H. S. Savithri

Genetic variability of begomoviruses associated with cotton leaf curl disease originating from India

Summary.Cotton leaf curl disease (CLCuD) causing viruses belong to the Begomovirus genus of the family Geminiviridae. Most begomoviruses are bipartite with two molecules of circular single stranded DNA (A and B) encapsidated in icosahedral geminate particles. However, the begomoviruses associated with CLCuD have DNA-β instead of DNA-B. In this communication we report the complete genomic sequence of DNA-A component of two CLCuD-causing begomoviruses, cotton leaf curl Kokhran virus-Dabawali (CLCuKV-Dab), tomato leaf curl Bangalore virus-Cotton [Fatehabad] (ToLCBV-Cotton [Fat]) and partial sequences of two other isolates cotton leaf curl Rajasthan virus-Bangalore (CLCuRV-Ban) and cotton leaf curl Kokhran virus-Ganganagar (CLCuKV-Gang). A phylogenetic analysis of these isolates along with other related begomoviruses showed that ToLCBV-Cotton [Fat] isolate was closest to the tomato leaf curl Bangalore virus-5 (ToLCBV-Ban5) where as CLCuKV-Dab isolate was close to the cotton leaf curl Kokhran virus-Faisalabad1 (CLCuKV-Fai1), cotton leaf curl Kokhran virus-72b (CLCuKV-72b) and cotton leaf curl Kokhran virus-806b (CLCuKV-806b) isolates from Pakistan. The phylogenetic analysis further showed that the ToLCBV-Cotton [Fat] and CLCuKV-Dab isolates along with CLCuKV-Fai1, CLCuKV-72b and CLCuKV-806b are closer to the ToLCBV, tomato leaf curl Gujarat virus (ToLCGV), tomato leaf curl Gujarat virus-Varanasi (ToLCGV-Var) and tomato leaf curl Sri Lanka virus (ToLCSLV) isolates, where as cotton leaf curl Alabad virus-804a (CLCuAV-804a), cotton leaf curl Multhan virus (CLCuMV) cluster with the isolates from cotton leaf curl Rajasthan virus (CLCuRV) and okra yellow vein mosaic virus (OYVMV). These results demonstrate the extensive variability observed in this group of viruses. The AC4 ORF is the least conserved among these viruses. In order to further asses the variability in the CLCuD-causing begomoviruses, the region showing minimum similarity in the DNA-A sequence was first determined by a comparison of segments of different lengths of the aligned sequences. The results indicated that region 2411–424 (771 nt) was the least conserved. A phylogenetic tree constructed using the sequences of all the CLCuD causing begomoviruses, corresponding to the least conserved region showed that they form two distinct clusters.

Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India.

Summary. Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae. The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV-AP) was cloned and expressed in Escherichia coli. The recombinant coat protein was used to raise high quality antiserum. The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India. The sequence of the cloned PCR products encoding 3′untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz. Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV-AP. The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level. This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV. In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

Complete nucleotide sequence of Sesbania mosaic virus: a new virus species of the genus Sobemovirus.

