Molecular bases for modulation of host cellular responses by porcine reproductive and respiratory syndrome virus non-structural protein 11
Porcine reproductive and respiratory syndrome (PRRS) is a swine disease causing severe economic losses in the pork industry worldwide. In the present study, the modulation of host cell responses by PRRS virus (PRRSV) has been investigated. Non-structural protein 11 (Nsp11) contains a nidovirus-specific domain designated as NendoU, and this domain is known to have a endoribonuclease activity. This RNase activity is important but not essential for viral replication in equine arteritis virus (EAV), suggesting that Nsp11 may target cellular RNAs and thus may function to counteract the host’s antiviral response. By luciferase reporter assays, PRRSV Nsp11 was shown to have potent inhibition of IFN-β, IRF3, and NF-κB activities. The suppression was mediated by the reduced phosphorylation of IRF3 and IκB, which might be resulted from the degradation of IPS-1 mRNA. Further studies indicated that the IPS-1 mRNA degradation was associated with the NendoU activity of Nsp11. The Nsp11 mutants that lost the NendoU activity was unable to degrade IPS-1 mRNA and thus unable to suppress IFN production. To study whether NendoU domain was essential for PRRSV replication, eight NendoU mutant viruses were constructed. The NendoU knockout mutant viruses were unable to replicate, indicating that the PRRSV NendoU activity plays an essential role for viral replication and modulation of innate immunity during infection. To determine if the IFN suppression was independent function from the NendoU activity of Nsp11, Nsp11 genes were cloned and sequenced from 16 different North American PRRSV isolates. Eleven of 16 Nsp11 sequences showed nucleotide sequence changes but the NendoU catalytic sites were conserved. Most of the Nsp11 proteins exhibited strong inhibition of type I IFN production, but 17401-Nsp11 showed weak suppression when compared to FL12-Nsp11 and other Nsp11s. To map the site(s) causing this difference, nine substitutions were constructed based on the sequence alignment, and alanine at position 160 (Ala160) in 17401-Nsp11 was identified as the responsible site. This may function as an additional mechanism besides the NendoU activity to impede the type I IFN production. In addition to the modulation of host innate immune response, Nsp11 was also found to participate in other cellular progresses to facilitate the virus replication. Using the retrovirus gene transfer system, a cell line was established to stably express the Nsp11 protein and an RNA microarray was conducted in these cells. Based on the microarray data, five major cellular pathways were identified to be regulated by Nsp11; histone-related, cell cycle and DNA replication, mitogen activated protein kinase (MAPK) signaling, complement, and ubiquitin-proteasome pathways. Among them, the cell cycle pathway was further examined. The flow cytometry and BrdU staining of the Nsp11-expressing cells showed that Nsp11 protein delayed the cell cycle progression at the S phase during infection. Taken together, these studies provide further insights into the molecular basis of the manipulation of host cells processes by PRRSV Nsp11.
Porcine reproductive and respiratory syndrome virus (PRRSV), Interferon (IFN), Non-structural protein 11 (Nsp11), NendoU, cell cycle
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign