The Barley stripe mosaic virus γb protein promotes chloroplast-targeted replication by enhancing unwinding of RNA duplexes
[['Positive sense ssRNA viruses consist of one or more highly structured genomic ( g ) RNAs .', 'Complex secondary motifs within viral gRNAs are required for efficient viral protein translation [1 , 2] , RNA synthesis [3 , 4] , cell-to-cell movement [5] and encapsidation [6 , 7] .', 'Current virology studies have shown that RNA viruses use several strategies to regulate secondary RNA structures during replication , including RNA helicase and RNA chaperone functions .', 'For example , conserved RNA helicase domains are found within many plant and animal viral proteins ( S1 Fig ) .', 'These helicases play major roles in establishing successful infections of RNA viruses [8] .', 'For example , the helicase domain of the Brome mosaic virus ( BMV ) 1a replicase subunit protein engages in interactions with the 2apol subunit to target viral replication complexes ( VRC ) to viral replication sites at the endoplasmic reticulum ( ER ) [9] .', 'In addition to membrane targeting functions , the C-terminal helicase domain of the BMV 1a subunit functions in recruitment of viral RNA templates to the ER and for efficient replication [9] .', 'An RNA chaperone activity has also been described for the Tomato bushy stunt virus ( TBSV ) p33 replication protein [10] that enables destabilization of the double-stranded RNA replicative form template [11] , and provides accessibility of the RNA genome to the viral RNA-dependent RNA polymerase ( RdRp ) for RNA synthesis [10] .', 'In addition , the cylindrical inclusion ( CI ) proteins of potyviruses also contain an RNA helicase domain that overlaps the N-terminal and central regions , and functions in RNA replication and cell-to-cell movement [5 , 12] .', 'Another strategy used by RNA viruses is to recruit host RNA helicases .', 'In the best studied of these , TBSV recruits multiple host RNA helicases , including DEAD-Box RNA helicases DED1 , RH20 , RH2 , and RH5 to assist in gRNA replication [13–15] , and suppression of recombination [16] .', 'In addition , genetic studies of Turnip mosaic virus ( TuMV ) infected Arabidopsis thaliana plants have also shown that various host RNA helicases are involved in potyviral infection [17] .', 'The hordeiviruses are positive sense RNA viruses exemplified by the type member , Barley stripe mosaic virus ( BSMV ) , which naturally infects barley , wheat and oat , and can also be transmitted to numerous other monocots and dicots by mechanical inoculation [18 , 19] .', 'Members of the genus Hordeivirus have rigid helical particles that encapsidate tripartite gRNAs designated RNAα , RNAβ , and RNAγ ( S2A Fig ) .', 'The BSMV genome encodes eight major proteins [20–22] .', 'Among these , the αa protein contains highly conserved methyltransferase ( MT ) and helicase ( HEL ) domains that function in capping of viral RNAs and unwinding of RNA duplexes during replication and transcription [23] .', 'RNAβ encodes the coat protein , which is responsible for virus particle formation , and the triple-gene block movement proteins ( TGB1 , TGB2 , and TGB3 ) , which are required for intra- and intercellular spread of the virus [24 , 25] .', 'RNAγ encodes the γa protein , which has an RdRp activity and interacts with the αa protein to promote RNA replication [26 , 27] .', 'Early studies showed that a second ORF translated from the RNAγ subgenomic RNA ( sgRNA ) encodes a 17 kDa cysteine-rich VSR protein designated γb that has pivotal roles in seed transmission in barley and modulates BSMV symptom severity [28 , 29] .', 'In addition , systemic movement in the dicot host Nicotiana benthamiana rarely occurs in the absence of γb [30] .', 'Subsequent studies have revealed that γb is a cysteine-rich VSR protein with a zinc finger-like region and protects viral dsRNA from degradation [31 , 32] .', 'Four regions in the γb protein are required for maintenance of functional integrity of γb ( S2A Fig ) .', 'Specifically the C1 and C2 regions of γb are responsible for zinc ion binding [33] , whereas the basic motif ( BM ) is critical for nonspecific binding of γb to single-stranded ( ss ) RNA [34] .', 'The coiled-coil domain in the C-terminal region participates in homologous interaction of γb , which is a prerequisite for RNA silencing suppression activities [31] .', 'Although