Search

Highlights • PRRSV induces the formation of bona fide stress granules (SGs) at late time points post-infection. • PRRSV-induced SGs are closely associated with VRCs, yet not required for efficient viral replication. • Arrest of cellular translation coincides with SG formation in PRRSV-infected cells. Abstract Stress granules (SGs) are dynamic sites of cytosolic mRNA storage that are formed in response to stress conditions, including viral infection. SGs have been implicated in regulating several aspects of the host immune response to various pathogens. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically-important global swine pathogen, reportedly induced SGs during replication, although the underlying mechanisms are poorly defined. In this study, we delineated the molecular mechanisms regulating the SG response to PRRSV infection. Using confocal microscopy, we first demonstrated that infection with PRRSV strain VR2385 induces an accumulation of the SG markers G3BP1, G3BP2, TIAR, eIF3b, and USP10 as well as mRNAs into punctate structures in the cytoplasm of infected host cells. Subsequently, we demonstrated that the PRRSV-induced SGs were in close proximity to viral replication complexes (VRCs) and processing bodies (P-bodies), and that SG formation was coordinated with inhibition of host cellular translation. Treatment of infected cells with cycloheximide disrupted the PRRSV-induced SGs. Furthermore, impairment of SG assembly by the shRNA-mediated knockdown of G3BP1, G3BP2 and USP10 did not affect viral replication. Collectively, these results demonstrate that PRRSV infection induces formation of SGs associated with VRCs, which is coordinated with the suppression of host cell protein synthesis. This is the first study to extensively characterize the formation and underlying mechanism of bona fide SGs during PRRSV infection. Our findings have important implications in understanding the mechanism of PRRSV-host interactions. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease, and has a complicated virus–host immunomodulation that often leads to a weak Th2 immune response and viral persistence. In this study, we identified a Src homology 3 (SH3) binding motif, PxxPxxP, that is conserved within the N protein of PRRSV strains. Subsequently, we identified five host cellular proteins [signal transducing adaptor molecule (STAM)I, TXK tyrosine kinase (TXK), protein tyrosine kinase fyn (Fyn), hematopoietic cell kinase (Hck), and cortactin] that interact with this SH3 motif. We demonstrated that binding of SH3 proteins with PRRSV N protein depends on at least one intact PxxP motif as disruption of P53 within the motif significantly reduced interaction of each of the 5 proteins. The first PxxP motif appears to be more important for STAMI–N protein interactions whereas the second PxxP motif was more important for Hck interaction. Both STAMI and Hck interactions with PRRSV N protein required an unhindered C-terminal domain as the interaction was only observed with STAMI and Hck proteins with N-terminal but not C-terminal fluorescent tags. We showed that the P56 residue within the SH3 motif is critical for virus lifecycle as mutation resulted in a loss of virus infectivity, however the P50 and P53 mutations did not abolish virus infectivity suggesting that these highly conserved proline residues within the SH3 motif may provide a selective growth advantage through interactions with the host rather than a vital functional element. These results have important implications in understanding PRRSV–host interactions. [ABSTRACT FROM AUTHOR]

• PRRSV induces UPR through all 3 signaling branches, including PERK, IRE1a and ATF6. • Induction of UPR significantly suppresses PRRSV replication and RNA synthesis. • The PERK arm of the UPR modulates the PRRSV-induced cytokine responses. Replication of most RNA viruses is closely associated with the endoplasmic reticulum (ER) within permissive cells. As such, viruses often induce tremendous amounts of stress on cells during viral replication. To cope with the stress, cells initiate the unfolded protein response (UPR) to promote cellular survival. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically important swine pathogen, was previously shown to induce cellular stress at late stages post-infection resulting in the formation of stress granules (SGs). Here in this study, we demonstrate that PRRSV also induces additional cellular response pathways, including the UPR. Confocal microscopy analysis demonstrated significant morphological changes in the ER of PRRSV-infected cells, indicative of pronounced ER stress. Further investigation revealed induction of all three branches of the UPR, including eukaryotic translation initiation factor 2-alpha kinase 3 (PERK), serine-threonine protein kinase/endoribonuclease (IRE1), and cyclic AMP-dependent transcription factor (ATF6). Activation of these sensors resulted in significant transcriptional upregulation of downstream UPR effectors. Additionally, UPR activation was shown to be detrimental to PRRSV replication, as treatment of cells with chemical ER stress inducers potently suppressed viral replication and RNA synthesis. Further investigation into the molecular mechanisms of UPR suppression of PRRSV replication revealed that PERK exacerbates the PRRSV-induced cytokine response. Collectively, these results demonstrate that PRRSV infection induces UPR activation through all three branches, and that UPR signaling may play a role in PRRSV pathogenesis. The results of this study further our understanding of the underlying molecular mechanisms of PRRSV replication and host-pathogen interactions. [ABSTRACT FROM AUTHOR]

