Yan-Jin Zhang

Porcine Reproductive and Respiratory Syndrome Virus Inhibits Type I Interferon Signaling by Blocking STAT1/STAT2 Nuclear Translocation

ABSTRACT Type I interferons (IFNs) IFN-α/β play an important role in innate immunity against viral infections by inducing antiviral responses. Porcine reproductive and respiratory syndrome virus (PRRSV) inhibits the synthesis of type I IFNs. However, whether PRRSV can inhibit IFN signaling is less well understood. In the present study, we found that PRRSV interferes with the IFN signaling pathway. The transcript levels of IFN-stimulated genes ISG15 and ISG56 and protein level of signal transducer and activator of transcription 2 (STAT2) in PRRSV VR2385-infected MARC-145 cells were significantly lower than those in mock-infected cells after IFN-α treatment. IFN-induced phosphorylation of both STAT1 and STAT2 and their heterodimer formation in the PRRSV-infected cells were not affected. However, the majority of the STAT1/STAT2/IRF9 (IFN regulatory factor 9) heterotrimers remained in the cytoplasm of PRRSV-infected cells, which indicates that the nuclear translocation of the heterotrimers was blocked. Overexpression of NSP1β of PRRSV VR2385 inhibited expression of ISG15 and ISG56 and blocked nuclear translocation of STAT1, which suggests that NSP1β might be the viral protein responsible for the inhibition of IFN signaling. PRRSV infection in primary porcine pulmonary alveolar macrophages (PAMs) also inhibited IFN-α-stimulated expression of the ISGs and the STAT2 protein. In contrast, a licensed low-virulence vaccine strain, Ingelvac PRRS modified live virus (MLV), activated expression of IFN-inducible genes, including those of chemokines and antiviral proteins, in PAMs without the addition of external IFN and had no detectable effect on IFN signaling. These findings suggest that PRRSV interferes with the activation and signaling pathway of type I IFNs by blocking ISG factor 3 (ISGF3) nuclear translocation.

A pneumo-virulent United States isolate of porcine reproductive and respiratory syndrome virus induces apoptosis in bystander cells both in vitro and in vivo

Evidence of apoptosis was detected for the United States porcine reproductive and respiratory syndrome virus (PRRSV) in ATCC CRL11171 cells inoculated with strain ATCC VR2385 and in the tissues of pigs infected with the same strain. Apoptosis was detected by agarose gel electrophoresis, transmission electron microscopy and terminal deoxytransferase dUTP nick end labelling (TUNEL) techniques. By electron microscopy and double-labelling techniques, apoptosis was detected primarily in uninfected bystander cells in the continuous cell line rather than the PRRSV-infected cells. In the lungs, the apoptotic cells were predominantly alveolar and pulmonary intravascular macrophages, and mononuclear cells in the alveolar septa. In the lymph nodes, the apoptotic cells were predominantly tingible body macrophages and mononuclear cells. The induction of apoptosis in a large number of mononuclear cells in the lungs and lymph nodes appears to be a mechanism of PRRSV pathogenesis and might be an explanation for a dramatic reduction in the number of alveolar macrophages and circulating lymphocytes and monocytes in PRRSV-infected pigs.

The Hepatitis E Virus Open Reading Frame 3 Product Interacts with Microtubules and Interferes with Their Dynamics

