Characterization of the interactome of the porcine reproductive and respiratory syndrome virus glycoprotein-5.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry, causing reproductive failure in sows and respiratory disorders in piglets. Glycosylated protein 5 (GP5) is a major envelope protein of the virus. It is essential for virus particle assembly and involved in viral pathogenesis. In the present study, we identified the host cellular proteins that interact with GP5 by performing immunoprecipitation in MARC-145 cells infected by a recombinant PRRSV containing a FLAG-tag insertion in GP5. In total, 122 cellular proteins were identified by LC-MS/MS. Gene Ontology and KEGG databases were used to map these proteins to different cellular processes, locations and functions. Interestingly, 10.24% of identified cellular proteins were involved in the process of translation. Follow up experiments demonstrated that expression of GP5 in transfected cells led to inhibition of translation of reporter genes. Interaction between GP5 and ATP synthase subunit alpha (ATP5A) was further confirmed by co-immunoprecipitation suggesting a possible role of GP5 in regulation of ATP production in cells. These data contribute to a better understanding of GP5's role in viral pathogenesis and virus-host interactions.

S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus infecting pigs of all ages with high morbidity and mortality among newborn piglets. Currently, there is no effective vaccine available to protect the pigs from PEDV. The N-terminal subunit of spike protein (S1) is responsible for virus binding to the cellular receptor and contains a number of neutralizing antibody epitopes. Thus, we expressed and produced recombinant S1 protein to protect newborn piglets by immunization of sows. METHODS: Affinity tagged PEDV S1 protein was expressed in a secretory form in yeast, insect and mammalian cells to identify the most suitable production system. Purified recombinant protein was analysed by SDS-PAGE, Western blot and deglycosylation assay. A pregnant sow was intramuscularly immunized three times with adjuvanted recombinant protein prior to farrowing. PEDV-specific immune responses in sera and colostrum of the sow and piglets were assayed by ELISA and virus neutralization assays. Piglets were challenged orally with PEDV, and clinical parameters were monitored for 6 days post-challenge. RESULTS AND CONCLUSION: Of three eukaryotic expression systems tested (yeast, insect-cell, and mammalian), expression by HEK-293 T cells gave the highest yield of protein that was N-glycosylated and was the most appropriate candidate for vaccination. Administration of the subunit vaccine in a sow resulted in induction of S1-specific IgG and IgA that were passively transferred to the suckling piglets. Also, high virus neutralization titres were observed in the serum of the vaccinated sow and its piglets. After PEDV challenge, piglets born to the vaccinated sow exhibited less severe signs of disease and significantly lower mortality compared to the piglets of a control sow. However, there were no significant differences in diarrhea, body weight and virus shedding. Thus, vaccination with S1 subunit vaccine failed to provide complete protection to suckling piglets after challenge exposure, and further improvements are needed for the development of a subunit vaccine that fully protects against PEDV infection.

Porcine reproductive and respiratory syndrome virus envelope (E) protein interacts with mitochondrial proteins and induces apoptosis.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses for the swine industry worldwide. The PRRSV E protein, encoded by ORF 2b, is one of the non-glycosylated minor structural proteins. In this study, we present evidence for the interaction of the E protein with mitochondrial proteins ATP5A (part of ATP synthase complex), prohibitin, and ADP/ATP translocase. We additionally demonstrate partial mitochondrial localization of the E protein in transfected cells. To functionally investigate these interactions, we infected MARC-145 cells with PRRSV or alphavirus replicon particles (VRPs) expressing PRRSV E protein. In infected cells, production of ATP was significantly reduced. The E protein also induced apoptosis by activating caspase-3, which results in PARP cleavage. Taken together, these data suggest that the PRRSV E protein interacts with mitochondrial proteins and induces apoptosis by inhibiting ATP production.

New insights into RNA packaging in porcine reproductive and respiratory syndrome virus.

While identifying whether the smallest packaged heteroclite subgenomic RNA (S9) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a packaging signal, we found that S9 was capable of binding to the basic amino acid-rich domain (synthetic peptide of aa 34-53) of the packaging protein (N). In addition, by using truncations at the 5' and 3' ends of S9, a minimal binding region of 35 nt was found to be essential for binding to both the synthetic peptide and to the full-length N protein. Furthermore, by using cell-culture experiments, we found that S9 was capable of packaging non-viral RNA sequence into PRRSV particles and that the 35 nt region was essential for this activity. Taken together, our data suggest that this 35 nt region might be an important element for packaging PRRSV genomic RNA into virus particles.

SARS coronavirus protein 7a interacts with human Ap4A-hydrolase.

The SARS coronavirus (SARS-CoV) open reading frame 7a (ORF 7a) encodes a 122 amino acid accessory protein. It has no significant sequence homology with any other known proteins. The 7a protein is present in the virus particle and has been shown to interact with several host proteins; thereby implicating it as being involved in several pathogenic processes including apoptosis, inhibition of cellular protein synthesis, and activation of p38 mitogen activated protein kinase. In this study we present data demonstrating that the SARS-CoV 7a protein interacts with human Ap4A-hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17). Ap4A-hydrolase is responsible for metabolizing the "allarmone" nucleotide Ap4A and therefore likely involved in regulation of cell proliferation, DNA replication, RNA processing, apoptosis and DNA repair. The interaction between 7a and Ap4A-hydrolase was identified using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation from cultured human cells transiently expressing V5-His tagged 7a and HA tagged Ap4A-hydrolase. Human tissue culture cells transiently expressing 7a and Ap4A-hydrolase tagged with EGFP and Ds-Red2 respectively show these proteins co-localize in the cytoplasm.

