Role Of Coat Protein Research Articles

Investigating the Roles of Coat Protein and Triple Gene Block Proteins of Potato Mop-Top Virus Using a Heterologous Expression System.

Potato mop-top virus (PMTV) is an emerging viral pathogen that causes tuber necrosis in potatoes. PMTV is composed of three single-stranded RNA segments: RNA1 encodes RNA-dependent RNA polymerase, RNA2 contains the coat protein (CP), and RNA3 harbors a triple gene block (TGB 1, TGB2, and TGB3). CP plays a role in viral transmission, while TGB is known to facilitate cell-to-cell and long-distance systemic movement. The role of CP in symptom development, specifically in the presence of TGB genes, was investigated using potato virus X (PVX) as a delivery vehicle to express PMTV genes in the model plant Nicotiana benthamiana. Plants expressing individual genes showed mild symptoms that included leaf curling and crumpling. Interestingly, symptom severity varied among plants infected with three different combinations: CP with TGB1, CP with TGB2, and CP with TGB3. Notably, the combination of CP and TGB3 induced a hypersensitive response, accompanied by stunted growth and downward curling and crumpling. These results suggest the potential role of TGB co-expressed with CP in symptom development during PMTV infection. Additionally, this study demonstrates the use of the PVX-based expression system as a valuable platform for assessing the role of unknown genes in viral pathogenicity.

Functional insights into the role of C-terminal disordered domain of Sesbania mosaic virus RNA-dependent RNA polymerase and the coat protein in viral replication in vivo

The C-terminal disordered domain of sesbania mosaic virus (SeMV) RNA-dependent RNA polymerase (RdRp) interacts with the viral protein P10. The functional significance of this interaction in viral replication was examined by a comparative analysis of genomic and sub-genomic RNA levels (obtained by quantitative real time PCR) in the total RNA extracted from Cyamopsis plants agro-infiltrated with wild-type or mutant forms of SeMV infectious cDNA (icDNA). The sgRNA copy numbers were found to be significantly higher than those of gRNA in the wild-type icDNA transfected plants. Transfection of a mutant icDNA expressing an RdRp lacking the C-terminal disordered domain led to a drastic reduction in the copy numbers of both forms of viral RNA. This could be due to the loss of interaction between the disordered domain of RdRp and P10 and possibly other viral/host proteins that might be required for the assembly of viral replicase. The C-terminal disordered domain also harbours the motif E which is essential for the catalytic function of RdRp. Mutation of the conserved tyrosine within this motif in the full length icDNA resulted in complete inhibition of progeny RNA synthesis in the transfected plants confirming the importance of motif E in the polymerase function in vivo. The role of coat protein (CP) in viral infection was also investigated by agro-infiltration of a CP start codon mutant icDNA which suggested that CP is essential for the encapsidation of viral progeny RNAs at later stages of infection.

Wheat streak mosaic virus coat protein is a host-specific long-distance transport determinant in oat

Viral determinants involved in systemic infection of hosts by monocot-infecting plant viruses are poorly understood. Wheat streak mosaic virus (WSMV, genus Tritimovirus, family Potyviridae) exclusively infects monocotyledonous crops such as wheat, oat, barley, maize, triticale, and rye. Previously, we reported that WSMV CP amino acids 36–84 are expendable for systemic infection of wheat, maize, barley and rye. In this study, the role of coat protein (CP) in systemic infection of oat by WSMV was examined by using a series of viable deletion mutants. WSMV bearing deletions within or encompassing all of amino acids 36–57 efficiently infected oat, indicating that these amino acids are dispensable for systemic infection of oat. However, WSMV mutants lacking CP amino acids 58–84 or 85–100 failed to systemically infect oat. Furthermore, green fluorescent protein-tagged WSMV mutants lacking CP amino acids 58–100 elicited local foci in oat but failed to enter the vasculature. These data suggest that CP amino acids 58–100 are required for systemic infection of oat by WSMV by specifically facilitating virus long-distance transport in oat.

VESICULAR TRANSPORT. A structure of the COPI coat and the role of coat proteins in membrane vesicle assembly.

Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.

An ACAP1 coat complex acting in endocytic recycling.

