Yan Zhou

A recombinant E1-deleted porcine adenovirus-3 as an expression vector.

Replication-defective E1-deleted porcine adenoviruses (PAVs) are attractive vectors for vaccination. As a prerequisite for generating PAV-3 vectors containing complete deletion of E1, we transfected VIDO R1 cells (fetal porcine retina cells transformed with E1 region of human adenovirus 5) with a construct containing PAV-3 E1B(large) coding sequences under the control of HCMV promoter. A cell line named VR1BL could be isolated that expressed E1B(large) of PAV-3 and also complemented PAV214 (E1A+E1B(small) deleted). The VR1BL cells could be efficiently transfected with DNA and allowed the rescue and propagation of recombinant PAV507 containing a triple stop codon inserted in the E1B(large) coding sequence. In addition, recombinant PAV227 containing complete deletion of E1 (E1A+E1B(small) + E1B(large)) could be successfully rescued using VR1BL cell line. Recombinant PAV227 replicated as efficiently as wild-type in VR1BL cells but not in VIDO R1 cells, suggesting that E1B(large) was essential for replication of PAV-3. Next, we constructed recombinant PAV219 by inserting green fluorescent (GFP) protein gene flanked by a promoter and a poly(A) in the E1 region of the PAV227 genome. We demonstrated that PAV219 was able to transduce and direct expression of GFP in some human cell lines.

Hepatitis C virus non-structural protein-2 activates CXCL-8 transcription through NF-κB

SummaryHepatitis C is a devastating disease worldwide. Proteins encoded by the etiologic agent, hepatitis C virus (HCV), are believed to play important roles in HCV-associated pathogenesis. However, the biological functions of the non-structural protein-2 (NS2) encoded by HCV are not well characterized. Here, we show that HCV NS2 protein activates CXCL-8 (interleukin-8, IL-8) transcription in HepG2 cells as measured by reverse transcription-polymerase chain reaction and IL-8 promoter-luciferase reporter assays. Furthermore, when the κB site on the IL-8 promoter was eliminated by mutagenesis or when intracellular NF-κB activity was suppressed by an inhibitor, NS2 did not activate the IL-8 promoter, suggesting a role of NF-κB in this process. These results prompted us to hypothesize that HCV NS2 might be able to activate NF-κB. This hypothesis was tested by determination of NF-κB-driven reporter gene expression and NF-κB p65 subunit subcellular localization after HCV NS2 expression. Indeed, NS2 could up-regulate NF-κB-driven luciferase activity and was associated with p65 nuclear localization. These results demonstrate that HCV NS2 up-regulates IL-8 transcription through NF-κB. This newly identified function increases our understanding of the role of HCV NS2 protein in virus-host interactions.

DDX3 Interacts with Influenza A Virus NS1 and NP Proteins and Exerts Antiviral Function through Regulation of Stress Granule Formation

ABSTRACT DDX3 belongs to the DEAD box RNA helicase family and is a multifunctional protein affecting the life cycle of a variety of viruses. However, its role in influenza virus infection is unknown. In this study, we explored the potential role of DDX3 in influenza virus life cycle and discovered that DDX3 is an antiviral protein. Since many host proteins affect virus life cycle by interacting with certain components of the viral machinery, we first verified whether DDX3 has any viral interaction partners. Immunoprecipitation studies revealed NS1 and NP as direct interaction partners of DDX3. Stress granules (SGs) are known to be antiviral and do form in influenza virus-infected cells expressing defective NS1 protein. Additionally, a recent study showed that DDX3 is an important SG-nucleating factor. We thus explored whether DDX3 plays a role in influenza virus infection through regulation of SGs. Our results showed that SGs were formed in infected cells upon infection with a mutant influenza virus lacking functional NS1 (del NS1) protein, and DDX3 colocalized with NP in SGs. We further determined that the DDX3 helicase domain did not interact with NS1 and NP; however, it was essential for DDX3 localization in virus-induced SGs. Knockdown of DDX3 resulted in impaired SG formation and led to increased virus titers. Taken together, our results identified DDX3 as an antiviral protein with a role in virus-induced SG formation. IMPORTANCE DDX3 is a multifunctional RNA helicase and has been reported to be involved in regulating various virus life cycles. However, its function during influenza A virus infection remains unknown. In this study, we demonstrated that DDX3 is capable of interacting with influenza virus NS1 and NP proteins; DDX3 and NP colocalize in the del NS1 virus-induced SGs. Furthermore, knockdown of DDX3 impaired SG formation and led to a decreased virus titer. Thus, we provided evidence that DDX3 is an antiviral protein during influenza virus infection and its antiviral activity is through regulation of SG formation. Our findings provide knowledge about the function of DDX3 in the influenza virus life cycle and information for future work on manipulating the SG pathway and its components to fight influenza virus infection.

Determination of bovine adenovirus-3 titer based on immunohistochemical detection of DNA binding protein in infected cells

DNA sequence coding for a portion of DNA binding protein (amino acids 3-58) of bovine adenovirus type-3 (BAV-3) was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase. The fusion protein was affinity purified and used to immunize rabbits. Immunoprecipitation and Western blot analysis showed that the antiserum could specifically recognize a protein of 48 kDa in BAV-3-infected cells, which was produced both in early and late phases of BAV-3 life cycle. Based on the ability of antiserum to recognize DNA binding protein, a novel assay for BAV-3 quantitation was established. The assay is less time consuming and can be performed on a wide variety of bovine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.

Characterization of DNA binding protein of porcine adenovirus type 3.