individual functional studies suggested that γb may have multifunctional roles in viral replication , defined functions of γb remain largely unexplored .', 'Observations of BSMV-infected leaf tissues by transmission electron microscopy have revealed vesicles resulting from invagination of the outer chloroplast membrane [35 , 36] .', 'Immunolocalization studies have also shown that double-stranded ( ds ) RNA is associated with the chloroplast vesicles , but the dsRNA was not specifically shown to be BSMV specific; nevertheless , the vesicles have been hypothesized to be virus replication factories [37] .', 'Consequently , Torrance et al . speculated that γb might have unspecified roles in replication because γb was found to localize to chloroplasts in BSMV-infected plants [35] .', 'Our current studies have answered several important questions arising from the previous research .', 'We have provided direct evidence that BSMV ssRNAs and dsRNAs and that BSMV αa and γa are specifically associated with the chloroplasts .', 'We also have found that the αa protein binds directly to the γb protein and facilitates associations of γb with the chloroplasts .', 'To extend these results , we conducted experiments showing that deletion of γb from the BSMV genome resulted in reduced levels of RNAα accumulation in infected N . benthamiana tissue .', 'We also tested the helicase activity of the use of the αa helicase motif in an in vitro cell-free RNA unwinding assay and carried out experiments to test whether αa-γb interactions might affect the αa helicase activity .', 'The results showed that γb alone does not possess helicase activity but suggested that γb greatly enhances αa unwinding of dsRNA duplexes when present in the assays .', 'Overall , these results broaden our understanding of the multifunctional roles of γb , and provide a novel mechanism whereby the γb VSR protein facilitates efficient production of progeny BSMV RNAs .'], ['To determine whether BSMV assembles viral replication complexes on chloroplast membranes , we conducted experiments to test the association of the αa and γa replicase subunits with chloroplasts and the subcellular localization of the BSMV plus- and minus-strand RNAs and dsRNA replicative intermediates .', 'The results at 3 days post-infiltration ( dpi ) of N . benthamiana leaf tissues with Agrobacterium tumefaciens strains harboring the pSuper1300-αaGFP or pSuper1300-γaGFP plasmid for expression of αa-GFP or γa-GFP respectively , showed that the αa and γa proteins colocalize with chloroplasts ( Fig 1A and 1B ) .', 'We also used a live-cell RNA localization system described by Tilsner et al . [38] to visualize the plus- and minus-sense RNAs generated during BSMV replication .', 'In these experiments , a consensus sequence that is specifically recognized by the Pumilio-based reporters was inserted downstream of the γb UAG stop codon in the plus or minus sequence orientations ( S2C Fig ) .', 'These manipulations provided bimolecular fluorescence complementation ( BiFC ) assays that rely on recruitment of Pumilio split fluorescent protein halves by plus- and minus-sense BSMV RNAs synthesized during replication [38] .', 'The specific binding of the Pumilio proteins to the BSMV RNAγ fragment inserted in either plus or minus sequence orientations enabled detection and localization of BSMV RNAs by confocal microscopy ( Fig 1C and 1D ) .', 'These experiments revealed bright fluorescent puncta representative of plus-sense BSMV RNAs associated with red chloroplast autofluorescence ( Fig 1C ) .', 'Additional fluorescence indicative of plus-strand BSMV RNAs could also be observed as puncta and as diffuse areas in the cytoplasm ( S3A Fig ) .', 'Furthermore , minus-strand RNA foci were detected in association with chloroplasts ( Fig 1D , arrowheads ) .', 'In control experiments , only low background cytoplasmic fluorescence or large nonspecific aggregates were visible in tissue infected with wild type ( wt ) BSMV lacking the Pumilio recognition sequence ( Fig 1E ) .', 'These results thus confirmed the specific binding of the Pumilio proteins to the engineered BSMV RNA strands .', 'We also used a dsRNA visualization system developed by Cheng et al . [39] to determine the distribution of BSMV-specific dsRNA replication intermediates .', 'In this system , two halves of the yellow fluorescence protein ( YFP ) were separately fused to