Mutations in a Highly Conserved Motif of nsp1β Protein Attenuate the Innate Immune Suppression Function of Porcine Reproductive and Respiratory Syndrome Virus

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1␤ (nsp1␤) is a multifunctional viral protein, which is involved in suppressing the host innate immune response and activating a unique ؊2/؊1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that the R128A or R129A mutation introduced into a highly conserved motif ( 123 GKYLQRRLQ 131 ) reduced the ability of nsp1␤ to suppress interferon beta (IFN-␤) activation and also impaired nsp1␤'s function as a PRF transactivator. Three recombinant viruses, vR128A, vR129A, and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild-type (WT) virus, vR128A and vR129A showed slightly reduced growth abilities, while the vRR129AA mutant had a significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, pigs infected with nsp1␤ mutants had lower levels of viremia than did WT virus-infected pigs. Compared to the WT virus in infected cells, all three mutated viruses stimulated high levels of IFN-␣ expression and exhibited a reduced ability to suppress the mRNA expression of selected interferon-stimulated genes (ISGs). In pigs infected with nsp1␤ mutants, IFN-␣ production was increased in the lungs at early time points postinfection, which was correlated with increased innate NK cell function. Furthermore, the augmented innate response was consistent with the increased production of IFN-␥ in pigs infected with mutated viruses. These data demonstrate that residues R128 and R129 are critical for nsp1␤ function and that modifying these key residues in the GKYL QRRLQ motif attenuates virus growth ability and improves the innate and adaptive immune responses in infected animals. IMPORTANCEPRRSV infection induces poor antiviral innate IFN and cytokine responses, which results in weak adaptive immunity. One of the strategies in next-generation vaccine construction is to manipulate viral proteins/genetic elements involved in antagonizing the host immune response. PRRSV nsp1␤ was identified to be a strong innate immune antagonist. In this study, two basic amino acids, R128 and R129, in a highly conserved GKYLQRRLQ motif were determined to be critical for nsp1␤ function. Mutations introduced into these two residues attenuated virus growth and improved the innate and adaptive immune responses of infected animals. Technologies developed in this study could be broadly applied to current commercial PRRSV modified live-virus (MLV) vaccines and other candidate vaccines. P orcine reproductive and respiratory syndrome (PRRS), a disease described in the United States in 1987 (1) and in Europe in 1990 (2), has caused tremendous economic losses to the swine industry since its appearance. Hallmark symptoms of PRRS are mild to severe respiratory disease in infected newborn and growing pigs and reproductive failure in pregnant sows. The etiologic agent, PRR...

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion

“…This finding was consistent with a previous report that once nsp1␤ was cleaved from nsp2 by autoproteolytic activity, PLP1␤ in nsp1␤ became inactive due to self-blocking by the conformational change of its own C-terminal extension (CTE) (76). A highly conserved domain had previously been identified within the PLP1␤ region (17,19), and L126 is one of the most critical residues that support nsp1␤ function (Fig. 4B) (19).…”

“…The SAP motif in nsp1␤ overlaps the highly conserved sequence 123 -GKYLQRRLQ-131 in PRRSV (18) and is also identified in lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV), but not in equine arteritis virus (EAV), in the family Arteriviridae. Mutations in the nsp1␤ SAP motif have conferred clinical attenuation of PRRSV in pigs (17,18,23). In our study, the SAP motif has been shown to be critical for nsp1␤-mediated host mRNA nuclear retention (19).…”

“…After translation, PRRSV nsp1␣ travels to the nucleus and triggers degradation of CREB (cyclic-AMP-responsive element binding)-binding protein (CBP), and this process causes the subversion of IFN production (12)(13)(14). In contrast, nsp1␤ is a multifunctional protein playing roles in viral genome replication (16), nsp2TF translational frame shifting (4), and type I IFN suppression (11,15,17,18). We have recently shown that nsp1␤ blocks the nuclear export of host mRNAs to the cytoplasm and causes a shutdown of host protein production and thus inhibition of host innate immunity (19).…”

Two Residues in NSP9 Contribute to the Enhanced Replication and Pathogenicity of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

“…The cell culture supernatant was harvested at 0, 24, 48, 72, and 96 hpi. The virus titers were measured by a microtitration assay using Marc-145 cells in 96-well plates and calculated as TCID 50 per milliliter according to the method of Reed and Muench (33). The sera from infected piglets were also probed by the same method.…”

The viral innate immune antagonism and an alternative vaccine design for PRRS virus

“…PRRSV whose PLP1b active sites are mutated is non-viable, indicating that PLP1b is essential for virus replication (Kroese et al, 2008). The PLP1b activity in nsp1b also regulates the generation of nsp2TF via À1/2 ribosomal-frameshifting (Fang et al, 2012;Li et al, 2016). A nuclease motif has been identified at K18-E32 in the N-terminal region of nsp1b (Xue et al, 2010), but this motif is irrelevant to IFN suppression (Han et al, 2014).…”