Prediction of putative regulatory elements in the subgenomic promoters of cucumber green mottle mosaic virus and their interactions with the RNA dependent RNA polymerase domain

Abstract

Characterization of the subgenomic RNA (sgRNA) promoter of many plant viruses is important to understand the expression of downstream genes and also to configure their genome into a suitable virus gene-vector system. Cucumber green mottle mosaic virus (CGMMV, genus Tobamovirus) is one of the RNA viruses, which is extensively being exploited as the suitable gene silencing and protein expression vector. Even though, characters of the sgRNA promoters (SGPs) of CGMMV are yet to be addressed. In the present study, we predicted the SGP for the movement protein (MP) and coat protein (CP) of CGMMV. Further, we identified the key regulatory elements in the SGP regions of MP and CP, and their interactions with the core RNA dependent RNA polymerase (RdRp) domain of CGMMV was deciphered. The modeled structure of core RdRp contains two palm (1-41 aa, and 63-109 aa), one finger (42-62 aa) subdomains with three conserved RdRp motifs that played important role in binding to the SGP nucleic acids. RdRp strongly preferred the double helix form of the stem region in the stem and loop (SL) structures, and the internal bulge elements. In MP-SGP, a total of six elements was identified; of them, the affinity of binding to - 26 nt to - 17 nt site (CGCGGAAAAG) was higher through the formation of strong hydrogen bonds with LYS16, TYR17, LYS19, SER20, etc. of the motif A in the palm subdomain of RdRp. Similar strong interactions were noticed in the internal bulge (CAACUUU) located at + 33 to + 39 nt adjacent to the translation start site (TLSS) (+ 1). These could be proposed as the putative core promoter elements in MP-SGP. Likewise, total five elements were predicted within - 114 nt to + 144 nt region of CP-SGP with respect to CP-TLSS. Of them, RdRp preferred to bind at the small hairpin located at - 60 nt to - 43 nt (UUGGAGGUUUAGCCUCCA) in the upstream region, and at the complex duplex structure spanning between + 99 and + 114 nt in the downstream region, thus indicating the distribution of core promoter within - 60 nt to + 114 nt region of CP-SGP with respect to TLSS (+ 1) of the CP; whereas, the - 114 nt to + 144 nt region of CP-SGP might be necessary for the full activity of the CP-SGP. Our in silico prediction certifies the gravity of these nucleotide stretches as the RNA regulatory elements and identifies their potentiality for binding with of palm and finger sub-domain of RdRp. Identification of such elements will be helpful to anticipate the critical length of the SGPs. Our finding will not only be helpful to delineate the SGPs of CGMMV but also their subsequent application in the efficient construction of virus gene-vector for the expression of foreign protein in plant.

Keywords: CGMMV; In-silico; RdRp; Regulatory elements; Tobamovirus; sgRNA promoters.

Fig. 1

Distribution of putative cis-elements within subgenomic promoter region (SGP) of movement protein (MP) and coat protein (CP) of different cucurbit infecting tobamoviruses identified using plant care database web-server. The colour indication is used to represent different elements. The coordinates of the elements in parenthesis are indicated with respect to the translation start site (green vertical arrow). a Showing the location of MP-SGP in the CGMMV genome and the distribution of cis-elements in MP-SGP. A total 15 different cis-elements were identified within MP-SGP regions of different tobamoviruses. The occurrence and distribution of similar regulatory elements within MP-SGP of CGMMV, CMoV, and WGMMV indicates the structural similarity among them, whereas CFMMV, ZGMMV and KGMMV have a distinct pattern. b Showing the location of CP-SGP in the CGMMV genome and the distribution of cis-elements in CP-SGP. A total 20 different cis-elements were identified in the CP-SGP region of six cucurbit-infecting tobamoviruses. CGMMV CP-SGP possesses only 7 cis elements; although they are common to others but their distribution pattern depicts significant variation (color figure online)

Fig. 2

RNA secondary structures of subgenomic promoter region (SGP) of movement protein (MP) and coat protein (CP) of different cucurbit-infecting tobamoviruses generated from M-fold web server using template RNA sequence. The arrow indicates the location of translation start site (TLSS) (+ 1) with AUG start codon (indicted with and arrow). a In MP-SGP of CGMMV, TLSS (+ 1) is located within a terminal loop of SL structure (SL-5) which is surrounded with other SL elements both in upstream (SL-1, SL-2, SL-3, SL-4) and downstream (SL-6, SL-7, SL-8, SL-9) regions that are indistinguishable from the secondary structures of CMoV, and WGMMV. The CFFMV, ZGMMV, and KGMMV shows different pattern of structural elements with localization of TLSS (+ 1) within internal buldge. b The secondary structure of CP-SGP of CGMMV has certain degree of similarity with that of CMoV and WGMMV. It contains two small hairpins emerged from a central loop located within 60 nt upstream with respect to TLSS (+ 1). These elements might have key regulatory function in sgRNA synthesis. ZGMMV also have similar pattern, but CFMMV and KGMMV have distinct features in this region. The start and end of the single stranded RNA are indicated with 5´ and 3´

Fig. 3

Three-dimensional model of core RNA dependent RNA polymerase domain (RdRp) of CGMMV replicase enzyme complex. a Genome map of CGMMV depicts the expression of 129 kDa (from ORF1) and 186 kDa (from ORF2) protein that polymerizes to form the replicase complex. The RdRP is part of the 186 kDa protein with its core catalytic sites are located within 1414aa to 1522aa. b The tertiary structure of core RdRp domain consisting of palm sub-domain (1-41aa) represented in yellow colour, finger sub-domain (42-62aa) represented in red colour, followed by another palm sub-domain (63-109aa) represented in yellow colour. The conserved GDD motif was shown in black colour. c The surface expose model of core RdRp with different sub domains (represented with different colour) to active protein surface (color figure online)

Fig. 4

Interaction of RdRp with the different structural elements of subgenomic promoter region (SGP) of movement protein (MP) and coat protein (CP) of CGMMV. The RNA secondary structures of the SGPs in negative strand were generated from M-fold web server, and used to present the interaction between 3D model of RNA and core RdRp. The arrow mark indicates the location of translation start site (TLSS) (+ 1) with UAC as the start codon (green colour, indicated with an arrow), and the number in roman designate the putative interacting sites. a A total six RdRp binding sites are predicted within MP-SGP of CGMMV; four (I to IV) are located in the upstream, and two (V and VI) in the downstream region of SGP. b A total five locations are predicted for the RdRp binding within CP-SGP of CGMMV, of them two putative sites (I, II) are in the upstream region and three (III, IV, V) are in the downstream region. The detail of the interacting nucleotides of RNA and amino acids of RdRp are presented in Tables 1 and 2, respectively. The nucleotides in yellow represent the different cis-elements (identified based on plant care database) distributed within SGP regions (color figure online)