Proteinas virales y celulares implicadas en la replicación del coronavirus

Résumé

Coronaviruses (CoVs) are enveloped plus-strand RNA viruses of the Nidovirales order, which cause a variety of enteric and respiratory diseases relevant in animal and human health, being of special interest the severe acute respiratory syndrome (SARS-CoV) in humans. CoV replication and transcription are complex processes that take place at cytoplasmic double membrane vesicles (DMVs) and involve coordinated processes of both continuous and discontinuous RNA synthesis. Both processes are mediated by a viral multienzymatic replicase complex encoded by the 20 Kb replicase gene, together with the participation of cellular factors, whose identity and role is largely unknown. The main objective of this thesis is the identification and the study of these viral and cell components of the CoV replication-transcription complex, using the SARS-CoV and the transmissible gastroenteritis coronavirus (TGEV) as models. To study the viral proteins involved in CoV RNA synthesis, a replicon of SARS-CoV was assembled into a bacterial artificial chromosome. The CoV replicase contains RNA dependent RNA polymerase (RdRp), RNA helicase, and protease activities (nsp3 and nsp5), which are common to positive-strand RNA viruses. In addition, the CoV replicase was predicted to contain the RNA-processing enzymes, 3¿-5¿ exoribonuclease, endoribonuclease, and 2¿-O-ribose methyltransferase, which are extremely rare or absent in other RNA viruses. Due the size of CoV genome (30 Kb) and the low fidelity of RdRp, these enzymatic activities might operate in a proof-reading mechanism to allow the stable maintenance of the CoV genome. Using the SARS-CoV replicon, we have shown that these RNA-processing enzymes were essential for efficient CoV replication and transcription. In addition to CoV replicons, the availability of replicase proteins specific antibodies may provide an excellent tool to study the precise strategies of CoV replication and transcription. To this end, specific antibodies against the TGEV RdRp and the replicase proteins nsp2, nsp3, nsp5 and nsp8 (primase) were generated and characterized. In the case of the RdRp, a set of six monoclonal antibodies (mAbs) was generated. These mAbs recognized four physically close linear epitopes located in a 62-amino acid region of the RdRp N-terminal domain, suggesting that this region may constitute an immunodominant domain. Using these antibodies, the expression kinetic and the subcellular localization of the RdRp and the nsps 2, 3, 5 and 8, were analyzed by Western blot and confocal microscopy, respectively. All of these proteins shown similar expression kinetics and colocalized in perinuclear vesicles, which were further identified as DMVs where the viral RNA synthesis takes place. Like many other RNA viruses, CoV may subvert several host factors to play a role in viral replication and transcription. The identification of these cellular factors was based of their interaction with either the TGEV genome ends, which contain essential cis-acting signals for viral RNA synthesis, or with the RdRp. Using the first approach, 10 cells proteins preferentially interacting with either the 5¿ or 3¿ ends of the TGEV genome were identified. Among these proteins, the polypyrimidine tract-binding protein (PTB) preferentially interacted with the 5¿ end of the genome, while a subset of 9 proteins, including several hnRNPs (A1, A0, A2B1, Q, and U), the glutamyl-prolyl-tRNA synthetase (EPRS), the arginyl-tRNA synthetase (RRS), the poly(A)-binding protein (PABP), and the p100 transcriptional co-activator, showed a preferential binding to the 3¿ end. Silencing studies using siRNAs were performed to analyze the relevance of these proteins on TGEV infection. A significant and highly reproducible reduction close to 3-fold in RNA synthesis and virus titers was found after silencing the expression of PABP, hnRNP Q and EPRS proteins, suggesting that these proteins play a positive role in TGEV infection. Interestingly, silencing of the control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) caused a 3-fold increase in TGEV RNA synthesis and virus titers, suggesting that this protein might have a role counteracting TGEV infection. Using the second approach, five cell proteins interacting with the RdRp were identified using the yeast two-hybrid system or by tandem-affinity-purification. These proteins included the HSP70, the ß-actin, the casein subunit 1¿, the splicing factor PUF60 and the transcription factor TRIM27. The relevance of these proteins in CoV RNA synthesis is being studied. In contrast to negative-strand RNA viruses, it is currently accepted that in plus-strand RNA viruses the replication complex is not encapsidated. However, a proteomic analysis of highly purified TGEV suggested the incorporation of the RdRp and several cell proteins into the viral particles. To further investigate whether the RdRp is encapsidated, its presence into viral particles was analyzed by Western blot. A polypeptide of 105 kDa, corresponding to RdRp, was specifically detected in highly purified TGEV virions even after treatment with proteinase K. To provide additional evidence of the RdRp encapsidation, purified TGEV was analyzed by confocal microscopy and immuno-electron microscopy, demonstrating that RdRp was incorporated into the virions. Taken into consideration that RdRp is a key enzyme of the replication-transcription complex, the encapsidation of other viral components of this complex, such us the nsps 2, 3 and 8, was also analyzed. The nsps 2 and 8 were also incorporated into the viral particles, and nsp3 was probably present in the viral envelope. Interestingly, several cell proteins involved in CoV RNA synthesis, such us the PABP, GAPDH, hnRNP Q and EPRS, were also detected within the viral particles, suggesting that the replication complex might be encapsidated in CoV. It could be postulated that this complex could act as a starting replication machinery, leading to a limited genome amplification before translation, to improve the efficiency of virus infection.