Roles Of RNA Secondary Structure In Dengue Virus C Gene And Capsid Protein In Viral Replication

Dengue virus type 1-4, members of genus flavivirus, family flaviviridae, can cause dengue fever (DF) and more severe dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) in humans. Dengue infections are becoming global public health concerns. The genome of dengue virus is a single-stranded RNA molecule of positive polarity comprising about 11,000 nucleotides, and it includes a long single open-reading frame, which is flanked by 5′and 3′untranslated region (UTR), encoding a polyprotein with about 3,300 ammo acids. Processing of the polyprotein results into 3 structural proteins(C, prM, and E) and at least 7 nonstructural proteins(NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).Capsid (C) protein forms viral nucleocapsid via its interactions with viral genome RNA. C protein of flavivirus is composed of 4α-helixes, and it has been suggested to have multiple functions. C proteins of several members of the flaviviridae family act as modulators of viral RNA replication and protein translation, and the modulation effect of C protein on viral RNA replication is associated with its nuclear localization. Like C proteins of some other members of the flaviviridae family, Dengue virus C protein can be localized in the nuclei and nucleoli of infected cells, which implies it has potential function in viral RNA replication. Other than C protein itself, the gene of C protein also plays important roles in viral RNA replication and translation. C gene of flavivirus is adjacent to the 5′-UTR of the genomic RNA, which contains conserved RNA secondary structures essential for viral RNA replication. And RNA elements, 5′CS and cHP, which are critical for viral RNA replication, have been found in C gene. However, the mechanisms of how flavivirus C protein and RNA elements in C gene modulate viral replication are not understood very clearly yet, and in dengue virus, whether C protein also plays a role in viral replication is still unknown, and there is no answer to whether any other RNA secondary structure inside C gene modulates viral replication.In the study, to investigate the effects of mutations in dengue virus C gene and its encoded protein on viral replication, a series of dengue virus replicon mutants were constructed and their replication efficiencies were compared. Furthermore, the role of RNA secondary structure in C gene in viral RNA replication was carefully studied in replicon system. Based on the correlation of dengue virus C protein's nuclear localization and viral RNA replication, we performed yeast two-hybrid screen for viral and host proteins that interact with dengue virus C protein. Main results are described below.1. Effects of C protein mutation on dengue virus RNA replicationIn order to analyze whether any structures of dengue virus C protein have effects on viral replication, and the possibility of RNA secondary structure inside the coding sequence of C protein can modulate viral replication, we introduced various mutations into C gene of dengue virus replicon: the fourα-helix of C protein was deleted individually, and different mutations were introduced into the bipartite NLS of C protein. The replication of replicons was compared by measuring the reporter Renilla luciferase activities. Mutations that disabled the NLS of C protein increased viral RNA replication moderately. These results indicate that the nuclear localization of dengue virus C protein correlates with viral RNA replication, and this is the first report on the function of dengue virus C protein's nuclear localization. Deletion ofα1-helix reduced viral RNA replication significantly, while deletion of the otherα-helixes did not affect viral RNA replication. Then by utilizing immunofluorescence assay and real-time quantitative RT-PCR, the effect ofα1-helix deletion on viral RNA replication was verified. Furthermore, we discovered the change of C protein structure did not account for the reduced replication ofΔα1 replicon by the co-transfection of a plasmid expressing C protein together withΔα1 replicon.2. Role of RNA secondary structure in dengue virus C gene in viral RNA replicationTo investigate whether the reduced replication ofΔα1 replicon was due to disruption of RNA secondary structures in C gene, first the structure of 5′220nt of dengue virus genome, including 5′-UTR and first 109nt of C gene, was predicted using mfold software. The 54-98nt region of DEN-4 C gene forms a long-stem-loop-like secondary structure which is conserved in all four dengue serotypes. This secondary structure, designated as cα1HP, covers the coding sequence of C proteinα1-helix and is composed of four sub-structures: the top hairpin, the left loop, the right loop and the bottom-paired region. A series of replicon mutants were constructed and reporter activities were measured to analyze the role of cα1HP in viral RNA replication and translation. The reporter activities of these mutants show no difference at 6h post transfection, which indicates disruption of cα1HP structure has no effect on viral translation. However, deletion of various parts of cHP, or the whole cα1HP structure reduces viral RNA replication, thus the integrity of cα1HP is required for normal viral RNA replication. Disruption of base pairs by introducing silent mutations into cα1HP top hairpin region reduces viral RNA replication, while restoration of cα1HP top hairpin recovers replication of the corresponding replicon. Analysis of replicon RNA copy numbers in transfected BHK-21 cells by quantitative RT-PCR consists with the results of reporter luciferase assay. Mutants such as MC69/70+82/83 and MC75/78, which don't change cα1HP secondary structure, replicate at the same level as wild type replicon. Thus the top hairpin structure (65-87nt in C gene) of cα1HP is important for normal viral RNA replication and its function is sequence-independent.The loop sequence in the middle of cα1HP is highly conserved in all four dengue serotypes. We introduced mutations into the left loop, the right loop and the bottom-paired region and observed the effect on viral RNA replication. Introducing silent mutations into left loop reduces viral RNA replication, while mutations in the right loop and the bottom-paired region have no effect on viral RNA replication. Thus the conserved sequence of left loop correlates with the maintaining of normal viral RNA replication.We also constructed a frame-shift replicon mutant by inserting a U between C73 and C74 and deleting the former C102. In this mutant, theα1-helix of C protein is frame-shifted, whereas the overall RNA structure of the corresponding region is only slightly changed. This mutant shows similar replication effiency with wild type replicon. This finding confirms that theα1-helix itself has no effect on viral RNA replication. Based on the afore mentioned results, we conclude that cα1HP can facilitate viral RNA replication and the conserved left loop correlates with its function in viral RNA replication, and the top hairpin of cα1HP also plays an important role in its regulation of viral RNA replication in a sequence-independent manner.3. Screening for viral and host proteins that interact with dengue virus C proteinPreviously, we have observed the nuclear localization of dengue virus C protein correlates with viral RNA replication. This implies C protein of dengue virus has multiple biological functions, including the modulation of viral replication. In order to investigate the role of C protein in viral replication, using CytoTrap yeast two-hybrid system, we performed a screen for viral and host proteins interacting with dengue virus C protein.Dengue virus C gene was cloned into pSos vector, and dengue nonstructural genes were cloned into pMyr vector. The resulted recombinant plasmids were co-transformed into cdc25H, a temperature sensitive mutant of S.cerevisiae yeast and the potential interactions between dengue virus C protein and nonstructural proteins were checked. However, no direct interactions between these proteins were discovered. Host proteins that interact with dengue virus C protein were screened from human umbilical cord blood endothelial cells cDNA library. After screening and re-transforming, two positive clones were obtained, one of which was identified as human tarbp2 gene. These findings will contribute to further understandings of the mechanism of how dengue virus C protein modulates viral replication.In summary, in this study we found deletion ofα1-helix of C protein reduces viral RNA replication using replicon system, and we have identified a new conserved secondary structure, the cα1HP, which plays an important role in viral RNA replication in C gene by RNA structure prediction and mutagenesis. And for the first time, we provided evidence that the nuclear localization of dengue virus C protein correlates with viral RNA replication, and dengue virus C protein interacts with TARBP2. This work contributes to the understanding of functions of dengue virus C protein and the replication mechanism of dengue virus.