ABSTRACT Hepatitis E virus (HEV) is the causative agent of hepatitis E, a major form of viral hepatitis in developing countries. The open reading frame 3 (ORF3) of HEV encodes a phosphoprotein with a molecular mass of approximately 13 kDa (hereinafter called vp13). vp13 is essential for establishing HEV infections in animals, yet its exact functions are still obscure. Our current study found evidence showing interaction between vp13 and microtubules. Live-cell confocal fluorescence microscopy revealed both filamentous and punctate distribution patterns of vp13 in cells transfected with recombinant ORF3 reporter plasmids. The filamentous pattern of vp13 was altered by a microtubule-destabilizing drug. The vp13 expression led to elevation of acetylated α-tubulin, indicating increased microtubule stability. Its association with microtubules was further supported by its presence in microtubule-containing pellets in microtubule isolation assays. Exposure of these pellets to a high-salt buffer caused release of the vp13 to the supernatant, suggesting an electrostatic interaction. Inclusion of ATP and GTP in the lysis buffer during microtubule isolation also disrupted the interaction, indicating its sensitivity to the nucleotides. Further assays showed that motor proteins are needed for the vp13 association with the microtubules because disruption of dynein function abolished the vp13 filamentous pattern. Analysis of ORF3 deletion constructs found that both of the N-terminal hydrophobic domains of vp13 are needed for the interaction. Thus, our findings suggest that the vp13 interaction with microtubules might be needed for establishment of an HEV infection.

Porcine Reproductive and Respiratory Syndrome Virus Nsp1β Inhibits Interferon-Activated JAK/STAT Signal Transduction by Inducing Karyopherin-α1 Degradation

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) inhibits the interferon-mediated antiviral response. Type I interferons (IFNs) induce the expression of IFN-stimulated genes by activating phosphorylation of both signal transducer and activator of transcription 1 (STAT1) and STAT2, which form heterotrimers (interferon-stimulated gene factor 3 [ISGF3]) with interferon regulatory factor 9 (IRF9) and translocate to the nucleus. PRRSV Nsp1β blocks the nuclear translocation of the ISGF3 complex by an unknown mechanism. In this study, we discovered that Nsp1β induced the degradation of karyopherin-α1 (KPNA1, also called importin-α5), which is known to mediate the nuclear import of ISGF3. Overexpression of Nsp1β resulted in a reduction of KPNA1 levels in a dose-dependent manner, and treatment of the cells with the proteasome inhibitor MG132 restored KPNA1 levels. Furthermore, the presence of Nsp1β induced an elevation of KPNA1 ubiquitination and a shortening of its half-life. Our analysis of Nsp1β deletion constructs showed that the N-terminal domain of Nsp1β was involved in the ubiquitin-proteasomal degradation of KPNA1. A nucleotide substitution resulting in an amino acid change from valine to isoleucine at residue 19 of Nsp1β diminished its ability to induce KPNA1 degradation and to inhibit IFN-mediated signaling. Interestingly, infection of MARC-145 cells by PRRSV strains VR-2332 and VR-2385 also resulted in KPNA1 reduction, whereas infection by an avirulent strain, Ingelvac PRRS modified live virus (MLV), did not. MLV Nsp1β had no effect on KPNA1; however, a mutant with an amino acid change at residue 19 from isoleucine to valine induced KPNA1 degradation. These results indicate that Nsp1β blocks ISGF3 nuclear translocation by inducing KPNA1 degradation and that valine-19 in Nsp1β correlates with the inhibition.

