiana (PB), Gujarat (GJ), Chandigarh (CH), Banglore (KK) and Chail (HP3). Isolates from UP, UA1, ArP, TN, and UK were distinct among the groups. Coat protein gene of a number of isolates viz. CHH, HSR, JK3, JK2, UA1, MP, SK, PB, BR, and MH showed the presence of 36 recombination events. Most of the recombination events occurred throughout the gene but concentrated on the 5' and 3'-end and was maximum in the UA1 isolate with thirteen events. Apart from CP gene, a replicase gene of 4.5 kb was sequenced for the first time.

Prunus necrotic ring spot virus (PNRSV)

Coat protein gene sequence showed 97-99% amino acid homology with other PNRSV isolates. It was concluded that rose and geranium are natural hosts of this virus.

CMV was identified and characterized on geranium grown in a greenhouse at IHBT on the basis of host range, aphid transmission, ELISA, DNA-RNA hybridization and RT-PCR. A complete CP gene was amplified using degenerate primers and sequenced. The gene showed nucleotide and amino acid homology of 97-98% and 96-99%, respectively with the sequences of CMV subgroup II. Also, the gene  showed 75-97% and 77-96% homology at nucleotide and amino acid level, respectively with the CMV Indian isolates infecting various crops. The sequence homologies inferred that CMV-infecting geraniums in India belonged to subgroup II.

Phylogenetic relationship of CMV-Gera with the strains of CMV subgroup I (A and B), II and Indian strains based on the amino acid alignment using ClustalW version 1.82 through TreeExplorer. Tomato aspermy virus (TAV) (Acc. No. AJ550020) was used as an outgroup. The bootstrapping and branch length values are above and below the joining lines, respectively.