two dsRNA binding proteins ( B2 of Flock house virus and VP35 of Marburg virus ) , to allow detection of dsRNAs in living cells by reconstruction of the YFP signal [39] .', 'The results of these experiments revealed fluorescent puncta representing the BSMV dsRNAs are intimately associated with chloroplasts in BSMV-infected N . benthamiana leaves ( Fig 1F and S3B Fig ) .', 'In contrast , fluorescent signals were not evident in the healthy control leaves ( Fig 1G ) .', 'In summary , our results show the colocalization of BSMV replication proteins , plus- and minus-strand BSMV RNAs and BSMV dsRNA intermediates within the chloroplasts , and unambiguously demonstrate that chloroplasts are the major sites of BSMV replication .', 'A previous study indicated that the γb protein localizes to chloroplasts , as well as the cytoplasm during BSMV infection [35] .', 'However , the functional VSR activities of the γb fluorescent protein fusions and their subcellular localization were not tested in these studies .', 'To extend these studies , a spot silencing assay was carried out [40 , 41] , and the results of this assay indicated that fusion of the GFP and RFP fluorescent proteins to the γb C-terminus had little , if any , effects on RNA silencing suppression ( S4 Fig ) .', 'We also used confocal microscopy to determine the subcellular localization of the γb-GFP fusion protein when expressed alone or co-expressed with other BSMV components in BSMV-infected N . benthamiana leaves .', 'In these experiments , only a low proportion of the γb-GFP protein localized with the chloroplast when only γb-GFP was transiently expressed , as only ~10% of the isolated chloroplasts contained γb-GFP punctata ( Fig 2B ) .', 'In contrast , γb-GFP associated with the ~90% of the isolated chloroplasts in cells infected with RNAs α , β , and γγb-GFP , and ~60% of γb-GFP fluorescence localized with the chloroplasts isolated from tissue infected RNAs α , and γγb-GFP ( Fig 2D and 2E ) .', 'In control experiments , the GFP protein localized predominantly in the cytoplasm and also diffused into the nuclei when expressed alone or with other BSMV components ( Fig 2A and 2C ) .', 'Notably , the free GFP fluorescence entered the nucleus because the 27 kDa GFP protein is sufficiently small to diffuse through nuclear pore complexes ( Fig 2A and S2B Fig ) , but the size of the 44 kDa γb-GFP fusion protein restricts passive nuclear diffusion ( Fig 2B ) .', 'Collectively , these results reveal strong associations of γb-GFP with chloroplasts in BSMV-infected tissues and verify that chloroplast associations of γb are greatly enhanced in the context of BSMV infections .', 'To explore mechanisms whereby γb is targeted to chloroplasts during BSMV infection , BiFC experiments were performed to examine possible interactions between γb and the chloroplast-localized BSMV replication proteins ( Fig 1A and 1B ) .', 'Different combinations ( as shown in Fig 3A ) of fusion proteins containing N- or C-terminally tagged YFP halves of BSMV αa , γa or γb were tested for possible in vivo interactions , which can be observed as reconstructed YFP signals by confocal laser scanning microscopy .', 'The BiFC assay provided convincing evidence that γb interacts with αa in chloroplasts ( Fig 3A , ii and', 'iii ) .', 'In the positive controls , homologous interactions of both αa and γb were observed ( Fig 3A , v and', 'vi ) , in agreement with previous reports [31 , 42] .', 'The αa self-interactions were exclusively associated with chloroplasts ( Fig 3A ,', 'v ) , whereas γb homologous interactions were largely restricted to the cytoplasm , and the fluorescent puncta typically were distinct from the chloroplasts ( Fig 3A ,', 'vi ) .', 'However , interactions between γb and γa were not detected in either BiFC combination ( Fig 3A , i and', 'iv ) .', 'In addition to the BSMV γb-αa interactions , Lychnis ringspot virus ( LRSV ) and Poa semilatent virus ( PSLV ) γb-αa interactions were also observed ( S5 Fig ) .', 'Thus , our results provide strong evidence that similar γb-αa interactions occur during infections of other members of the genus Hordeivirus .', 'As a complement to the BiFC assays , total protein samples from N . benthamiana leaves infiltrated with the BiFC vectors were subjected to Western blot analysis with antibodies against GFP and Actin .', 'The results showed that all half-YFP fusion proteins