Summary. The complete nucleotide sequence of the Sesbania mosaic virus (SeMV) genomic RNA was determined by sequencing overlapping cDNA clones. The SeMV genome is 4149 nucleotides in length and encodes four potential overlapping open reading frames (ORFs). Comparison of the nucleotide sequence and the deduced amino acid sequence of the four ORFs of SeMV with that of other sobemoviruses revealed that SeMV was closest to southern bean mosaic virus Arkansas isolate (SBMV-Ark, 73% identity). The 5′ non-coding regions of SeMV, SBMV and southern cowpea mosaic virus (SCPMV) are nearly identical. However ORF1 of SeMV which encodes for a putative movement protein of Mr 18370 has only 34% identity with SBMV-Ark. ORF 2 encodes a polyprotein containing the serine protease, genome linked viral protein (VPg) and RNA dependent RNA polymerase domains and shows 78% identity with SBMV-Ark. The N-terminal amino acid sequence of VPg was found to be TLPPELSIIEIP, which mapped to the region 326–337 of ORF2 product and the cleavage site between the protease domain and VPg was identified to be E325-T326. The cleavage site between VPg and RNA dependent RNA polymerase was predicted to be E445-T446 based on the amino acid sequence analysis of the polyprotein from different sobemoviruses. ORF3 is nested within ORF2 in a − 1 reading frame. The potential ribosomal frame shift signal and the downstream stem-loop structure found in other sobemoviruses are also conserved in SeMV RNA sequence, indicating that ORF3 might be expressed via − 1 frame shifting mechanism. ORF4 encodes the coat protein of SeMV, which shows 76 and 66% identity with SBMV-Ark and SCPMV, respectively. Thus the comparison of the non-coding regions and the ORFs of SeMV with other sobemoviruses clearly revealed that it is not a strain of SBMV. Phylogenetic analysis of six different sobemoviruses, including SeMV, suggests that recombination event is not frequent in this group and that SeMV is a distinct member of the genus sobemovirus. The analysis also shows sobemoviruses infecting monocotyledons and dicotyledons fall into two distinct clusters.

Molecular characterization and interviral relationships of a flexuous filamentous virus causing mosaic disease of sugarcane (Saccharum officinarum L.) in India.

SummaryA virus isolate causing mosaic disease of commercial sugarcane was purified to homogeneity. Electron microscopy revealed flexuous filamentous virus particles of ca 890 × 15 nm. The virus isolate reacted positively with heterologous antiserum to narcissus latent virus form UK, but failed to react with potyvirus group specific antiserum. N-terminal sequencing of the intact coat protein (CP) and the tryptic peptides indicated that the virus was probably a potyvirus but distinct from several reported potyviruses. Comparison of the 3′-terminal 1084 nucleotide sequence of the RNA genome of this virus revealed 93.6% sequence identity in the coat protein coding region with the recently described sugarcane streak mosaic virus (Pakistani isolate). The molecular weight of the coat protein (40 kDa) was higher than that deduced from the amino acid sequence (34 kDa). The apparent increase in size was shown to be due to glycosylation of the coat protein which has not been reported thus far in the family, Potyviridae. This is the first report on the molecular characterization of a virus causing mosaic disease of sugarcane in India and the results demonstrate that the virus is a strain of sugarcane streak mosaic virus, a member of the Tritimovirus genus of the Potyviridae. We have named it sugarcane streak mosaic virus – Andhra Pradesh isolate (SCSMV-AP).

Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate

In Escherichia coli and Salmonella typhimurium, L-threonine is cleaved non-oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or pyruvate formate-lyase), phosphotransacetylase and propionate kinase. In the anaerobic condition, L-threonine is converted to the energy-rich keto acid and this is subsequently catabolised to produce ATP via substrate-level phosphorylation, providing a source of energy to the cells. Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by the anaerobically regulated tdc operon. In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups. Besides detailed structural and functional insights, these studies have also shown the similarities and differences between the other related enzymes present in the metabolic network. In this paper, we review the structural and biochemical studies carried out on these enzymes.

Taxonomic position of sugarcane streak mosaic virus in the family Potyviridae

Summary. A cDNA library was generated from purified RNA of sugarcane streak mosaic virus – Andhra Pradesh (SCSMV-AP). Two overlapping clones covering 3160 nucleotides encoding partial CI, complete 6K2, VPg-NIa and NIb genes were sequenced. A comparison of this sequence along with the 3′ terminal 1315 nucleotides of SCSMV-AP determined earlier with the other members of the family Potyviridae indicated that it had only 30% identity at the amino acid level for the partial polyprotein open reading frame (ORF) with the members of Ipomovirus and Tritimovirus genera. Further, in the most conserved NIb region also there was only 40% identity with the type members of these genera. Based on this analysis, we suggest the taxonomic affiliation of SCSMV-AP into an undescribed new genus in the family Potyviridae.