Hepatitis E Virus Inhibits Type I Interferon Induction by ORF1 Products

ABSTRACT Hepatitis E virus (HEV) causes both endemic and epidemic human hepatitis by fecal-oral transmission in many parts of the world. Zoonotic transmission of HEV from animals to humans has been reported. Due to the lack of an efficient cell culture system, the molecular mechanisms of HEV infection remain largely unknown. In this study, we found that HEV replication in hepatoma cells inhibited poly(I·C)-induced beta interferon (IFN-β) expression and that the HEV open reading frame 1 (ORF1) product was responsible for this inhibition. Two domains, X and the papain-like cysteine protease domain (PCP), of HEV ORF1 were identified as the putative IFN antagonists. When overexpressed in HEK293T cells, the X domain (or macro domain) inhibited poly(I·C)-induced phosphorylation of interferon regulatory factor 3 (IRF-3), which is the key transcription factor for IFN induction. The PCP domain was shown to have deubiquitinase activity for both RIG-I and TBK-1, whose ubiquitination is a key step in their activation in poly(I·C)-induced IFN induction. Furthermore, replication of a HEV replicon containing green fluorescent protein (GFP) (E2-GFP) in hepatoma cells led to impaired phosphorylation of IRF-3 and reduced ubiquitination of RIG-I and TBK-1, which confirmed our observations of X and PCP inhibitory effects in HEK293T cells. Altogether, our study identified the IFN antagonists within the HEV ORF1 polyprotein and expanded our understanding of the functions of several of the HEV ORF1 products, as well as the mechanisms of HEV pathogenesis. IMPORTANCE Type I interferons (IFNs) are important components of innate immunity and play a crucial role against viral infection. They also serve as key regulators to evoke an adaptive immune response. Virus infection can induce the synthesis of interferons; however, viruses have evolved many strategies to antagonize the induction of interferons. There is little knowledge about how hepatitis E virus (HEV) inhibits induction of host IFNs, though the viral genome was sequenced more than 2 decades ago. This is the first report of identification of the potential IFN antagonists encoded by HEV. By screening all the domains in the open reading frame 1 (ORF1) polyprotein, we identified two IFN antagonists and performed further research to determine how and at which step in the IFN induction pathway they antagonize host IFN induction. Our work provides valuable information about HEV-cell interaction and pathogenesis.

Peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication

Abstract Porcine reproductive and respiratory syndrome (PRRS) has been devastating the global swine industry for more than a decade, and current strategies to control PRRS are inadequate. In this study we characterized the inhibition of PRRS virus (PRRSV) replication by antisense phosphorodiamidate morpholino oligomers (PMO). Of 12 peptide-conjugated PMO (PPMO), four were found to be highly effective at inhibiting PRRSV replication in cell culture in a dose-dependant and sequence-specific manner. PPMO 5UP2 and 5HP are complementary to sequence in the 5′ end of the PRRSV genome, and 6P1 and 7P1 to sequence in the translation initiation regions of ORF6 and ORF7, respectively. Treatment of cells with 5UP2 or 5HP caused a 4.5log10 reduction in PRRSV yield, compared to a control PPMO. Combination of 6P1 and 7P1 led to higher level reduction than 6P1 or 7P1 alone. 5UP2, 5HP, and a combination of 6P1 and 7P1 inhibited PRRSV replication in porcine alveolar macrophages and protected the cells from PRRSV-induced cytopathic effect. Northern blot and real-time RT-PCR results demonstrated that the effective PPMO led to a reduction of PRRSV RNA level. 5UP2 and 5HP inhibited virus replication of 10 other strains of PRRSV. Results from this study suggest potential applications of PPMO for PRRS control.

Induction of type I interferons by a novel porcine reproductive and respiratory syndrome virus isolate

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is known to interfere with the signaling of type I interferons (IFNs). Here we found PRRSV A2MC2 induced type I IFNs in cultured cells. A2MC2 replication in MARC-145 cells resulted in the synthesis of IFN-α2 protein, transcript elevation of the IFN-stimulated genes ISG15 and ISG56, and the proteins of the signal transducer and activator of transcription 2 (STAT2) and ISG56. A2MC2 infection of primary porcine pulmonary alveolar macrophages (PAMs) also led to the elevation of the two proteins, but had little cytopathic effect. Furthermore, A2MC2 infection of MARC-145 or PAM cells had no detectable inhibitory effect on the ability of IFN-α to induce an antiviral response. Sequencing analysis indicated that A2MC2 was closely related to VR-2332 and Ingelvac PRRS MLV with an identity of 99.8% at the nucleotide level. The identification of this IFN-inducing PRRSV isolate may be beneficial for vaccine development against PRRS.

