Plant viral suppressors of RNA silencing

RNA silencing is an ancient eukaryotic pathway in which double stranded RNA (dsRNA) triggers destruction of related RNAs in the cell. Early studies in plants pointed to a role for this pathway as a defense against viruses. Most known plant viruses have RNA genomes and replicate via dsRNA intermediates, thereby serving as potent inducers of RNA silencing early in replication and as silencing targets later in infection. Because RNA silencing is an antiviral mechanism, it is not surprising that many plant viruses encode suppressors of RNA silencing. This review focuses on the currently known plant virus encoded suppressors of silencing and the functional assays used to identify these proteins. Because they interfere with silencing at different points in the pathway, these viral suppressors are powerful tools to help unravel the mechanism of RNA silencing in plants.     

Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase

The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn(2+) concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp.     

Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon

Launching the Beet yellows virus (BYV) minireplicon by agrobacterial delivery resulted in an unexpectedly low number of infected cells per inoculated leaf. This effect was due to a strong RNA silencing response in the agroinfiltrated leaves. Strikingly, ectopic co-expression of p21, a BYV RNA silencing suppressor, increased minireplicon infectivity by three orders of magnitude. Mutational analysis demonstrated that this effect correlates with suppressor activity of p21. Five diverse, heterologous viral suppressors were also active in this system, providing a useful approach for a dramatic, up to 10,000-fold, increase of the efficiency of agroinfection. The minireplicon agroinfection assay was also used to identify a new suppressor, a homolog of BYV p21, derived from Grapevine leafroll-associated virus-2. In addition, we report preliminary data on the suppressor activity of the p10 protein of Grapevine virus A and show that this protein belongs to a family of Zn-ribbon-containing proteins encoded by filamentous plant RNA viruses from three genera. The members of this family are predicted to have RNA silencing suppressor activity.     

Estimation of the functions of viral RNA silencing suppressors by apple latent spherical virus vector

Virus Genes - Apple latent spherical virus (ALSV) is a latent virus with wide host range of plant species. In the present study, we prepared ALSV vectors expressing RNA silencing suppressors (RSSs)...     

Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step

RNA silencing suppressors from different plant viruses are structurally diverse. In addition to inhibiting the antiviral silencing response to condition susceptibility, many suppressors are pathogenicity factors that cause disease or developmental abnormalities. Here, unrelated suppressors from multiple viruses were shown to inhibit microRNA (miRNA) activities and trigger an overlapping series of severe developmental defects in transgenic Arabidopsis thaliana. This suggests that interference with miRNA-directed processes may be a general feature contributing to pathogenicity of many viruses. A normally labile intermediate in the miRNA biogenesis/RNA-induced silencing complex (RISC) assembly pathway, miRNA*, accumulated specifically in the presence of suppressors (P1/HC-Pro, p21, or p19) that inhibited miRNA-guided cleavage of target mRNAs. Both p21 and p19, but not P1/HC-Pro, interacted with miRNA/miRNA* complexes and hairpin RNA-derived short interfering RNAs (siRNAs) in vivo. In addition, p21 bound to synthetic miRNA/miRNA* and siRNA duplexes in vitro. We propose that several different suppressors act by distinct mechanisms to inhibit the incorporation of small RNAs into active RISCs.     

Biochemical characterization of a recombinant SARS coronavirus nsp12 RNA-dependent RNA polymerase capable of copying viral RNA templates

The severe acute respiratory syndrome coronavirus (SARS-CoV) RNA genome is replicated by a virus-encoded RNA replicase, the key component of which is the nonstructural protein 12 (nsp12). In this report, we describe the biochemical properties of a full-length recombinant SARS-CoV nsp12 RNA-dependent RNA polymerase (RdRp) capable of copying viral RNA templates. The purified SARS-CoV nsp12 showed both primer-dependent and primer-independent RNA synthesis activities using homopolymeric RNA templates. The RdRp activity was strictly dependent on Mn²⁺. The nsp12 preferentially copied homopolymeric pyrimidine RNA templates in the absence of an added oligonucleotide primer. It was also able to initiate de novo RNA synthesis from the 3’-ends of both the plus- and minus-strand genome of SARS-CoV, using the 3’-terminal 36- and 37-nt RNA, respectively. The in vitro RdRp assay system established with a full-length nsp12 will be useful for understanding the mechanisms of coronavirus replication and for the development of anti-SARS-CoV agents.     