A key function of coat proteins is the sorting of protein cargoes into intracellular transport pathways. For many years, however, it has been unclear whether this role of coat proteins would apply to pathways of endocytic recycling. This issue has been clarified in recent years through the identification of multiple coat complexes acting in the recycling pathways. Leading this charge have been studies on a coat complex defined by ACAP1 (adenosine diphosphate ribosylation factor GTPase-activating proteins with Coiled-coil, Ankryin repeat and PH domains 1), which acts in the sorting of cargoes at the recycling endosome for their return to the plasma membrane. This chapter describes the methods used to characterize this role of ACAP1.

A Sobemovirus coat protein gene complements long-distance movement of a coat protein-null Dianthovirus

Red clover necrotic mosaic virus (RCNMV; genus Dianthovirus) and Turnip rosette virus (TRoV; genus Sobemovirus) are taxonomically and ecologically distinct plant viruses. In addition, the two genera differ in the role of coat protein (CP) in cell-to-cell movement. However, both are small icosahedral viruses requiring CP for systemic movement in the host vasculature. Here, we show that the TRoV CP gene is capable of facilitating the vascular movement of a Dianthovirus. Substitution of the RCNMV CP gene with the TRoV CP gene permits movement of the resulting chimeric virus to non-inoculated leaves. RCNMV lacking a CP gene or containing a non-translatable TRoV CP gene do not move systemically. This report introduces the molecular characterization of TRoV and describes the unprecedented complementation of systemic movement function by intergenic complete substitution of a plant virus CP gene.

RNAs 1 and 2 of Alfalfa mosaic virus, expressed in transgenic plants, start to replicate only after infection of the plants with RNA 3.

RNAs 1 and 2 of the tripartite genome of Alfalfa mosaic virus (AMV) encode the two viral replicase subunits. Full-length DNA copies of RNAs 1 and 2 were used to transform tobacco plants (R12 lines). None of the transgenic lines showed resistance to AMV infection. In healthy R12 plants, the transcripts of the viral cDNAs were copied by the transgenic viral replicase into minus-strand RNAs but subsequent steps in replication were blocked. When the R12 plants were inoculated with AMV RNA 3, this block was lifted and the transgenic RNAs 1 and 2 were amplified by the transgenic replicase together with RNA 3. The transgenic expression of RNAs 1 and 2 largely circumvented the role of coat protein (CP) in the inoculum that is required for infection of nontransgenic plants. The results for the first time demonstrate the role of CP in AMV plus-strand RNA synthesis at the whole plant level.

A conformational switch at the 3' end of a plant virus RNA regulates viral replication.

3' untranslated regions of alfamo- and ilar-virus RNAs fold into a series of stem-loop structures to which the coat protein binds with high affinity. This binding plays a role in initiation of infection ('genome activation') and has been thought to substitute for a tRNA-like structure that is found at the 3' termini of related plant viruses. We propose the existence of an alternative conformation of the 3' ends of alfamo- and ilar-virus RNAs, including a pseudoknot. Based on (i) phylogenetic comparisons, (ii) in vivo and in vitro functional analyses of mutants in which the pseudoknot has been disrupted or restored by compensatory mutations, (iii) competition experiments between coat protein and viral replicase, and (iv) investigation of the effect of magnesium, we demonstrate that this pseudoknot is required for replication of alfalfa mosaic virus. This conformation resembles the tRNA-like structure of the related bromo- and cucumo-viruses. A low but specific interaction with yeast CCA-adding enzyme was found. The existence of two mutually exclusive conformations for the 3' termini of alfamo- and ilar-virus RNAs could enable the virus to switch from translation to replication and vice versa. The role of coat protein in this modulation and in genome activation is discussed.

Molecular Studies on Bromovirus Capsid Protein

To determine whether the role of coat protein (CP) in cell-to-cell movement of dicot-adapted cowpea chlorotic mottle bromovirus (CCMV) is distinct from that of monocot-adapted brome mosaic bromovirus (BMV), two reporter genes, β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP), were substituted for the CP in a biologically active clone of CCMV RNA3 (C3). Primary leaves ofNicotiana benthamiana, Chenopodium quinoa,and cowpea were co-inoculated with wild-type (wt) CCMV RNA 1 and -2 and either C3/ΔCP-GUS or C3/ΔCP-EGFP and analyzed for GUS activity or the presence of green fluorescence. The visual appearance of infections caused by GUS or EGFP variants indicated that, in CCMV, epidermal cell-to-cell movement can occur without a functional CP. By contrast, inoculation of MP defective variants of C3/ΔCP-GUS or C3/ΔCP-EGFP resulted in subliminal infections. Additional experiments examining the infectivity of wt BMV RNA 1 and -2 and a BMV RNA3 variant bearing the EGFP in the place of CP (B3/ΔCP-EGFP) confirmed previous observations that, unlike CCMV, epidermal cell-to-cell movement of BMV is dependent on the expression of a functional CP. Taken together, the results demonstrate that BMV and CCMV use different mechanisms for initial epidermal cell-to-cell spread, and the individual role played by the respective CP genes in this active process is discussed.

