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

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) blocks host mRNA nuclear export to the cytoplasm, and nonstructural protein 1 beta (nsp1␤) of PRRSV has been identified as the protein that disintegrates the nuclear pore complex. In the present study, the molecular basis for the inhibition of host mRNA nuclear export was investigated. Nucleoporin 62 (Nup62) was found to bind to nsp1␤, and the region representing the C-terminal residues 328 to 522 of Nup62 was determined to be the binding domain for nsp1␤. The nsp1␤ L126A mutant in the SAP domain did not bind to Nup62, and in L126A-expressing cells, host mRNA nuclear export occurred normally. The vL126A mutant PRRSV generated by reverse genetics replicated at a lower rate, and the titer was lower than for wild-type virus. In nsp1␤-overexpressing cells or small interfering RNA (siRNA)-mediated Nup62 knockdown cells, viral protein synthesis increased. Notably, the production of type I interferons (IFN-␣/␤), IFN-stimulated genes (PKR, OAS, Mx1, and ISG15 genes), IFNinduced proteins with tetratricopeptide repeats (IFITs) 1 and 2, and IFN regulatory factor 3 decreased in these cells. As a consequence, the growth of vL126A mutant PRRSV was rescued to the level of wild-type PRRSV. These findings are attributed to nuclear pore complex (NPC) disintegration by nsp1␤, resulting in increased viral protein production and decreased host protein production, including antiviral proteins in the cytoplasm. Our study reveals a new strategy of PRRSV for immune evasion and enhanced replication during infection. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS and is known to effectively suppress host innate immunity. The PRRSV nsp1␤ protein blocks host mRNA nuclear export, which has been shown to be one of the viral mechanisms for inhibition of antiviral protein production. nsp1␤ binds to the cellular protein nucleoporin 62 (Nup62), and as a consequence, the nuclear pore complex (NPC) is disintegrated and the nucleocytoplasmic trafficking of host mRNAs and host proteins is blocked. We show the dual benefits of Nup62 and nsp1␤ binding for PRRSV replication: the inhibition of host antiviral protein expression and the exclusive use of host translation machinery by the virus. Our study unveils a novel strategy of PRRSV for immune evasion and enhanced replication during infection.KEYWORDS Nuclear pore complex, nucleoporin, Nup62, arterivirus, nsp1, nucleocytoplasmic trafficking, porcine reproductive and respiratory syndrome virus P orcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that belongs to the family Arteriviridae in the order Nidovirales (https://talk.ictvonline .org/taxonomy). The PRRSV genome is a single-stranded positive-sense RNA approximately 15 kb in length with a 5= cap and a 3= polyadenylated tail and thus is translated FIG 8 Nup62 binding by nsp1 subunits of different arteriviruses and nuclear rim disintegration. (A) HeLa cells were cotransfected with the HA-Nup62 gene together with the indicated plasmids...

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

“…This activity is known to be associated with coronavirus nsp1, which inhibits nuclear export by interacting with the nuclear export factor NXF1 in SARS-CoV or the nuclear pore complex component Nup93 in SARS-CoV-2 [111,112] . Similarly, PRRSV nsp1β causes imprisonment of host mRNAs in the nucleus, by binding Nup62 to cause the disintegration of the nuclear pore complex 113,114 . This inhibition of host mRNA nuclear export has been found to reduce synthesis of interferon-stimulated genes 114 , and mutations in nsp1β which ablate this activity lead to reduced viral load and increased neutralising antibody titres in pigs 115 .…”

“…Similarly, PRRSV nsp1β causes imprisonment of host mRNAs in the nucleus, by binding Nup62 to cause the disintegration of the nuclear pore complex 113,114 . This inhibition of host mRNA nuclear export has been found to reduce synthesis of interferon-stimulated genes 114 , and mutations in nsp1β which ablate this activity lead to reduced viral load and increased neutralising antibody titres in pigs 115 . These findings suggest that nsp1β-mediated nuclear export inhibition may be responsible for the translational repression seen in our host differential gene expression analyses, which, analogously to conclusions drawn for SARS-CoV-2 [109,110] , may be a key mechanism by which PRRSV evades the host response to infection.…”

“…We detected heteroclite sgRNAs as early as 3 hpi, consistent with the finding that they are packaged into PRRSV virions 44,62 , and our results suggest they are present throughout infection. If they do produce functional proteins, this could serve as a mechanism to increase the levels of nsp1α and nsp1β, generally considered the most potent innate immune suppressors encoded by PRRSV [113][114][115][147][148][149][150][151] , from early timepoints onwards to evade immune activity.…”

The Function of the PRRSV–Host Interactions and Their Effects on Viral Replication and Propagation in Antiviral Strategies

“…Besides the abovementioned host factors, other cellular components are also involved in interacting with PRRSV. The cellular protein nucleoporin 62 (Nup62) interacts with nsp1β, leading to inhibition of host antiviral protein expression, revealing a new strategy of immune escape [ 47 ]. In addition, research found that cellular retinoblastoma protein (pRb) interacts with the nsp9 of genotype 2 PRRSV, which will benefit the replication of PRRSV-2 [ 49 ].…”

Porcine Reproductive and Respiratory Syndrome Virus Antagonizes PCSK9’s Antiviral Effect via Nsp11 Endoribonuclease Activity

“…Numerous host factors have been reported as either facilitating or suppressing virus infection and/or replication. These factors encompass cell surface receptors for PRRSV infection such as CD163, sialoadhesin, heparin sulfate, vimentin, CD151, and CD209 [24] and other cellular factors such as MYH9 [25], heparinase [26], CH25H [27,28], nucleoporin 62 [29], annexin A2 [30], ZAP [31], DHX36 [32], and so forth. Likewise, studies from our laboratory show that membrane proteins including MOV10, galectin-1, and galectin-3 can influence the replication of PRRSV [33][34][35].…”