Inhibition of Replication and Transcription Activator and Latency-Associated Nuclear Antigen of Kaposi’s Sarcoma-Associated Herpesvirus by Morpholino Oligomers

Abstract Kaposis sarcoma-associated herpesvirus (KSHV) is associated with Kaposis sarcoma and primary effusion lymphoma (PEL). The KSHV replication and transcription activator (RTA) and latency-associated nuclear antigen (LANA) play key roles in activating KSHV lytic replication and maintaining KSHV latency, respectively. Phosphorodiamidate morpholino oligomers (PMO) are similar to short single-stranded DNA oligomers, but possess a modified backbone that confers highly specific binding and resistance to nucleases. In this study, RTA and LANA mRNA in PEL cells were targeted by antisense peptide-conjugated PMO (P-PMO) in an effort to suppress KSHV replication. Highly efficient P-PMO uptake by PEL cells was observed. Treatment of PEL cells with a RTA P-PMO (RP1) reduced RTA expression in a dose-dependent and sequence-specific manner, and also caused a significant decrease in several KSHV early and late gene products, including vIL-6, vIRF-1, and ORF-K8.1A. KSHV viral DNA levels were reduced both in cells and culture supernatants of RP1 P-PMO-treated cells, indicating that KSHV lytic replication was suppressed. Treatment of BCBL-1 cells with P-PMO against LANA resulted in a reduction of LANA expression. Cell viability assays detected no cytotoxicity from P-PMO alone, within the concentration range used for the experiments in this study. These results suggest that RP1 P-PMO can specifically block KSHV replication, and further study is warranted.

Suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a contagious disease characterized by reproductive failure in sows and respiratory disease in piglets. This infectious disease results in significant losses in the swine industry and specific anti-PRRSV drugs are needed. In this study, we evaluated a novel class of antisense compounds, peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs), for their ability to suppress PRRSV replication in cell culture. P-PMOs are analogs of single-stranded DNA and contain a modified backbone that confers highly specific binding to RNA and resistance to nucleases. Of six P-PMOs tested, one (‘5UP1’), with sequence complementary to the 5′-terminal 21 nucleotides of the PRRSV genome, was found to be highly effective at reducing PRRSV replication in a specific and dose-dependent manner in CRL11171 cells in culture. 5UP1 treatment generated up to a 4.5log reduction in infectious PRRSV yield, while a control P-PMO had no effect on viral titer. Immunofluorescence assay with an anti-PRRSV monoclonal antibody confirmed the titer observations. The sequence-specificity of 5UP1 effect was confirmed in part by a cell-free luciferase reporter assay system, which showed that 5UP1-mediated inhibition of translation decreased if the target-RNA contained mispairings in relation to the 5UP1 P-PMO. Real-time RT-PCR showed that the production of PRRSV negative-sense RNA was reduced if 5UP1 was added to cells at up to 6h post-virus inoculation. Cell viability assays detected no cytotoxicity of 5UP1 within the concentration-range of this study. These results indicate that P-PMO 5UP1 has potential as an anti-PRRSV agent.

Antagonizing Interferon-Mediated Immune Response by Porcine Reproductive and Respiratory Syndrome Virus

Interferons (IFNs) are important components in innate immunity involved in the first line of defense to protect host against viral infection. Porcine reproductive and respiratory syndrome virus (PRRSV) leads to severe economic losses for swine industry since being first identified in early 1990s. PRRSV interplays with host IFN production and IFN-activated signaling, which may contribute to the delayed onset and low level of neutralizing antibodies, as well as weak cell-mediated immune response in infected pigs. PRRSV encodes several proteins that act as antagonists for the IFN signaling. In this review, we summarized the various strategies used by PRRSV to antagonize IFN production and thwart IFN-activated antiviral signaling, as well as the variable interference with IFN-mediated immune response by different PRRSV strains. Thorough understanding of the interaction between PRRSV and host innate immune response will facilitate elucidation of PRRSV pathogenesis and development of a better strategy to control PRRS.