Cassava Brown Streak Viruses express second 6-kilodalton (6K2) protein with varied polarity and three dimensional (3D) structures: Basis for trait discrepancy between the virus species.

Cassava Brown Streak Virus (CBSV) and Ugandan Cassava Brown Streak Virus (UCBSV) are the two among six virus species speculated to cause the most catastrophic Brown Streak Disease of Cassava (CBSD) in Africa and Asia. Cassava Brown Streak Virus (CBSV) is hard to breed resistance for compared to Ugandan Cassava Brown Streak Virus (UCBSV) species. This is exemplified by incidences of CBSV species rather than UCBSV species in elite breeding line, KBH 2006/0026 at Bagamoyo, Tanzania. It is not yet understood as to why CBSV species could breakdown CBSD-resistance in the KBH 2006/0026 unlike the UCBSV species. This marks the first in silico study conducted to understand molecular basis for the trait discrepancy between CBSV and UCBSV species from structural biology view point. Following ab initio modelling and analysis of physical-chemical properties of second 6-kilodalton (6K2) protein encoded by CBSV and UCBSV species, using ROBETTA server and Protein Parameters tool, respectively we report that; three dimensional (3D) structures and polarity of the protein differs significantly between the two virus species. (95% and 5%) and (85% and 15%) strains of 20 CBSV and 20 UCBSV species respectively, expressed the protein in homo-trimeric and homo-tetrameric forms, correspondingly. 95% and 85% of studied strain population of the two virus species expressed hydrophilic and hydrophobic 6K2, respectively. Based on findings of the curent study, we hypothesize that; (i) The hydrophilic 6K2 expressed by the CBSV species, favour its faster systemic movement via vascular tissues of cassava host and hence result into higher tissue titres than the UCBSV species encoding hydrophobic form of the protein. t and (ii) The hydrophilic 6K2 expressed byCBSV species have additional interaction advantage with Nuclear Inclusion b protease domain (NIb) and Viral genome-linked protein (VPg), components of Virus Replication Complex (VRC) and hence contributing to faster replication of viral genome than the hydrophobic 6K2 expressed by the UCBSV species. Experimental studies are needed to resolve the 3D structures of the 6K2, VPg and NIb and comprehend complex molecular interactions between them. We suggest that, the 6K2 gene should be targeted for improvement of RNA interference (RNAi)-directed transgenesis of virus-resistant cassava as a more effective way to control the CBSD besides breeding.

Wheat streak mosaic virus coat protein is a determinant for vector transmission by the wheat curl mite.

Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae), is transmitted by the wheat curl mite (Aceria tosichella Keifer). The requirement of coat protein (CP) for WSMV transmission by the wheat curl mite was examined using a series of viable deletion and point mutations. Mite transmission of WSMV was completely abolished with deletions comprising CP amino acids 58-100. In contrast, the amino-proximal (amino acids 6-27 and 36-57) and carboxy-terminal (14 amino acids) regions of CP were expendable for mite transmission. Mutation of aspartic acid residues at amino acid positions 289 or 326 (D289A or D326A) at the carboxy-proximal region of CP significantly reduced mite transmission. Remarkably, every wheat plant infected by mutants D289A or D326A through mite transmission but not with in vitro transcripts contained a second-site mutation of R131C and N275H, respectively. Collectively, these data demonstrate for the first time that CP is a determinant for an eriophyid-transmitted plant virus.

The Potyviridae P1a leader protease contributes to host range specificity.

The P1a protein of the ipomovirus Cucumber vein yellowing virus is one of the self-cleavage serine proteases present in Potyviridae family members. P1a is located at the N-terminal end of the viral polyprotein, and is closely related to potyviral P1 protease. For its proteolytic activity, P1a requires a still unknown host factor; this might be linked to involvement in host specificity. Here we built a series of constructs and chimeric viruses to help elucidate the role of P1a cleavage in host range definition. We demonstrate that host-dependent separation of P1a from the remainder of the polyprotein is essential for suppressing RNA silencing defenses and for efficient viral infection. These findings support the role of viral proteases as important determinants in host adaptation.

Wheat streak mosaic virus infects systemically despite extensive coat protein deletions: identification of virion assembly and cell-to-cell movement determinants.