were expressed in the infiltrated leaf tissues ( S6 Fig ) .', 'Our results described above suggested that αa might have a direct role in subcellular localization of γb during BSMV infection .', 'To provide additional evidence to support this possibility , transient expression of RFP-tagged γb ( γb-RFP ) and GFP-tagged αa ( αa-GFP ) was carried out in N . benthamiana leaves .', 'The results showed that γb-RFP was redistributed from the cytosol to the chloroplasts when co-expressed with αa-GFP , but remained primarily in the cytoplasm when co-expressed with GFP ( Fig 3B , i and ii; Note: Chloroplast autofluorescnce is expressed as a false blue color ) .', 'In contrast , the chloroplast localization of αa-GFP was not altered during coexpression of γb-RFP ( Fig 3B , compare iii with i ) .', 'These results clearly demonstrate a key role for αa in γb chloroplast targeting .', 'To further verify αa and γb interactions , yeast two-hybrid ( Y2H ) and co-immunoprecipitation ( co-IP ) assays were conducted ( Fig 3C and 3D ) .', 'As a positive control , Y2H analyses confirmed the αa and γb self-interactions suggested by the BiFC experiments ( Fig 3A , v and', 'vi ) .', 'Yeast cells transformed with the αa-activating domain ( AD-αa ) and the γb-binding domain ( BD-γb ) plasmids grew well on SD-Trp-Leu-His-Ade drop-out plates as evidenced by the blue color of the streaks ( Fig 3C ) .', 'However , yeast cells with the reciprocal AD-γb and BD-αa plasmids failed to grow on the drop-out plates ( Fig 3C ) , but this may be due to protein topology changes introduced by the γb and αa AD or BD fusions .', 'Yeast cells harboring combinations of the empty vectors ( pGBKT7 or pGAD ) , and the corresponding GAL4 AD or BD fusion constructs failed to grow on the drop-out plates , thus excluding the possibility of auto-activation ( Fig 3C ) .', 'In addition , interactions between αa and γb were also revealed by immuno-precipitation ( IP ) assays ( Fig 3D ) .', 'The γb-3×Flag and αa-GFP fusions were transiently expressed via agro-infiltration under control of the 35S and Super promoters , respectively .', 'Total proteins were solubilized in GTEN buffer containing 1% Triton X-100 and 0 . 15% NP-40 , and subjected to IP assays with FLAG-beads .', 'In these experiments , αa-GFP co-precipitated with γb-3×Flag ( Fig 3D , lane 3 ) , whereas appreciable GFP-fusion proteins were not detected in negative controls when either γb-3×Flag was replaced by GUS-3×Flag , or αa-GFP was replaced by HA-GFP ( Fig 3D , lanes 1–2 ) .', 'Taken together , these results demonstrate that αa engages in specific physical interaction with γb , and that αa-γb interactions result in the recruitment of γb to chloroplasts .', 'To identify which regions of γb and αa are responsible for γb-αa interactions , we constructed a series of γb and αa deletion mutants for yeast two-hybrid assays ( Fig 4A and 4C ) .', 'Fragments of γb were expressed as fusion proteins with the GAL4 DNA-binding domain and tested for their possible interactions with full-length αa fused with the GAL4 activation domain ( AD-αa ) .', 'In these tests , yeast expressing the BD-γb86-152 containing C-terminal 86–152 amino acids ( aa ) of γb grew well on Trp , Leu , His , and Ade drop-out plates , whereas yeast expressing BD-γb1-85 failed to grow ( Fig 4B , left panel ) .', 'Truncations of the γb C-terminal sequences further indicated that γb amino acids 86–127 retained the ability to interact with αa , whereas γb128-152 was unable to participate in γb-αa binding ( Fig 4B , left panel ) .', 'These data indicate that the γb C-terminal region between amino acids 86 and 127 is required for γb-αa interactions .', 'We also mapped the regions of αa that interact with γb by constructing GAL4AD fusions to different αa truncations ( Fig 4C ) and tested for interactions with the BD-γb .', 'Y2H analysis showed that methyltransferase ( MT ) with hinge-like domain was able to participate in γb-αa binding , whereas the C-terminal helicase ( HEL ) domain was unable to interact with γb ( Fig 4D , left panel ) .', 'As negative controls , various constructs harboring the αa and γb-derived mutants were co-transformed with empty yeast vectors .', 'The results show that these controls are unable to grow on dropout medium lacking Ade , Trp , Leu , and His ( Fig 4B and 4D , right panels ) .', 'We next conducted experiments to assess whether the