To identify and characterize the protein encoded by the E2A region of porcine adenovirus (PAV)-3, DNA sequence coding for a portion (amino acids 102–457) of the DNA binding protein (DBP) open reaching frame was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase protein of Schistosoma japonica. The affinity-purified fusion protein was used to immunize rabbits. Immunoprecipitation/Western blot analysis demonstrated that the antisera specifically recognized a protein of 50 kD in PAV-3-infected cells. Immunoperoxidase staining detected the DBP protein predominantly in the nucleus of the cells. Western blot analysis demonstrated that DBP was detected as early as 6 h after infection and remained detectable throughout the infection. Based on these results, a novel assay for quantitation of PAV-3 was established. The assay is less time consuming and can be performed in different porcine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.

Networks of Host Factors that Interact with NS1 Protein of Influenza A Virus

Pigs are an important host of influenza A viruses due to their ability to generate reassortant viruses with pandemic potential. NS1 protein of influenza A viruses is a key virulence factor and a major antagonist of innate immune responses. It is also involved in enhancing viral mRNA translation and regulation of virus replication. Being a protein with pleiotropic functions, NS1 has a variety of cellular interaction partners. Hence, studies on swine influenza viruses (SIV) and identification of swine influenza NS1-interacting host proteins is of great interest. Here, we constructed a recombinant SIV carrying a Strep-tag in the NS1 protein and infected primary swine respiratory epithelial cells (SRECs) with this virus. The Strep-tag sequence in the NS1 protein enabled us to purify intact, the NS1 protein and its interacting protein complex specifically. We identified cellular proteins present in the purified complex by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and generated a dataset of these proteins. 445 proteins were identified by LC-MS/MS and among them 192 proteins were selected by setting up a threshold based on MS parameters. The selected proteins were analyzed by bioinformatics and were categorized as belonging to different functional groups including translation, RNA processing, cytoskeleton, innate immunity, and apoptosis. Protein interaction networks were derived using these data and the NS1 interactions with some of the specific host factors were verified by immunoprecipitation. The novel proteins and the networks revealed in our study will be the potential candidates for targeted study of the molecular interaction of NS1 with host proteins, which will provide insights into the identification of new therapeutic targets to control influenza infection and disease pathogenesis.

NS1 Protein of 2009 Pandemic Influenza A Virus Inhibits Porcine NLRP3 Inflammasome-Mediated Interleukin-1 Beta Production by Suppressing ASC Ubiquitination

ABSTRACT The inflammasome represents a molecular platform for innate immune regulation and controls proinflammatory cytokine production. The NLRP3 inflammasome is comprised of NLRP3, ASC, and procaspase-1. When the NLRP3 inflammasome is activated, it causes ASC speck formation and caspase-1 activation, resulting in the maturation of interleukin-1β (IL-1β). The NLRP3 inflammasome is regulated at multiple levels, with one level being posttranslational modification. Interestingly, ubiquitination of ASC has been reported to be indispensable for the activation of the NLRP3 inflammasome. Influenza A virus (IAV) infection induces NLRP3 inflammasome-dependent IL-1β secretion, which contributes to the host antiviral defense. However, IAVs have evolved multiple antagonizing mechanisms, one of which is executed by viral NS1 protein to suppress the NLRP3 inflammasome. In this study, we compared IL-1β production in porcine alveolar macrophages in response to IAV infection and found that the 2009 pandemic H1N1 induced less IL-1β than swine influenza viruses (SIVs). Further study revealed that the NS1 C terminus of pandemic H1N1 but not that of SIV was able to significantly inhibit NLRP3 inflammasome-mediated IL-1β production. This inhibitory function was attributed to impaired ASC speck formation and suppression of ASC ubiquitination. Moreover, we identified two target lysine residues, K110 and K140, which are essential for both porcine ASC ubiquitination and NLRP3 inflammasome-mediated IL-1β production. These results revealed a novel mechanism by which the NS1 protein of the 2009 pandemic H1N1 suppresses NLRP3 inflammasome activation. IMPORTANCE Influenza A virus (IAV) infection activates the NLRP3 inflammasome, resulting in the production of IL-1β, which contributes to the host innate immune response. ASC, an adaptor protein of NLRP3, forms specks that are critical for inflammasome activation. Here, we report that the NS1 C terminus of the 2009 pandemic H1N1 has functions to suppress porcine IL-1β production by inhibiting ASC speck formation and ASC ubiquitination. Furthermore, the ubiquitination sites on porcine ASC were identified. The information gained here may contribute to an in-depth understanding of porcine inflammasome activation and regulation in response to different IAVs, helping to further enhance our knowledge of innate immune responses to influenza virus infection in pigs.

Nuclear-resident RIG-I senses viral replication inducing antiviral immunity

The nucleus represents a cellular compartment where the discrimination of self from non-self nucleic acids is vital. While emerging evidence establishes a nuclear non-self DNA sensing paradigm, the nuclear sensing of non-self RNA, such as that from nuclear-replicating RNA viruses, remains unexplored. Here, we report the identification of nuclear-resident RIG-I actively involved in nuclear viral RNA sensing. The nuclear RIG-I, along with its cytoplasmic counterpart, senses influenza A virus (IAV) nuclear replication leading to a cooperative induction of type I interferon response. Its activation signals through the canonical signaling axis and establishes an effective antiviral state restricting IAV replication. The exclusive signaling specificity conferred by nuclear RIG-I is reinforced by its inability to sense cytoplasmic-replicating Sendai virus and appreciable sensing of hepatitis B virus pregenomic RNA in the nucleus. These results refine the RNA sensing paradigm for nuclear-replicating viruses and reveal a previously unrecognized subcellular milieu for RIG-I-like receptor sensing.RIG-I senses cytoplasmic viral RNA, resulting in induction of an antiviral response. Here, the authors identify nuclear RIG-I and show that it binds nuclear influenza A virus RNA, resulting in a cooperative interferon induction along with its cytoplasmic counterpart.