Functional Analysis And Applications Of The Noncoding Regions Of Hantavirus And A Preliminary Exploration On The Mucosal Immunity

Hantavirus is one of the Members of the Bunyaviridae family, which comprises more than 300 viruses grouped into the five genera (Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus), is enveloped viruses with a tripartite, single-stranded RNA genome of negative polarity. The L segment encodes the RNA-dependent RNA polymerase (L), the M segment encodes the two glycoproteins , and the S segment encodes the nucleoprotein (N).Hantavirus share several common features with other negative-strand RNA viruses. The templates for viral polymerase-catalyzed transcription and replication are the ribonucleoproteins (RNP), which for hantvirus consist of the full-length RNA segments associated with the nucleoprotein N and the viral polymerase L. The genomic viral RNA (vRNA) segments contain genes in antisense orientation flanked by stretches of noncoding nucleotides. The noncoding regions (NCRs) of segmented negative-stranded RNA viruses contain cis-acting sequences important for regulating RNA encapsidation, transcription, replication, and packaging of the viral genome segments. The terminal nucleotides are genus specific and highly conserved, and because of their partial inverted complementarities they can form double-stranded regions leading to circular RNAs, providing the functional promoter region for interaction with the viral polymerase. Chimeric minigenomes with flanking NCRs from different genome segments revealed that the number of total base pairs within the inverted, partially complementary ends was important for transcription and replication. The function of the remaining nucleotides of the NCRs is still not well understood.In this study, the green florescent protein (GFP) gene was respectively flanked with the noncoding regions (NCR) of Hantavirus L, M and S segments (strain A9 and L99). Then, these constructions were subcloned into the pHH21 vector by anti-sense orientation with the appropriate restriction endonucleases. The pHH21 vector has the RNA polymerase I (pol I) promoter and terminator sequences. These chimeric cDNAs (pol I expression cassettes) were cloned between the pol I promoter and the terminator. The 3 plasmids were transfected into Vero-E6 cells that were infected with A9 or L99 virus 7 days ago. The expression level of GFP was observed 24 to 48 hours after transfection, and mRNA levels of GFP were detected by quantitative real time polymerase chain reaction.In our study, comparison of the different segments showed that all NCRs were sufficient to mediate transcription/replication of a minigenome, M minigenomes showed the strongest GFP activity, followed by the L-segment-based construct and the S-segment-based construct. The mRNA levels of GFP of the three minigenomes was found also to be M > L > S by RT-PCR. Our study demonstrated decreased promoter strength of hantavirus NCRs in the order M > L > S.The terminal nucleotides of hantavirus are genus specific and highly conserved, because of their partial inverted complementarities they can form double-stranded regions leading to circular RNAs, providing the functional promoter region for interaction with the viral polymerase. The GFP gene was flanked with the NCR of Hantavirus (strain A9) M segment, subcloned between the RNA polymerase I promoter and terminator sequences by anti-sense orientation, and then inserted into the multiple cloning site of vector pRTE2hyg with the appropriate restriction endonucleases. A transfection solution was prepared by adding a mixture of pTRE2hyg-M-NCR-GFP and of Lipofectin reagent (Life Technologies) and incubated at room temperature for 30 min. The transfection solution was added to the cell culture. After incubation at 37 8C for 24 h, the solution was then replaced with DMEM. Cells were cultured in DMEM containing 300μg/ml of Hygromycin B for 3 weeks. The Hygromycin B -resistant colonies were selected and transferred to a 96 well plate, then were further selected. GFP of the bioluminescent jellyfish Aequorea victoria is a marker for gene expression with several promising features: small size of 238 amino acids, heat stability, and emission of green light after exposure to ultraviolet light without extrinsic labeling or fixation. In addition, GFP requires no cofactor and lacks apparent toxicity for eukaryotic cells. After infected by HV, the GFP in the cell line is expressed and thereby the viral infection can be visualized by UV microscopy. Since the hantavirus N protein trimers have species-specific character within genus hantavirus in recognition of the "panhandle" structure, we construct