Viral coat proteins function in virion assembly and virus biology in a tightly coordinated manner with a role for virtually every amino acid. In this study, we demonstrated that the coat protein (CP) of Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) is unusually tolerant of extensive deletions, with continued virion assembly and/or systemic infection found after extensive deletions are made. A series of deletion and point mutations was created in the CP cistron of wild-type and/or green fluorescent protein-tagged WSMV, and the effects of these mutations on cell-to-cell and systemic transport and virion assembly of WSMV were examined. Mutants with overlapping deletions comprising N-terminal amino acids 6 to 27, 36 to 84, 85 to 100, 48 to 100, and 36 to 100 or the C-terminal 14 or 17 amino acids systemically infected wheat with different efficiencies. However, mutation of conserved amino acids in the core domain, which may be involved in a salt bridge, abolished virion assembly and cell-to-cell movement. N-terminal amino acids 6 to 27 and 85 to 100 are required for efficient virion assembly and cell-to-cell movement, while the C-terminal 65 amino acids are dispensable for virion assembly but are required for cell-to-cell movement, suggesting that the C terminus of CP functions as a dedicated cell-to-cell movement determinant. In contrast, amino acids 36 to 84 are expendable, with their deletion causing no obvious effects on systemic infection or virion assembly. In total, 152 amino acids (amino acids 6 to 27 and 36 to 100 and the 65 amino acids at the C-terminal end) of 349 amino acids of CP are dispensable for systemic infection and/or virion assembly, which is rare for multifunctional viral CPs.

Mechanistic divergence between P1 proteases of the family Potyviridae.

P1a and P1b are two serine proteases of Cucumber vein yellowing virus (an ipomovirus). They belong to the group of P1 factors present at the N terminus of the polyproteins of most members of the family Potyviridae. The present work compares the protease activities of P1a and P1b in different experimental systems. The findings made regarding how these two proteases work, such as the requirement for a host factor by P1a but not by P1b, underscore important differences in their catalytic activity that point towards their undergoing divergent evolution involving the acquisition of mechanistic variations. The expression of several truncated forms of P1b in bacteria and in planta helped define the protease domain of P1b, along with other important features such as its apparently in cis mode of action. Recent phylogenetic data, together with the present results, allow an appealing hypothesis to be proposed regarding P1 evolution and its involvement in potyvirid speciation.

Characterization and genetic diversity of sugarcane streak mosaic virus causing mosaic in sugarcane.

Sixty-three sugarcane leaf samples were collected from fifty-eight sugarcane varieties, evolved from eleven major sugarcane growing states in India, Australia, South Africa and USA. In RT-PCR, using gene specific primers for sugarcane streak mosaic virus (SCSMV)-CP, 58 of 63 sugarcane samples were found positive to the virus infection and rest of the five samples were negative. Partial CP gene sequences of 42 SCSMV isolates including an isolate from aphid colony (Melanaphis indosacchari) infested on sugarcane variety from this study were characterized after cloning and sequencing for selective isolates represented by at least one isolate from each location. The new sequences identified in the study were named as SCSMV-CB isolates. Fifty two sequences including the 10 database sequences (complete CP cds) deposited earlier from this institute were compared with each other as well as GenBank database sequences of Potyviridae members viz., Rymovirus, Potyvirus, Ipomovirus, Tritimovirus and eight sequences of SCSMV reported from elsewhere. Among the SCSMV-CB isolates sequenced in the study, 85.7-100% (nucleotide) and 89.9-100% (amino acid) sequence identities were observed and with the other data base sequences of SCSMV, the respective identities were 82.2-97.5 and 89.7-98.6%. Grouping of the isolates by the maximum likelihood with molecular clock model, distributed 60 SCSMV sequences including the eight database sequences deposited by other SCSMV working groups from India and USA in 16 different phylogenetic groups. Although the isolates of SCSMV were relatively close to Ipomovirus and Tritimovirus, they were sandwiched between Rymovirus and Ipomovirus. The sequence comparison and phylogenetic studies revealed that the relatedness of SCSMV with the potyviral related genera was comparatively low to consider it as a member of earlier described potyviral genera, hence the genus "Susmovirus" (sugarcane streak mosaic virus) has been proposed, with SCSMV as the sole species to be included. The 52 SCSMV-CB isolates from this institute were distributed in 14 phylogenetic groups and the grouping pattern revealed that the virus isolates could not be grouped based on geographical origin of the host varieties or longevity of the host variety.

Recombination and gene duplication in the evolutionary diversification of P1 proteins in the family Potyviridae.

Genome structure and sequence are notably conserved between members of the family Potyviridae. However, some genomic regions of these viruses, such as that encoding the P1 protein, show strikingly high variability. In this study, some partially conserved motifs were identified upstream of the quite well-conserved protease domain located near the P1 C terminus. The irregular distribution of these motifs suggests that the potyviral P1 proteins have undergone complex evolutionary diversification. Evidence was found of recombination events in the P1 N-terminal region, similar to those reported in potyviruses of the bean common mosaic virus subgroup, but also affecting other potyviruses. Moreover, intergeneric recombination events affecting potyviruses and ipomoviruses were also observed. Evidence that these recombination events could be linked to host adaptation is provided. Specific sequence features and differences in net charge help to classify the P1 proteins of members of the family Potyviridae into two groups: those from potyviruses and rymoviruses and those from tritimoviruses. The ipomovirus Cucumber vein yellowing virus has two P1 copies arranged in tandem, the most N-terminal one being of the potyvirus type and the other being of the tritimovirus type. These findings suggest that both recombination and gene duplication have contributed to P1 evolution and helped to facilitate successful adaptation of members of the family Potyviridae to a wide range of host species.