Sesbania mosaic virus (SeMV) infectious clone: possible mechanism of 3' and 5' end repair and role of polyprotein processing in viral replication

Abstract

Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.

Figure 1. Genome organization.

(a) SeMV is a single stranded RNA virus with genome size of 4147 nt. The 5′ end of the genome is covalently linked to VPg and the 3′ end lacks polyA tail. ORF 1 encodes movement protein and ORF 3 encodes the coat protein which is expressed through a subgenomic RNA (sgRNA). The ORF 2 codes for two polyproteins 2a and 2ab. The numbers indicate the position of start and stop codons in each of the ORFs. The polyprotein 2a contains N-terminal membrane anchor (MA)-protease-VPg-p10-p8 domains. The polyprotein 2ab contains N-terminal membrane anchor (MA)-protease-VPg-RdRp. The RdRp is expressed through a −1 ribosomal frame shifting mechanism. The numbers in the polyproteins 2a and 2ab indicate the cleavage site positions. (b) Features of infectious clone: The infectious construct was initially made in pBluescript SK+ vector and later subcloned into pRD400 vector. The infectious construct consists 2×35S Promoter-SeMV cDNA-sTobRV RZ (Ribozyme)-Nos terminator. It has additional 4 nt at the 5′ end (5′ CCTC 3′) and 21 nt at the 3′end. The 3′ terminal two nucleotides present in the wild type viral RNA are absent in this clone (5′ AAA T 3′ instead of 5′ AAA TGT 3′). The ribozyme self cleavage site is shown by a curved arrow.

Figure 2. RT-PCR and Western blotting analysis of Sesbania grandiflora infiltrated with SeMV icDNA.

(a) RT-PCR of total RNA isolated from systemically infected Sesbania leaves 21 dpi with SeMV icDNA: The RT-PCR was carried out with SeMV RdRp reverse and coat protein forward primer as described in the methods section. lanes 1 & 2, two different plants infiltrated with SeMV icDNA, lane 3, 100 bp ladder, lane 4, RT-PCR with SeMV genomic RNA, lane 5, RT(−)-control. (b) Western blot analysis of Sesbania plants Agrobacterium infiltrated with SeMV icDNA clone using CP specific antibodies: lanes 1 and 2 correspond to mock agroinfiltrated Sesbania leaf samples; lanes 3 and 4 leaf extracts of systemically infected leaves 21 dpi; lane 5 protein molecular mass marker.

Figure 3. Agroinfiltration of SeMV icDNA on Cyamopsis tetragonoloba plants and analysis of time course of SeMV icDNA infection.

(a) Mock and SeMV icDNA infiltrated Cyamopsis leaves (b) Western blot of SeMV icDNA infected Cyamopsis tetragonoloba plants. Lane 1, mock agroinfiltrated leaf extract; lanes 2–6, SeMV icDNA infected systemic leaves from five independent plants showing symptoms; lane 7, protein molecular mass markers; lane 8, positive control (native virus infected leaf extract). (c) Time course of SeMV icDNA infection on Cyamopsis plants: western blot analysis using CP antibodies. Lanes 1–3, 3 dpi; lanes 4–8, 6 dpi; lanes 9–13, 9 dpi; lane 14, is a positive control; lanes 15–16 are mock agroinfiltrated samples; lane 17 is a protein molecular mass marker.

Figure 4. Northern analysis of SeMV icDNA infiltrated cotyledon leaves of Cyamopsis tetragonoloba plants.

(a) 0.8% TBE agarose gel (EtBr staining) and northern blot analysis of total RNA extracted from SeMV icDNA agroinfiltrated leaves 6 dpi from three different plants. The negative sense ³²P labelled probe used for hybridization was complementary in sequence to the (+) sgRNA. (b) 0.8% TBE agarose gel analysis (EtBr staining) and northern blot analysis of total RNA extracted from SeMV icDNA agroinfiltrated leaves 6 dpi. The positive sense ³²P labelled probe corresponding in sequence to that of (+) sgRNA was used for hybridization.