We previously demonstrated that the C-terminus of the capsid gene of porcine circovirus type 2 (PCV2) is an immune reactive epitope displayed on the surface of virions. Insertion of foreign epitope tags in the C-terminus produced infectious virions that elicited humoral immune responses against both PCV2 capsid and the inserted epitope tags, whereas mutation in the N terminus impaired viral replication. Since the non-pathogenic porcine circovirus type 1 (PCV1) shares similar genomic organization and significant sequence identity with pathogenic PCV2, in this study we evaluated whether PCV1 can serve as a vaccine delivery virus vector. Four different antigenic determinants of porcine reproductive and respiratory syndrome virus (PRRSV) were inserted in the C-terminus of the PCV1 capsid gene, the infectivity and immunogenicity of the resulting viruses are determined. We showed that an insertion of 12 (PRRSV-GP2 epitope II, PRRSV-GP3 epitope I, and PRRSV-GP5 epitope I), and 14 (PRRSV-GP5 epitope IV) amino acid residues did not affect PCV1 replication. We successfully rescued and characterized four chimeric PCV1 viruses expressing PRRSV linear antigenic determinants (GP2 epitope II: aa 40–51, ASPSHVGWWSFA; GP3 epitope I: aa 61–72, QAAAEAYEPGRS; GP5 epitope I: aa 35–46, SSSNLQLIYNLT; and GP5 epitope IV: aa 187–200, TPVTRVSAEQWGRP). We demonstrated that all chimeric viruses were stable and infectious in vitro and three chimeric viruses were infectious in vivo . An immunogenicity study in pigs revealed that PCV1-VR2385 EPI chimeric viruses elicited neutralizing antibodies against PRRSV-VR2385. The results have important implications for further evaluating PCV1 as a potential vaccine delivery vector. [ABSTRACT FROM AUTHOR]

Co-infection of pigs in the field with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) is common and poses a major concern in effective control of PCV2 and PRRSV. We previously demonstrated that insertion of foreign epitope tags in the C-terminus of PCV2 ORF2 produced infectious virions that elicited humoral immune responses against both PCV2 capsid and inserted epitope tags. In this study, we aimed to determine whether the non-pathogenic chimeric virus PCV1-2a, which is the basis for the licensed PCV2 vaccine Fostera™ PCV, can express PRRSV antigenic epitopes, thus generating dual immunity as a potential bivalent vaccine against both PCV2 and PPRSV. Four different linear B-cell antigenic epitopes of PRRSV were inserted into the C-terminus of the capsid gene of the PCV1-2a vaccine virus. We showed that insertion of 12 (PRRSV-GP2 epitope II, PRRSV-GP3 epitope I, and PRRSV-GP5 epitope I), and 14 (PRRSV-GP5 epitope IV) amino acid residues did not impair the replication of the resulting PCV1-2a-PRRSV EPI chimeric viruses in vitro . The four chimeric PCV1-2a viruses expressing PRRSV B-cell linear epitopes were successfully rescued and characterized. An immunogenicity study in pigs revealed that two of the four chimeric viruses, PCV1-2a-PRRSV EPI GP3IG and PCV1-2a-PRRSVEPI EPI GP5IV, elicited neutralizing antibodies against PRRSV VR2385 as well as PCV2 (strains PCV2a, PCV2b, and mPCV2b). The results have important implications for exploring the potential use of PCV1-2a vaccine virus as a live virus vector to develop bivalent MLVs against both PCV2 and PRRSV. [ABSTRACT FROM AUTHOR]

Highlights: [•] The genetic variability of PRRSV during serial passage in PCV2-infected pigs was investigated. [•] PRRSV structural genes ORF6 and ORF7 mutated at various degrees over time in vivo. [•] A significantly higher mutation rate was observed when pigs were co-infected with PCV2. [Copyright &y& Elsevier]

Abstract: A highly pneumovirulent strain of porcine reproductive and respiratory syndrome virus (PRRSV), ATCC VR2385, was isolated from a pig exhibiting typical PRRS in the early 90s. While passaging the virus in monkey kidney cells, we identified a large spontaneous deletion of a 435-bp in the nsp2 gene. To assess the biological significance of this spontaneous deletion, we first determined the full-length genomic sequence of this virus and established a DNA-launched infectious clone of the passage 14 virus containing the 435-bp nsp2 deletion (designated as pIR-VR2385-CA). The full-length viral genome engineered with two ribozyme elements at both ends was placed under the control of the eukaryotic CMV promoter. The infectious virus was successfully rescued from pIR-VR2385-CA DNA-transfected BHK-21 cells. To characterize the biological and pathological significance of this large nsp2 deletion, we subsequently constructed another DNA-launched infectious clone, pIR-VR2385-R, in which we restored the deleted 435-bp nsp2 sequence back to the pIR-VR2385-CA backbone. The growth characteristics of the two rescued viruses (VR2385-CA and VR2385-R) were compared, and the results showed that the VR2385-CA virus with the nsp2 deletion replicated more efficiently in vitro (1.0–1.5 log titer higher) than the VR2385-R virus with the restored nsp2 sequence but the VR2385-CA virus exhibited a significantly reduced serum viral RNA load in vivo. A comparative pathogenicity study in pigs (n =10) revealed that the nsp2 deletion had no effect on virus virulence, and the restored nsp2 sequence in the VR2385-R virus remains stable during virus replication in pigs. The results from this study indicates that the spontaneous nsp2 deletion plays a role for enhanced PRRSV replication in vitro but has no effect on the pathogenicity of the virus. [Copyright &y& Elsevier]