Muhammad Shah Nawaz-ul-Rehman

Evolution of geminiviruses and their satellites.

Geminiviruses and their satellites have circular single stranded DNA genomes, infecting many crops and weeds across the globe. To successfully invade new hosts, break host resistance, move virus particles within and between plants, geminiviruses and their satellites have evolved a coordinated network of protein interactions, showing a possible evolutionary path. Humans have played an important role in the last century to promote the emergence of many geminivirus diseases, thereby impacting their evolution. The greatest molecular diversity of geminiviruses and their satellites resides in Southeast Asia revealing a possible center of origin. This minireview leads us to a possible general grand scheme of their evolution.

Post-transcriptional gene silencing suppressor activity of two non-pathogenic alphasatellites associated with a begomovirus.

Alphasatellites and betasatellites are begomovirus-associated single-stranded circular DNA molecules. Two distinct alphasatellites, Gossypium darwinii symptomless alphasatellite and Gossypium mustelinium symptomless alphasatellite, were previously isolated from Gossypium davidsonii and G.mustelinium. Here we show that the replication-associated proteins (Rep: a rolling-circle replication initiator protein) encoded by these alphasatellites interact with the Rep and C4 proteins encoded by their helper begomovirus, Cotton leaf curl Rajasthan virus (CLCuRaV), in a yeast two-hybrid assay. Both the alphasatellite-encoded Reps were found to have strong gene silencing suppressor activity, in contrast to the betasatellite-encoded betaC1 and CLCuRaV-encoded C2, C4 and V2 proteins. The presence of alphasatellites maintained suppression of gene silencing in the youngest, actively growing tissue of CLCuRaV-betasatellite-infected plants. This is the first demonstration of a rolling-circle replication initiator protein with suppressor of gene silencing activity and provides a possible explanation for the selective advantage provided by the association of alphasatellites with begomovirus-betasatellite complexes.

Maintenance of an Old World Betasatellite by a New World Helper Begomovirus and Possible Rapid Adaptation of the Betasatellite

ABSTRACT Begomoviruses (family Geminiviridae) cause major losses to crops throughout the tropical regions of the world. Begomoviruses originating from the New World (NW) and the Old World (OW) are genetically distinct. Whereas the majority of OW begomoviruses have monopartite genomes and whereas most of these associate with a class of symptom-modulating satellites (known as betasatellites), the genomes of NW begomoviruses are exclusively bipartite and do not associate with satellites. Here, we show for the first time that a betasatellite (cotton leaf curl Multan betasatellite [CLCuMuB]) associated with a serious disease of cotton across southern Asia is capable of interacting with a NW begomovirus. In the presence of CLCuMuB, the symptoms of the NW cabbage leaf curl virus (CbLCuV) are enhanced in Nicotiana benthamiana. However, CbLCuV was unable to interact with a second betasatellite, chili leaf curl betasatellite. Although CbLCuV can transreplicate CLCuMuB, satellite accumulation levels in plants were low. However, progeny CLCuMuB isolated after just one round of infection with CbLCuV contained numerous mutations. Reinoculation of one such progeny CLCuMuB with CbLCuV to N. benthamiana yielded infections with significantly higher satellite DNA levels. This suggests that betasatellites can rapidly adapt for efficient transreplication by a new helper begomovirus, including begomoviruses originating from the NW. Although the precise mechanism of transreplication of betasatellites by begomoviruses remains unknown, an analysis of betasatellite mutants suggests that the sequence(s) required for maintenance of CLCuMuB by one of its cognate begomoviruses (cotton leaf curl Rajasthan virus) differs from the sequences required for maintenance by CbLCuV. The significance of these findings and, particularly, the threat that betasatellites pose to agriculture in the NW, are discussed.