a cell line to monitor hantavirus with high specificity. There were no detectable GFP activities in uninfected cells, or following inoculations with several viruses other than hantavirus.In summary, the establishment of a stable cell line with a hantavius M-NCR-driven GFP reporter gene has provided a fast, easy, inexpensive, and accurate tool for monitoring and investigating HV infection, anti-HV drug activity, and cellular signaling. The unique feature of visualization, direct counting, and the possibility of sorting fluorescent cells without the addition of external substrate or labels are also of value compared with traditional methods.Route of transmission for hantavirus consist of respiratory tract and digestivetract, both covered with mucous membrane. The mature heat-labile enterotoxin (LT) from enterotoxic Escherichia coli consists of a single A subunit and five B subunits with each one being synthesized as a precursor polypeptide. After cleavage of the signal peptide, these two components of LT are released into the periplasmic space where they spontaneously assemble into a mature holotoxin. The LT is one of the most powerful, known mucosal adjuvants. Nowadays the B subunit of E. coli heat-labile enterotoxin (LTB) has shown to elicit a profound immunological response to co-administered antigens. Therefore, great efforts have been invested in the preparation and application of recombinant heat-labile enterotoxin B subunit (rLTB). Some studies have focused on the production of rLTB through eukaryotic expression systems, including yeast, transgenic tobacco systems and edible plants, but none is as convenient or high-yielding as the E. coli system.E. coli is one of the most convenient and extensively-used hosts for the expression of recombinant proteins in vitro. However, it has been difficult for a variety of proteins to be produced in an active form by E. coli. The pET System is the most powerful technique available for the cloning and expression of recombinant proteins by E. coli. One member of this system, pET22b (+) expression vectors harbor a pelB signal peptide at the protein N-terminal, which can direct the protein into the periplasmic space. The transfer of protein to the periplasmic region has numerous advantages, such as promoting separation from cytoplasmic proteins, providing an oxidizing medium, which is suitable for the formation of disulfide bonds, allowing for the refolding of the protein, and causing the formation of an active biological structure. In addition, the localization of the recombinant protein in the periplasm can simplify downstream stages of separation and purification. Genes of interest can be cloned into the pET plasmids, transformed into BL21 (DE3), and expressed under the inducer of isopropylβ-D-1-thiogalactopyranoside (IPTG). Lactose is transformed into allolactose in E. coli, and, as an analog of IPTG, is capable of inducing lac and its derivative promoters for the expression of genes of interest. Low cost and lack of toxicity make lactose an appropriate inducer for both vaccine and drug engineering applications.In this study, we obtained the LTB gene from pathogenic E. coli, cloned it into the pET22b (+) prokaryotic expression vector, and expressed it as a fusion protein with His tag in E. coli BL21 (DE3). The recombinant LTB was expressed and purified by Ni2+ affinity chromatography. The biological activity of the purified recombinant LTB was assayed in a series of monosialotetrahexosylganglioside (GM1)-ELISA experiments. The recombinant LTB (rLTB) was efficiently expressed under the induction of 10g/L lactose at 37oC for 6h and yielded up to 31% of the total bacterial protein. Fused with pelB signal peptide, rLTB was successfully localized to the periplasmic space. GM1-ELISA experiments showed that the rLTB obtained retains strong GM1 ganglioside-binding activity. The ELISA result of hantavirus nucleoprotein-specific secretory immunoglobulin A (sIgA) and IgG showed that intranasal administration of inactivated hantavirus with rLTB significantly increased the levels of hantavirus-specific sIgA (P<0.01) and IgG (P<0.01) in comparison with inactivated hatavirus alone.In summary, we compared the promoter activities of NCRs of three segments, established a stable cell line with a hantavius M-NCR-driven GFP reporter gene and provided a fast, easy, inexpensive, and accurate tool for monitoring and investigating HV infection, developed a method for the efficient secretory expression and purification of rLTB, and demonstrated that the inactivated hantavirus coadministered intranasally with rLTB could effectively induce both mucosal and humoral immune responses specific to hantavirus.