mapped γb region responsible for interactions of γb with αa contributes to chloroplast targeting of γb during BSMV infection .', 'The plasmid pCaBS-γγb-GFP reporter construct was mutagenized to retain or eliminate γb residues 86–127 that constitute the γb-αa interaction region ( S2C Fig ) .', 'N . benthamiana leaves were coinfiltrated with Agrobacterium strains containing pCaBS-α , pCaBS-β , and individual pCaBS-γγb-GFP derivatives to assess γb-GFP subcellular localization during infection .', 'In comparison with wt γb-GFP , which formed fluorescent rings around the chloroplasts , and fluorescent puncta on the chloroplasts ( Fig 4E , i , Note arrowheads ) , deletion of the αa-interacting region within γb ( aa 86–127 ) resulted in reduced chloroplast associated fluorescence ( Fig 4E , ii , γbΔ86-127-GFP ) that were similar to the GFP substitutions for the γb ORF in RNAγ ( Fig 4E ,', 'iii ) .', 'In contrast , the αa-interacting region of γb-GFP alone ( aa 86–127 ) was able to localize to the chloroplasts during BSMV replication ( Fig 4E ,', 'iv ) .', 'These results thus provide persuasive evidence that physical interactions of γb residues 86 to 127 are required to mediate αa binding and recruitment of γb to chloroplasts .', 'Because RNAβ encodes the coat protein and the triple gene block proteins that are essential for virion formation and movement ( S2A Fig ) , these processes could interfere with studies of RNA replication per se .', 'Therefore , to avoid these problems , we eliminated pCaBS-β for the experiments below and infiltrated N . benthamiana leaves with Agrobacterium strains harboring pCaBS-α and various pCaBS-γ mutant derivatives .', 'This procedure eliminated BSMV cell-to-cell movement and ensured that replication was restricted only to the initially infected cells , and that virion morphogenesis and movement processes did not affect BSMV RNA accumulation .', 'Moreover , the high percentages of leaf cells transiently expressing BSMV RNA and proteins after agroinfiltration [43] , resulted a high proportion of infected cells in the infiltrated leaf tissue , while also enhancing the likelihood that infected cells were undergoing synchronous replication at 3 dpi .', 'One question arising from our results is whether the γb protein participates directly in the replication of BSMV RNAs , or if the effects are due to an RNA segment within the γb ORF .', 'To address this issue , we generated two mutants in the RNAγ infectious clone ( pCaBS-γ ) to differently affect γb protein or RNA synthesis .', 'The first mutant ( designated γΔγb ) was engineered by deleting the γb ORF in order to eliminate both γb protein and RNA synthesis , and the second mutation ( γγbATGm ) , generated to maintain the γb ORF RNA sequence , was constructed by introducing a single site specific mutation ( AUG→UUG ) into the γb start codon ( S2C Fig ) .', 'We co-infiltrated BSMV RNAα and the RNAγ derivatives to initiate BSMV replication .', 'At 3 dpi , total RNA from the infiltrated regions was extracted and subjected to Northern blot analysis by using the 3′-untraslated region ( UTR ) probe or the RNAα-specific probe to monitor accumulation of BSMV RNAs .', 'Due to the high nucleotide sequence identity amongst the 3′-UTR regions of the genomic RNAs [44] , accumulation of α and γ plus-strand RNAs and sgRNAγ could be detected simultaneously .', 'The RNAα-specific probe enabled specific detection of the RNAα plus-strand , and a BSMV RNAα probe for detection of minus-strand RNAα was also used .', 'As expected , N . benthamiana leaves infiltrated with pCaBS-α or pCaBS-γ alone resulted in barely detectable RNA accumulation at 3 dpi ( Fig 5B and 5C; panels I and II , lanes 1 and 2 ) , suggesting that BSMV was not replicating in the infiltrated tissue .', 'In contrast , high-levels of wt BSMV gRNAs and sgRNAγ accumulation were detected at 3 dpi after co-infiltrations of pCaBS-α and pCaBS-γ ( Fig 5B and 5C , panels I and II , lane 3 ) .', 'However , co-infiltrations with pCaBS-α and pCaBS-γΔγb , in which the γb ORF had been deleted , or pCaBS-α and pCaBS-γγbATGm , which only eliminated γb AUG codon , both resulted in substantially impaired accumulation of BSMV plus-strand RNAα ( Fig 5B , panels I and II , compare lanes 4 and 5 with lane 3 ) , but not the minus-strand RNAα ( Fig 5B and 5C , panel III; compare lane 4 or 5 with lane 3 ) .', 'These results thus strongly