The Study Of Interaction Between FMDV 3C(D)^pro And Host Factor Fascin-1 In BHK-21 Cells

Foot-and-mouth disease (FMD) is one of the most highly contagious diseases of animals or humans, and the causative agent foot-and-mouth disease virus (FMDV) rapidly replicates and spreads within the infected animal, among in-contact susceptible animals, and by aerosol. FMDV is the prototype member of the Aphthovirus genus of the family Picornaviridae. The virus exists in the form of seven different serotypes:A, O, C, Asial, and South African Territories 1 (SAT1), SAT2, and SAT3, but a large number of subtypes have evolved within each serotype. Its genome is a single-stranded, positive-sense RNA which is over 8000 bases in length covalently bound at 5'-terminus to a 23-24 amino acid residue genome-linked protein, 3B. Replication of the FMDV genome RNA follows the same general pattern as replication of other members in the Picornaviridae family which occurs via a complementary negative strand RNA intermediate. To efficiently complete the life cycle of FMDV, not only viral proteins are involved, but also some host factors play important roles in most steps of viral infection, and identification of such host factors and their contributions has long been recognized as an important frontier.1. Construction of FMDV full-length cDNA clone.To stabilize FMDV genome and its genetical information, we successfully constructed type O FMDV full-length cDNA clone vector, pBluescript-FMDV. Basing on three incision enzyme sites within viral genome, four fragments were cloned into pBluescriptâ…¡KS(+) vector sequentially and then integrated which has artificial 25 poly(A) in 3'-terminal and 15 poly(C) within 5'UTR. 2. Establishment of a duplex quantitative real-time RT-PCR assay for simultaneous detection and quantitation of foot-and-mouth disease viral positive-stranded RNAs and negative-stranded RNAs.Simplified, cost-effective, two-step duplex quantitative real-time RT-PCR assay was shown to detect and quantify foot-and-mouth disease virus positive-stranded RNAs and negative-stranded RNAs simultaneously for improved investigation of the state of virus infection and replication. The primers and Taqman probes were selected from the coding regions of 2B gene and 3D gene respectively, which have the least variations among serotypes. Cells infected acutely, tissue samples and single cell samples were used for evaluation of the assay. At the early stages of virus infection in vitro, the replication level reached a peak at 9 h.p.i. and the negative strands were detectable until 3 h.p.i. The kinetics of ratios of positive strands to negative strands (+RNA/-RNA) in vivo in the liver, kidney and spleen were similar, which demonstrated that the replication dynamics were similar in the three organs.55 single cell samples out of 187 were positive by both positive strands qPCR and negative strands qPCR, the ratios (+RNA/-RNA) ranged from 15.6 to 1463.4 which showed considerable difference among single cell samples, indicating that active viral replication differs greatly in single cells. A duplex quantitative real-time RT-PCR was validated as effective and reliable.3. Interaction between FMDV 3CDpro and host factor Fascin-1 in BHK-21 cells.Both virus-encoded proteins and cellular factors are involved in foot-and-mouth disease virus (FMDV) replication and pathogenesis. Tandem affinity purification was used in our study to purify cellular interactors of FMDV recombined 3 CD premature protein,â–³3CDpro (deletion of C-terminal 30 amino acids of 3C fragment in order to inactivate its auto-cleaving function), one of which was then identified as Fascin-1 by mass spectrum. The interaction between Fascin-1 andâ–³3CDpro orâ–³3Cpro rather than 3Dpro was conformed by co-immunoprecipitation assays. Results of confocal immunofluorescence analysis showed that they were well co-located in the cytoplasm of BHK-21 cells. GST pull down assay demonstrated further that the fascin directly interact with the 3CDpro as well as one of its mature protein 3Cpro in vitro. Overexpression or down-regulated expression of fascin-1 could inhibit or promote the replication of FMDV in BHK-21 rather than PV in Vero cells, especially synthesis of negative-strand RNAs. Furthermore, the levels of fascin mRNA decreased in early step of FMDV infection (maybe the initiation of replication).