De novo initiation of viral RNA-dependent RNA synthesis

RNA viruses use several initiation strategies to ensure that their RNAs are synthesized in appropriate amounts, have correct termini, and can be translated efficiently. Many viruses with genomes of single-stranded positive-, negative-, and double-stranded RNA initiate RNA synthesis by a de novo (primer-independent) mechanism. This review summarizes biochemical features and variations of de novo initiation in viral RNA replication.     

No viral gene is able to elicit RNA-dependent RNA polymerase in cucumber mosaic virus-infected cucumber cotyledons

Infection of cucumber cotyledons by cucumber mosaic virus, a divided genome virus, leads to the early appearance of considerable RNA-dependent RNA polymerase activity. When the genome components were tested separately for this effect, no single one was found active, and different combinations of two components caused varying lesser stimulations than when the three components necessary for infectivity were used. These intermediate effects paralleled the infectivities of these two-component mixtures. It appears probable that the stimulation of enzyme production is the consequence of viral infection, rather than being attributable to one or more viral genes.     

Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase

RNA-dependent RNA polymerase (RdRp) was solubilized from cellular membranes of brome mosaic virus (BMV)-infected barley. The solubilized enzyme was subsequently purified by glycerol gradient centrifugation and DEAE ion-exchange chromatography. The purified enzyme proved to be highly stable and both dependent on and specific for BMV RNAs. The enzyme is inhibited by high template RNA concentrations. This inhibition indicates feedback regulation of minus-strand synthesis. The nonstructural viral protein P1 was found to be a component of the RdRp complex (R. Quadt, H.J.M. Verbeek, and E.M.J. Jaspars, 1988, Virology 165, 256-261). Using antibodies directed against a C-terminal peptide of P1 a complex of seven 125I-labeled proteins was precipitated. This indicates that the P1 protein is associated with at least six proteins in the infected cell.     

Enhanced resistance and neutralization of defense responses by suppressors of RNA silencing

The effects of transgenic expression of the potato virus Y (PVY) HCPro silencing suppressor in tobacco were examined on infection by several viruses. Infection by tobacco mosaic virus (TMV) was reduced at 25 °C, but not at 33 °C. By contrast, systemic infection at 33 °C by the TMV expressing green fluorescent protein was promoted by the HCPro. Infection by tobacco rattle virus (TRV) was restricted to local necrotic lesions by the PVY HCPro. However, this resistance was neutralized by expression of the cucumber mosaic virus (CMV) 2b protein from TRV. By contrast, infection by either wild-type CMV or CMV with a deletion of the 2b gene was not affected. Similarly, infection by cauliflower mosaic virus, red clover necrotic mosaic virus (both limited to infection of the inoculated leaves of tobacco) or tomato bushy stunt virus (systemically infecting tobacco) was not altered by the expression of PVY HCPro. Therefore, it appeared that the PVY HCPro was able to induce defense responses at 25 °C, but not at 33 °C, where it actually neutralized a pre-existing defense response. Moreover, the CMV 2b protein was able to neutralize a defense response activated by HCPro in combination with TRV.     