implicate γb protein functions in accumulation of plus-strand RNAα .', 'Nevertheless , accumulation of RNAγ appeared not to be substantially affected by the γb deletion/mutations .', 'This suggests a model whereby replication of RNAα , but not RNAγ , is cis-preferential [27] , and that γb-αa protein interactions are required for efficient cis-preferential replication of the plus-strand of RNAα ( see discussion ) .', 'To provide additional information about the effects of γb on BSMV infection , other BSMV mutants were constructed in pCaBS-γ .', 'These consist of RNAγγb-GFP , which contained an N-terminal GFP fusion , a truncated γb derivative , ( RNAγγb1-127-GFP ) designed to eliminate suppression of RNA silencing and a null mutant ( RNAγΔγb-GFP ) generated by substitution of the gfp ORF for the γb ORF ( S7A Fig ) .', 'These RNAγ derivatives were agroinfiltrated into plant leaves along with wt RNAα ( S7B Fig ) , and the results at 3 dpi showed that γb1-127-GFP , which has a deletion of 25 amino acids from the γb C-terminus and lacks VSR activity , had much lower amounts of GFP in the infiltrated regions than the full length γb-GFP protein and the amounts of GFP in the null mutant regions were even lower than those γb1-127-GFP ( S7C and S7D Fig ) .', 'These results thus provide additional evidence that , in addition to its VSR activities , γb has other functions that participate in efficient BSMV replication .', 'A previous report also showed that deletion of the γb ORF from RNAγ affected BSMV pathogenesis in barley [29] , but these experiments did not determine directly whether deletion of the sequences affected BSMV pathogenesis in N . benthamiana .', 'Therefore , we co-infiltrated pCaBS-α and pCaBS-β along with pCaBS-γ , or the pCaBS-γ mutants γbATGm , or Δγb to determine whether presence of the RNA sequence encoding the γb protein could ameliorate the pathogenic effects that occur when the γb ORF is deleted .', 'Symptoms arising from both the γbATGm and the Δγb mutant co-infiltrations elicited very mild symptoms in infected N . benthamiana ( S8A Fig ) and also destroyed γb expression ( S8B and S8D Fig ) .', 'Systemic BSMV symptoms in barley also were severely affected by both mutants ( S8C Fig ) , and resulted in the “Null” chlorotic streak phenotype previously reported when the γb ORF was deleted from RNAγ [29] .', 'In contrast , expression of wtRNAγ in combination with RNAβ and RNAγ resulted in typical chlorotic or mosaic symptoms in systemically infected N . benthamiana and barley leaves and nearly 100% of the inoculated plants were infected ( S8A and S8C Fig ) .', 'Thus , our results verify that the BSMV wild-type phenotypic effects in both barley and N . benthamiana depend on expression of the γb protein , and that sequences within the γb ORF in RNAγ can not compensate for the absence of γb .', 'Considering that γb is a multifunctional protein [29 , 31 , 34] , we extended the analyses to more clearly define γb mutant activities that inhibit replication of RNAα .', 'Previous studies indicated that arginine and lysine residues at amino acid ( aa ) residues 25 and 26 in the γb basic motif of γb are essential for single-strand RNA binding activity [34] , and that seven residues within positions 102 to 130 in the C-terminal region of the γb protein are critical for maintenance of RNA silencing suppressor activity [31] .', 'Therefore , two γb mutants similar to those previously reported [31] were introduced into pCaBS-γ to determine their effects on silencing suppressor activities .', 'Specifically , these mutants included γb1-127 , whose product contains a C-terminal 25 aa residue truncation , and γb1-85 , which contains a 67 aa truncated product that lacks the αa-interacting region present within γb1-127 ( S2B and S2C Fig ) , and largely maintains the RNA binding activity [45] .', 'Addition mutants included γb1-85m , to produce substitutions ( 25RK26 to 25QN26 ) of adjacent basic motif residues in the γb1-85 protein that affect RNA binding [29 , 31 , 34] .', 'We also constructed a single substitution mutant ( γbI130G ) whose product contains a Gly residue at the end of the coiled-coil motif that inhibits RNA silencing suppressor activities of the wt γb protein ( S2B and S2C Fig ) .', 'The results of spot silencing experiments with these γb derivatives showed that the γb1-85 , γb1-85m and γb1-127 proteins completely failed to