Subcellular Immunolocalization of the Coat Protein of Two Potexviruses in InfectedChenopodium quinoa

The production of coat protein is necessary for cell-to-cell transport of potexviruses in plants. To investigate the role of coat protein in the movement process, the intracellular distribution of coat protein in tissues infected with either of two potexviruses was studied using immunocytochemical procedures. We report that coat protein antigens are associated with the plasmodesmata of infected cells.

The role of coat proteins in the biosynthesis of secretory proteins

The biosynthesis of secretory proteins requires vesicle-mediated transport between the organelles of the secretory pathway. Biochemical and genetic analysis of the secretory pathway has identified two non-clathrin coats—COPI and COPII — that drive the formation of vesicles that mediate transport between the endoplasmic reticulum and the Golgi apparatus, and through the compartments of the Golgi. Recently, a molecular description of the subunits of these coats and the development of biochemical reagents to study their function has yielded new information on how these proteins share the task of organizing vesicle traffic early in the secretory pathway.

Targeting to the endoplasmic reticulum by dilysine motifs: role of coat proteins.

Targeting to the endoplasmic reticulum by dilysine motifs: role of coat proteins.

Association of TMV coat protein with chloroplast membranes in virus-infected leaves

The polypeptide composition of extracts of chloroplasts from tobacco leaves systemically infected with different strains of Tobacco Mosaic Virus (TMV) was analyzed by one- and two-dimensional gel electrophoresis. There were no changes in the protein profiles of chloroplasts from infected leaves when compared to control leaves except for the presence of coat protein (CP) of TMV, identified by immunoblotting. When protease-treated intact chloroplasts isolated on Percoll gradients were osmotically disrupted the CP could be detected in both stroma and membrane fractions. The majority of the CP associated with the thylakoid membranes (about 1-5% of the total thylakoid proteins) was in the form of free molecules while stroma contained aggregated or assembled CP (about 0.1% of the soluble proteins). Thylakoid-associated CP was insensitive to protease digestion unless the membranes were first treated with a detergent, indicating that the CP was embedded inside or otherwise complexed with the thylakoid membranes.Chloroplasts isolated from leaves infected with TMV-PV42, a symptomless strain, contained approximately 10-50 times less CP than did chloroplasts isolated from leaves bearing mosaic symptoms induced by other strains of TMV (U1, PV230 or PV39). A possible role of CP in symptom development is discussed.

The effect of inhibitors of protein biosynthesis on the infectivity of alfalfa mosaic virus: Role of coat protein

Cycloheximide, when present in the inoculum at a concentration of 50 μg/ml decreases the infectivity of nucleoproteins of strain 425 of alfalfa mosaic virus (AMV) by more than 90%. Infectivity of the nucleoproteins of the AMV strain yellow spot mosaic virus (YSMV) and the Strasbourg strain were much less sensitive to cycloheximide; at 50 μg/ml of the antibiotic 60–80% of the normal infectivity was found. However, when chloramphenicol and cycloheximide were given simultaneously, the infectivity of these strains was as much reduced as that of AMV 425 in the presence of cycloheximide alone. As was shown earlier, infectious RNA preparations consist of 4 RNA species, 3 large RNAs constituting the complete genome, and a small monocistronic RNA, the top component a RNA. When the latter is removed, the RNA preparation is no longer infectious. A mixture of bottom, middle, and top component b RNAs can be activated by the coat protein. The infectivity of the 4 RNAs from YSMV and AMV 425 was equally sensitive to cycloheximide. A combination of YSMV RNA activated by AMV 425 coat protein was as sensitive to cycloheximide as AMV 425 nucleoprotein. This suggests that the coat protein plays a role in the localization of translation, which is in accordance with the previous finding that sensitivity to cycloheximide is determined by the top component b RNA, which contains the genetic information for the coat protein.