The multifunctional plant viral suppressor of gene silencing P19 interacts with itself and an RNA binding host protein

Tomato bushy stunt virus (TBSV) is an RNA plant virus encoding a protein of approximately 19 kDa (P19) that is involved in various activities important for pathogenicity, including virus transport and suppression of gene silencing. In this study, we provide evidence in vivo and in vitro that P19 specifically interacts with itself to predominantly form dimers, and with a novel host protein, Hin19. Hin19 has a high degree of similarity with a class of RNA-binding proteins of which many are involved in RNA processing. The binding of P19 to itself and to Hin19 both depend on a structurally important central region of P19 that was previously shown critical for its biological function in plants. Our findings provide evidence for a model in which virus spread through suppression of defense-related gene silencing involves the formation of a complex that includes P19 dimers and a newly identified host RNA-binding protein.     

Identification of two RNA silencing suppressors from banana bunchy top virus

In order to suppress RNA silencing, many plant and some animal viruses encode RNA silencing suppressors to achieve infection. In this study, we report that B3 and B4, encoded by DNA3 and DNA4 of banana bunchy top virus (BBTV), exhibit RNA silencing suppression activity. B3 and B4 were able to increase the transient expression of green fluorescent protein (GFP) and dramatically enhanced the pathogenicity of potato virus X (PVX) in Nicotiana benthamiana. B4 was able to reverse established gene silencing on an inoculated leaf or on an upper leaf. B3, however, was only active during infection of an inoculated leaf. Furthermore, B4, but not B3, was able to enhance GFP expression in the transgenic N. benthamiana line 16c. In conclusion, B3 and B4 are the RNA silencing suppressors of BBTV, and they may act at different steps in the RNA silencing pathways.     

The characteristics of the RNA-dependent DNA polymerase of baboon endogenous virus

The characteristics of the virion-associated RNA-dependent DNA polymerase (RDPase) of a baboon endogenous virus, M7, were studied extensively; the optimal conditions for the exogenous RDPase reaction were obtained with 0.4 mM-Mn2+, 110 mM-NaCl, 24 microgram/ml poly(rA). oligo(dT)12-18, at pH 7.6 in the presence of 0.01 % Brij-58. Under these conditions, the incorporation of 3H-TMP proceeded up to 90 min at a speed of 0.1 pmol TMP/microgram virus protein/min. Poly(rC). oligo (dG)12-18 and poly(rCm). oligo (dG)12-18 served as the template-primers in the exogenous reaction with 3 mM-Mg2+ and 0.4 mM-Mn2+, respectively. Polyuridylic acid, bleomycin, rifampicin, spermidine and inorganic phosphate significantly inhibited the RDPase activity of BaEV. The RDPase of BaEV requires a higher concentration of NaCl, a lower pH and milder conditions of detergent treatment than those of other mammalian retroviruses.     

Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro

Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3'-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution ("far-Northwestern blotting"). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals.     

Determination of the mutation rate of poliovirus RNA-dependent RNA polymerase

The fidelity of poliovirus RNA-dependent RNA polymerase (3D(pol)) was determined using a system based on the fidelity of synthesis of the alpha-lac gene which codes for a subunit of beta-galactosidase. Synthesis products are screened for mutations by an alpha-complementation assay, in which the protein product from alpha-lac is used in trans to complement beta-galactosidase activity in bacteria that do not express alpha-Lac. Several polymerases have been analyzed by this approach allowing comparisons to be drawn. The assay included RNA synthesis by 3D(pol) on an RNA template that coded for the N-terminal region of alpha-Lac. The product of this reaction was used as a template for a second round of 3D(pol) synthesis and the resulting RNA was reverse transcribed to DNA by MMLV-RT. The DNA was amplified by PCR and inserted into a vector used to transform Escherichia coli. The bacteria were screened for beta-galactosidase activity by blue-white phenotype analysis with white or faint blue colonies scored as errors made during synthesis on alpha-lac. Results showed a mutation rate for 3D(pol) corresponding to approximately 4.5x10(-4) errors per base (one error in approximately 2200 bases). Analysis of mutations showed that base substitutions occurred with greater frequency than deletions and insertions.