exhibit RNA silencing suppressor activity , and that the γbI130G γb point mutant protein resulted in greatly reduced levels of RNA silencing suppressor activity ( Fig 5A ) .', 'In addition , the effects of the γb mutations on BSMV replication were tested by Northern blotting .', 'Control comparisons revealed that plus-strand RNAα accumulation in the RNAα + RNAγ infiltrations was more than two-fold greater than those of the RNAα + RNAγΔγb infiltrations ( Fig 5B , panels I and II , lanes 3 and 4 , ) .', 'The results also indicated that accumulation of plus-strand RNAα in plants with BSMV expressing γb1-127 and the γbI130G mutants was ~70% of the level of the wt γb control ( Fig 5B , panels I and II , compare lanes 6 and 7 with lane 3 ) .', 'In addition , plus-strand RNAα accumulation in infections with the γb1-127 and the γbI130G mutants ( both of which eliminate suppression of RNA silencing ) was more than two-fold higher than RNAα accumulation in the RNAα + RNAγΔγb or RNAα + RNAγγbATGm infections ( Fig 5B , panels I and II , compare lanes 6 and 7 with lanes 4 and 5 ) .', 'The results also show that the mutants do not have substantial effects on the accumulation of minus-strand RNAα ( Fig 5B , panel III ) .', 'Thus , these results suggest that regions within the γb N-terminal 127 aa may have a role in RNAα accumulation , and that γb RNA silencing suppressor activity per se is not entirely responsible for efficient replication of BSMV plus-strand RNAα .', 'Next , we compared wt γb and RNAγγb1-127 with more extensive γb C-terminal deletion mutants ( RNAγγb1-85 , and RNAγγb1-85m ) that eliminate the αa interacting region in γb ( Fig 5C , panels I and II ) .', 'Comparisons with RNAγγb1-127 revealed that plus-strand RNAα accumulation was nearly 70% of wt γb control and that RNAα accumulation in RNAγγb1-85 was at least 50% of wt γb ( Fig 5C , panels I and II , compare lanes 3 , 5 and 6 ) .', 'Again , none of the mutants had substantial effects on the accumulation of minus- strand RNAα ( Fig 5C , panel III ) .', 'These results suggest that γb interactions with αa are important for maintenance of the normal accumulation of viral plus-strand RNA during BSMV replication .', 'In separate experiments , when the BM motif ( 25RK26→25QN26 ) , which is involved in RNA binding , was mutated in γb1-85 ( γb1-85m ) , the plus-strand RNAα levels decreased to about one-half of that in RNAα + RNAγγb1-85 and were nearly the same levels as in RNAα + RNAγΔγb infected cells ( Fig 5C , panels I and II , compare lanes 4 and 6 with lane 7 ) .', 'Taken together , these results suggest that multifunctional activities of the γb protein affect RNAα accumulation .', 'The γb RNA silencing suppressor activity has a limited positive effect on RNAα replication and additional γb functions involving γb-αa and RNA binding activities may interact synergistically in accumulation of BSMV RNAα .', 'To determine whether the compromised γb VSR activity in the γb-deficient BSMV mutants can be complemented by other known VSRs , we expressed the TBSV p19 protein , a well-studied RNA silencing suppressor [46 , 47] , in cis and in trans to investigate whether this heterologous VSR protein can rescue BSMV replication defects elicited by the γb mutants .', 'First , we cultivated transgenic N . benthamiana plants , and conducted genomic PCR analysis to verify γb or p19 transformation .', 'The results of growth experiments revealed that compared to non-transgenic plants , the p19-transgenic plants have a stunted growth phenotype as well as mild leaf curling ( S9A Fig ) , and PCR experiments showed that all of the tested transgenic plants were positive for genomic γb or p19 DNA integration ( S9B Fig ) .', 'In addition , spot silencing experiments indicated that the RNA silencing suppression activity by γb- and p19-transgenic N . benthamiana were both moderate as indicated by comparisons of the GFP fluorescence of infiltrated leaves with agrobacterium strains harboring γb or p19 ( Fig 6A ) .', 'However , the suppressor activity in the transgenic leaves was much more pronounced than with agrobacterium control infiltrations with the empty vector ( EV ) plasmid ( Fig 6A ) .', 'Western blot analysis of protein extracts from agro-infiltrated leaf areas also confirmed the gfp silencing phenotypes observed under UV light ( Fig 6B ) .', 'Next , Agrobacterium strains for expression of RNAα together