Figure 5. Western blot analysis with membrane enriched fraction of SeMV icDNA infected cotyledon leaves.

Western blot analysis with SeMV VPg polyclonal antibodies: Leaf samples were collected at 9 dpi; Lane 1, protein molecular weight marker; Lane 2, crude membrane fraction (25,000 g) from mock infiltrated cotyledon leaves. Lanes 3 & 4, Crude membrane fractions obtained at 10,000 g and 25,000 g respectively from SeMV icDNA infiltrated cotyledon leaves.

Figure 6. In vivo 3′ end repair of SeMV icDNA.

(a) Comparison of nucleotide sequence corresponding to the 3′ end of the genomic RNA from native virus (SeMV Wild type), genomic RNA from virus purified from SeMV icDNA infiltrated sample (SeMV icDNA inf 1 & 2 ) and sequence of SeMV icDNA clone (SeMV icDNA clone). (b) Comparison of nucleotide sequence corresponding to the 5′ end of the genomic RNA from native virus (SeMV wild type), genomic RNA from virus purified from SeMV icDNA infiltrated sample (SeMV icDNA inf) and sequence of SeMV icDNA clone (SeMV icDNA clone).

Figure 7. Mutational analysis of 5′ and 3′ terminus of SeMV genome.

(a) Western blot analysis of 3′ and 5′ end deletion mutants of SeMV icDNA infiltrated plants. Lanes 1–3 represent 3′ UTR Δ3 nt icDNA; lanes 4–6 corresponds to 3′UTR Δ4 nt icDNA; lanes 7–9 represent 3′UTR Δ5 nt icDNA; lanes10–12 corresponds to 5′UTR Δ1 nt icDNA; lanes 13–15 represents for 5′UTR Δ3 nt icDNA; lanes 16–18 corresponds to 5′UTR Δ5 nt icDNA and lane 19 is a positive control (SeMV native virus).

Figure 8. Western blot analysis of SeMV icDNA cleavage site mutants coinfiltrated with pEAQ ORF2.

Each mutant was tested in 20 independent plants but three were used for western blotting. Lanes 1–3, SeMV icDNA E132A cleavage site mutant coinfiltrated with pEAQ ORF2; lanes 4–6, SeMV icDNA E325A mutant coinfiltrated with pEAQ ORF2; lanes 7–9, SeMV icDNA E402A mutant coinfiltrated with pEAQ ORF2; lanes 10–12, SeMV icDNA E498A mutant coinfiltrated with pEAQ ORF2.

Figure 9. A possible mechanism for 5′ and 3′ end repair in SeMV.

VPg is shown as a small circle at the 5′ end of the (+/−) gRNA, (+/−) sgRNA and primer nucleotides. The 5′ end of the (+) gRNA and (+) sgRNA begins with 5′ACAA3′ sequence. Step1, The VPg-ACAA or VPg-ACA primers could be synthesized by RdRp using unknown internal sequence element (shown as stem-loop) (presence of different VPg forms in the western blots supports this possibility). Step 2, these primers realign at the 3′ end of the (+) gRNA even in the absence of complementarity (Note that initial RNA formed from SeMV icDNA lacks complementarity with the VPg-ACA/VPg-ACAA primer at the 3′ end). Alignment or positioning of primers at the genomic termini could be determined by cis acting elements. The RNA chain could be elongated to synthesize full length negative strand or terminated prematurely to synthesize subgenomic length negative strand (the full length genomic negative strand in replicative form (ds gRNA) and (−) ss sgRNA or ds sgRNA are indeed detected in northern blots). Step 3, the VPg-ACAA/VPg-ACA primers align at the 3′ end of these negative stranded genomic and subgenomic RNAs which could be elongated to synthesize positive stranded genomic and subgenomic RNA respectively.