Proteome-Wide Overexpression of Host Proteins for Identification of Factors Affecting Tombusvirus RNA Replication: an Inhibitory Role of Protein Kinase C

ABSTRACT To identify host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model positive-stranded RNA virus, we overexpressed 5,500 yeast proteins individually in Saccharomyces cerevisiae, which supports TBSV replication. In total, we identified 141 host proteins, and overexpression of 40 of those increased and the remainder decreased the accumulation of a TBSV replicon RNA. Interestingly, 36 yeast proteins were identified previously by various screens, greatly strengthening the relevance of these host proteins in TBSV replication. To validate the results from the screen, we studied the effect of protein kinase C1 (Pkc1), a conserved host kinase involved in many cellular processes, which inhibited TBSV replication when overexpressed. Using a temperature-sensitive mutant of Pkc1p revealed a high level of TBSV replication at a semipermissive temperature, further supporting the idea that Pkc1p is an inhibitor of TBSV RNA replication. A direct inhibitory effect of Pkc1p was shown in a cell-free yeast extract-based TBSV replication assay, in which Pkc1p likely phosphorylates viral replication proteins, decreasing their abilities to bind to the viral RNA. We also show that cercosporamide, a specific inhibitor of Pkc-like kinases, leads to increased TBSV replication in yeast, in plant single cells, and in whole plants, suggesting that Pkc-related pathways are potent inhibitors of TBSV in several hosts.

Yeast screens for host factors in positive-strand RNA virus replication based on a library of temperature-sensitive mutants

RNA viruses exploit host cells by altering cellular pathways, recruiting host factors, remodeling intracellular membranes and escaping host antiviral responses. Model hosts, such as Saccharomyces cerevisiae (yeast), are valuable to identify host factors involved in viral RNA replication. The many advantages of using yeast include the availability of various yeast mutant libraries, such as (i) single gene-deletion library; (ii) the essential gene library (yTHC); and (iii) the yeast ORF over-expression library. Here, we have used a novel temperature-sensitive (ts) mutant library of essential yeast genes to identify 118 host proteins affecting replication of Tomato bushy stunt virus, in yeast model host. Testing 787 ts mutants led to the identification of host factors, of which 72 proteins facilitated TBSV replication in yeast and 46 proteins were inhibitory. Altogether, ~85% of the identified proteins are novel host factors affecting tombusvirus replication. The ts mutant library screen also led to the identification of 17 essential genes, which have been documented before, thus confirming the importance of these genomic screens. Overall, we show the power of ts mutant library in identification of host factors for RNA virus replication.

A Melting Pot of Old World Begomoviruses and Their Satellites Infecting a Collection of Gossypium Species in Pakistan

CLCuD in southern Asia is caused by a complex of multiple begomoviruses (whitefly transmitted, single-stranded [ss]DNA viruses) in association with a specific ssDNA satellite; Cotton leaf curl Multan betasatellite (CLCuMuB). A further single ssDNA molecule, for which the collective name alphasatellites has been proposed, is also frequently associated with begomovirus-betasatellite complexes. Multan is in the center of the cotton growing area of Pakistan and has seen some of the worst problems caused by CLCuD. An exhaustive analysis of the diversity of begomoviruses and their satellites occurring in 15 Gossypium species (including G. hirsutum, the mainstay of Pakistans cotton production) that are maintained in an orchard in the vicinity of Multan has been conducted using φ29 DNA polymerase-mediated rolling-circle amplification, cloning and sequence analysis. The non-cultivated Gossypium species, including non-symptomatic plants, were found to harbor a much greater diversity of begomoviruses and satellites than found in the cultivated G. hirsutum. Furthermore an African cassava mosaic virus (a virus previously only identified in Africa) DNA-A component and a Jatropha curcas mosaic virus (a virus occurring only in southern India) DNA-B component were identified. Consistent with earlier studies of cotton in southern Asia, only a single species of betasatellite, CLCuMuB, was identified. The diversity of alphasatellites was much greater, with many previously unknown species, in the non-cultivated cotton species than in G. hirsutum. Inoculation of newly identified components showed them to be competent for symptomatic infection of Nicotiana benthamiana plants. The significance of the findings with respect to our understanding of the role of host selection in virus diversity in crops and the geographical spread of viruses by human activity are discussed.

Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly.

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions.

A High‐Throughput Approach for Studying Virus Replication in Yeast

Viruses are intracellular pathogens that are dependent on viral and host factors for multiplication. Model hosts, such as yeast, can be very valuable in identifying host factors involved in viral replication. Yeast is also useful for studies on functional interactions of host factors with viral proteins and/or virus nucleic acids. The advantages of using yeast include the availability of a single gene–deletion library and the essential gene library (yTHC); the controllable small‐ or large‐scale expression of viral proteins and nucleic acids; and the rapid growth of yeast strains. Procedures that facilitate high‐throughput analysis of host factors and plant and animal RNA virus replication in yeast, with a plant virus (tombusvirus; TBSV) and an animal virus (nodavirus; FHV) as examples, are described. Curr. Protoc. Microbiol. 19:16J.1.1‐16J.1.15.

Diversity, Mutation and Recombination Analysis of Cotton Leaf Curl Geminiviruses

The spread of cotton leaf curl disease in China, India and Pakistan is a recent phenomenon. Analysis of available sequence data determined that there is a substantial diversity of cotton-infecting geminiviruses in Pakistan. Phylogenetic analyses indicated that recombination between two major groups of viruses, cotton leaf curl Multan virus (CLCuMuV) and cotton leaf curl Kokhran virus (CLCuKoV), led to the emergence of several new viruses. Recombination detection programs and phylogenetic analyses showed that CLCuMuV and CLCuKoV are highly recombinant viruses. Indeed, CLCuKoV appeared to be a major donor virus for the coat protein (CP) gene, while CLCuMuV donated the Rep gene in the majority of recombination events. Using recombination free nucleotide datasets the substitution rates for CP and Rep genes were determined. We inferred similar nucleotide substitution rates for the CLCuMuV-Rep gene (4.96X10-4) and CLCuKoV-CP gene (2.706X10-4), whereas relatively higher substitution rates were observed for CLCuMuV-CP and CLCuKoV-Rep genes. The combination of sequences with equal and relatively low substitution rates, seemed to result in the emergence of viral isolates that caused epidemics in Pakistan and India. Our findings also suggest that CLCuMuV is spreading at an alarming rate, which can potentially be a threat to cotton production in the Indian subcontinent.

Structure activity relationship (SAR) and quantitative structure activity relationship (QSAR) studies showed plant flavonoids as potential inhibitors of dengue NS2B-NS3 protease

BackgroundDue to dengue virus disease, half of the world population is at severe health risk. Viral encoded NS2B-NS3 protease complex causes cleavage in the nonstructural region of the viral polyprotein. The cleavage is essentially required for fully functional viral protein. It has already been reported that if function of NS2B-NS3 complex is disrupted, viral replication is inhibited. Therefore, the NS2B-NS3 is a well-characterized target for designing antiviral drug.ResultsIn this study docking analysis was performed with active site of dengue NS2B-NS3 protein with selected plant flavonoids. More than 100 flavonoids were used for docking analysis. On the basis of docking results 10 flavonoids might be considered as the best inhibitors of NS2B-NS3 protein. The interaction studies showed resilient interactions between ligand and receptor atoms. Furthermore, QSAR and SAR studies were conducted on the basis of NS2B-NS3 protease complex docking results. The value of correlation coefficient (r) 0.95 shows that there was a good correlation between flavonoid structures and selected properties.ConclusionWe hereby suggest that plant flavonoids could be used as potent inhibitors of dengue NS2B-NS3 protein and can be used as antiviral agents against dengue virus. Out of more than hundred plant flavonoids, ten flavonoid structures are presented in this study. On the basis of best docking results, QSAR and SAR studies were performed. These flavonoids can directly work as anti-dengue drug or with little modifications in their structures.