with individual RNAγ derivatives shown in S2C Fig were co-infiltrated into leaves of non-transgenic , p19-transgenic and γb-transgenic N . benthamiana plants , respectively .', 'Amongst the infiltrated leaves , RNAγ with an amino acid mutation in the GDD motif ( GDD→GAD ) that abolishes RdRp activity of γa served as a negative control .', 'At 3 dpi , total RNA was extracted from agro-infiltrated leaves followed by Northern blot analyses with either the RNAα- or 3′-UTR-specific probe .', 'As shown in Fig 6C , the RNAα plus RNAγγaGDDm agroinfiltrations had undetectable levels of the BSMV RNAs ( Fig 6C , panels I and II , lane 1 ) , whereas in the RNAα + RNAγ positive controls , highly abundant plus-strand RNAs could be detected in both the transgenic and non-transgenic N . benthamiana plants ( Fig 6C , panels I and II , lanes 2 , 5 and 8 ) .', 'The non-transgenic N . benthamiana leaf discs that had been agroinfiltrated to express RNAα + RNAγΔγb had significantly reduced levels of BSMV replication compared to the RNAα + RNAγ controls ( Fig 6C , panels I and II , lane 3 ) , in agreement with the results shown in Fig 5 .', 'In contrast , when TBSV p19 was expressed in cis by replacing the γb ORF in RNAγ ( γΔγb-P19 ) , the BSMV plus-strand RNA signaling intensity was similar to that of the γb-deficient ( RNAα + RNAγΔγb ) infiltrated tissue ( Fig 6C , panels I and II , compare lane 3 with 4 ) .', 'Similar trends were also evident when these BSMV derivatives were agroinfiltrated into p19-transgenic N . benthamiana ( Fig 6C , panels I and II , lanes 6 and 7 ) .', 'Compared to viral replication in non-transgenic plants , expression of the p19 transgene also failed to enhance the accumulation of either the γb-deficient virus ( RNAα + RNAγΔγb ) or accumulation of RNAα + RNAγΔγb-P19 ( Fig 6C , panels I and II , compare lanes 6 and 7 with lane 2 ) .', 'Surprisingly , replication of the wt RNAα + RNAγ controls was moderately inhibited in p19-transgenic N . benthamiana ( Fig 6C , panels I and II
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles .', 'These proteins function as RNA helicases , methyltransferases , RNA-dependent RNA polymerases , RNA silencing suppressors , RNA chaperones , movement proteins , and so on .', 'Although many of the proteins bind the viral RNA genome during different stages of infection , our knowledge about the coordination of their functions is limited .', 'In this study , we describe a novel role for the Barley stripe mosaic virus ( BSMV ) γb as an enhancer of αa RNA helicase activity , and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication .', 'Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand ( + ) RNAα accumulation , but γb mutations abolishing viral suppressor of RNA silencing ( VSR ) activity did not completely eliminate genomic RNA replication .', 'In addition , cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions , indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions .', 'These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated , as well as formation of viral replication complexes .']
['In order to maximize reproduction , RNA viruses often encode multifunctional proteins to perform different activities during replication .', 'We report that the Barley stripe mosaic virus γb VSR protein is recruited to viral replication sites through interactions with the αa replicase subunit protein where it enhances αa helicase activity to increase the levels of BSMV replication .', 'Our discovery of the γb RNA helicase enhancement activity provides a novel function associated with a viral suppressor of RNA silencing that enhances understanding of multifunctional strategies whereby virus-encoded proteins contribute to genome replication and counter host defense mechanisms .']
['plant anatomy', 'rna-binding proteins', 'nucleic acid synthesis', 'rna interference', 'enzymes', 'plant cell biology', 'rna extraction', 'microbiology', 'enzymology', 'chloroplasts', 'plant science', 'epigenetics', 'rna synthesis', 'cellular structures and organelles', 'extraction techniques', 'chemical synthesis', 'research and analysis methods', 'genetic interference', 'proteins', 'gene expression', 'viral replication', 'leaves', 'biosynthetic techniques', 'biochemistry', 'rna', 'plant cells', 'helicases', 'cell biology', 'nucleic acids', 'virology', 'genetics', 'biology and life sciences', 'cellular types']