The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is a multidomain protein and has been shown to undergo remarkable genetic variation, primarily in its middle region, while exhibiting high conservation in the N-terminal putative protease domain and the C-terminal predicted transmembrane region. A reverse genetics system of PRRSV North American prototype VR-2332 was developed to explore the importance of different regions of nsp2 for viral replication. A series of mutants with in-frame deletions in the nsp2 coding region were engineered, and infectious viruses were subsequently recovered from transfected cells and further characterized. The results demonstrated that the cysteine protease domain (PL2), the PL2 downstream flanking sequence (amino acids [aa] 181 to 323), and the putative transmembrane domain were critical for replication. In contrast, the segment of nsp2 preceding the PL2 domain (aa 13 to 35) was dispensable for viral replication, and the nsp2 middle hypervariable region (aa 324 to 813) tolerated 100-aa or 200-aa deletions but could not be removed as a whole; the largest deletion was about 400 aa (nsp2Δ324-726). Characterization of the mutants demonstrated that those with small deletions possessed growth kinetics and RNA expression profiles similar to those of the parental virus, while the nsp2Δ324-726 mutant displayed decreased cytolytic activity on MARC-145 cells and did not develop visible plaques. Finally, the utilization of the genetic flexibility of nsp2 to express foreign genes was examined by inserting the gene encoding green fluorescent protein (GFP) in frame into one nsp2 deletion mutant construct. The recombinant virus was viable but impaired and unstable and gradually gained parental growth kinetics by the loss of most of the GFP gene.

Glycoprotein E (gE) of HSV plays a key role in cell-to-cell spread and virus-induced cell fusion. Here, we report that this function of gE requires the cooperation of tegument proteins UL11, UL16, and UL21. We found that the four proteins come together with very high efficiency to form a complex in transfected cells and in a manner that is regulated and coordinated. In particular, the inefficient interaction of UL16 with each membrane protein (UL11 and gE) observed in pairwise transfections became efficient when other binding partners were present. The significance of these interactions was revealed in studies of viral mutants, which showed that each of these tegument proteins is critical for processing, transport, and biological activity of gE. These findings provide insights into the mechanisms of how gE executes its function and also have implications in understanding HSV assembly and budding.

The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed bothtrans- andcis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr⁴⁷to Cys²⁴⁰) and aa 47 to 323 (Tyr⁴⁷to Leu³²³), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys⁵⁵- His¹²⁴catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys¹¹¹, Cys¹⁴², and Cys¹⁴⁷). The conserved aspartic acids (e.g., Asp⁸⁹) were essential for the PL2 proteasetrans-cleavage activity. Reverse genetics revealed that the PL2trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 proteasetransfunction, but not thecisactivity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G¹¹⁹⁶|G¹¹⁹⁷dipeptide.

The unfolded protein response (UPR) in the endoplasmic reticulum (ER) constitutes a critical component of host innate immunity against microbial infections. In this report, we show that porcine reproductive and respiratory syndrome virus (PRRSV) utilizes the UPR machinery for its own benefit. We provide evidence that the virus targets the UPR central regulator GRP78 for proteasomal degradation via a mechanism that requires viral glycoprotein GP2a, while both IRE1-XBP1s and PERK-eIF2α-ATF4 signaling branches of the UPR are turned on at early stage of infection. The activated effector XBP1s was found to enter the nucleus, but ATF4 was unexpectedly diverted to cytoplasmic viral replication complexes by means of nonstructural proteins nsp2/3 to promote viral RNA synthesis. RNAi knockdown of either ATF4 or XBP1s dramatically attenuated virus titers, while overexpression caused increases. These observations reveal attractive host targets (e.g., ATF4 and XBP1s) for antiviral drugs and have implications in vaccine development. Porcine reproductive and respiratory syndrome virus (PRRSV) poses a major threat to the worldwide swine industry, but no effective vaccines or antiviral drugs are available. A better understanding of the pathogen-host interactions that support PRRSV replication is essential for understanding viral pathogenesis and the development of preventive measures. Here we report that PRRSV utilizes unconventional strategies to reprogram the unfolded protein response (UPR) of the host to its own advantage. The virus targets GRP78 for partial degradation to create a favorable environment for UPR induction and hijacks ATF4 into cytoplasmic replication complexes to promote viral RNA synthesis. The data also reveal potential targets (e.g., ATF4 and XBP1s) for antiviral drugs and have implications in vaccine development.