Surface-exposed amino- and carboxy-terminal residues are crucial for the initiation of assembly in Pepper vein banding virus: a flexuous rod-shaped virus.

The mechanism of assembly of flexuous viruses, such as potyviruses, is poorly understood. Using a recombinant system, we provide evidence that disassembly and reassembly of Pepper vein banding virus (PVBV), a member of the genus potyvirus, proceeds via a ring-like intermediate, and show that electrostatic interactions may be pivotal in stabilizing the particles. Although the surface-exposed N- and C-terminal residues can be removed from the virus-like particles (VLPs) by limited trypsinization without affecting their stability, such truncated CP subunits are unable to form VLPs. To further evaluate importance of these residues, N- and C-terminal deletion mutants were generated and their assembly behavior was investigated. N-terminal 53 and C-terminal 23 amino acids were found to be crucial for the intersubunit interactions involved in the initiation of virus assembly. These segments are surface exposed in the ring-like intermediate and dispensable for further interactions that result in the formation of the VLPs.

Processing of SeMV polyproteins revisited

Processing of Sesbania mosaic virus (SeMV) polyprotein 2a and 2ab was reanalyzed in the view of the new genome organization of sobemoviruses. Polyprotein 2a when expressed in E. coli, from the new cDNA clone, got cleaved at the earlier identified sites E325-T326, E402-T403 and E498-S499 to release protease, VPg, P10 and P8, respectively. Additionally, a novel cleavage was identified within the protease domain at position E132-S133, which was found to be essential for efficient polyprotein processing. Products, corresponding to cleavages identified in E. coli, were also detected in infected Sesbania leaves. Interestingly, though the sites are exactly the same in polyprotein 2ab, it got cleaved between Protease-VPg but not between VPg-RdRp. This indicates to a differential cleavage preference, governed probably by the conformation of 2ab. Also, the studies revealed that, in SeMV, processing is regulated by mode of cleavage and context of the cleavage site.

Determination of 3′-terminal nucleotide sequence of pepper\break vein banding virus RNA and expression of its coat protein in Escherichia coli

Summary. Pepper vein banding virus (PVBV) is an important virus infecting chilli pepper in south India. Earlier reports suggested it to be a distinct potyvirus. The nucleotide sequence of PVBV RNA from the 3′-end (3862 nt) was determined. Analysis of the nucleotide and deduced amino acid sequence revealed that it encompasses a partial open reading frame encoding the partial sequence of VPg, NIa-protease, NIb, coat protein (CP) and 3′-untranslated region (UTR). Comparison of the amino acid sequence of CP and the nucleotide sequence of 3′-UTR with those of other potyviruses confirmed an earlier observation that PVBV is a distinct member of the Potyvirus sub-group and it had significant similarity to a recently characterized virus infecting chilli pepper, chilli vein-banding mottle virus (CVbMV), from Thailand. The analysis showed that both PVBV and CVbMV might represent strains of the same virus. Further, the PVBV CP gene was overexpressed in E. coli, which assembled into potyvirus-like particles (PVLPs). The assembled particles were shown to encapsidate the CP mRNA.

Complete genomic sequence of Pepper vein banding virus (PVBV): a distinct member of the genus Potyvirus

Summary.The complete genomic sequence of Pepper vein banding virus (PVBV), a potyvirus infecting chilli and other solanaceous plants in south India, was determined and compared with those of other potyviruses. The viral genome contained 9711 nucleotides, excluding the poly-A tail. The length of the 5′- and 3′-untranslated regions (UTR) were 163 and 281 nucleotides respectively. As for other potyviruses, the PVBV genome has a single open reading frame (ORF) starting at nucleotide 164 and ending at nt 9430, which encodes a polyprotein of 3088 amino acid residues. There are nine putative conserved cleavage sites within the polyprotein, which can result in ten functionally distinct protein products. Phylogenetic analysis of the potyviral polyprotein sequences showed that PVBV is a distinct species of this genus.