Intracellular localization of the SARS coronavirus protein 9b: evidence of active export from the nucleus.

Open reading frame 9b (ORF 9b) encodes a 98 amino acid group-specific protein of severe acute respiratory syndrome (SARS) coronavirus (CoV). It has no homology with known proteins and its function in SARS CoV replication has not been determined. The N-terminal part of the 9b protein was used to raise polyclonal antibodies in rabbits, and these antibodies could detect 9b protein in infected cells. We analyzed the sub-cellular localization of recombinant 9b protein using fluorescence microscopy of live transfected cells and indirect immunofluorescence of transfected fixed cells. Our findings indicate that the 9b protein is exported outside of a cell nucleus and localizes to the endoplasmic reticulum. Our data also suggest that the 46-LRLGSQLSL-54 amino acid sequence of 9b functions as a nuclear export signal (NES).

Optimization of a DNA vaccine against SARS.

Severe acute respiratory syndrome coronavirus (SARS-CoV) first appeared in Southern China in November 2002, and then quickly spread to 33 countries on five continents along international air travel routes. Although the SARS epidemic has been contained, there is a clear need for a safe and effective vaccine should an outbreak of a SARS-CoV infection reappear in human population. In this study, we tested four DNA-vaccine constructs: (1) pLL70, containing cDNA for the SARS-CoV spike (S) gene; (2) pcDNA-SS, containing codon-optimized S gene for SARS-CoV S protein (residues 12-1255) fused with a leader sequence derived from the human CD5 gene; (3) pcDNA-St, containing the gene encoding the N-portion of the codon-optimized S gene (residues 12-532) with the CD5 leader sequence; (4) pcDNA-St-VP22C, containing the gene encoding the N-portion of the codon-optimized S protein with the CD5 leader sequence fused with the C-terminal 138 amino acids of the bovine herpesvirus-1 (BHV-1) major tegument protein VP22. Each of these plasmids was intradermally administered to C57BL/6 mice in three separate immunizations. Analysis of humoral and cellular immune responses in immunized mice demonstrated that pcDNA-SS and pcDNA-St-VP22C are the most immunogenic SARS vaccine candidates.

Construction of capsid-modified recombinant bovine adenovirus type 3.

Adenoviruses have become a popular vehicle for gene transfer into animal and human cells. However, wide prevalence of preexisting immunity to human adenovirus (HAdV) and the promiscuous nature of the virus have made the use of nonhuman adenoviruses an attractive alternative. Moreover, readministration of viral vectors is often required to maintain therapeutic levels of transgene expression, resulting in vector-specific immune responses. Although a number of features of bovine adenovirus (BAdV)-3 make it attractive for use as a vector in human vaccination, BAdV-3 transduces nonbovine cells, including human cells, poorly. However, genetic modification of capsid proteins (e.g., fiber, pIX) has helped in increasing the utility of BAdV-3 as a vector for transducing nonbovine cells. Here, we will describe the methods used to construct recombinant BAdV-3 expressing chimeric fiber or chimeric pIX proteins.

Porcine reproductive and respiratory syndrome virus envelope (E) protein interacts with tubulin.

Porcine reproductive and respiratory syndrome virus (PRRSV) encodes the small envelope (E) protein which is a minor structural component of the virion that is important for virus infectivity. To better understand the biological functions of the E protein, we studied interactions between E and PRRSV cellular proteins. Using immunoprecipitation-coupled mass spectrometry approach, we previously identified tubulin-α as an interacting partner of E. In this study, we confirmed this interaction using co-immunoprecipitation and co-localization assays. In addition, we demonstrated that the 25-residue C-terminal endodomain of E was essential for its interaction with tubulin-α. Over-expression of the E protein in cultured cells led to microtubule depolymerisation. Similarly, we observed that microtubule depolymerisation occurs in MARC-145 cells at the late stage of PRRSV replication. Also, depolymerisation of microtubules by colcemid significantly inhibited PRRSV replication in MARC-145 cells at early time points but the effect was not as dramatic at the late stage of infection. These data suggest that PRRSV infection of MARC-145 cells requires the microtubules network to facilitate early phase of infection whereas microtubules depolymerisation occurs at the late stage of PRRSV replication. Interaction between E and tubulin-α may contribute to microtubules depolymerisation.

Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses.

The recent introduction of the porcine epidemic diarrhea virus (PEDV) into the North American swine herd has highlighted again the need for effective vaccines for swine coronaviruses. While vaccines for transmissible gastroenteritis virus (TGEV) have been available to producers around the world for a long time, effective vaccines for PEDV and deltacoronaviruses were only recently developed or are still in development. Here, we review existing vaccine technologies for swine coronaviruses and highlight promising technologies which may help to control these important viruses in the future.

Exploration of New Sites in Adenovirus Hexon for Foreign Peptides Insertion.

Adenoviral vectors are now being explored as vaccine carriers to prevent infectious diseases in humans and animals. There are two strategies aimed at the expression of a vaccine antigen by adenoviral vectors. The first includes an insertion of the foreign gene expression cassette into the E1 region. The second strategy is antigen incorporation into the viral capsid proteins. To extend this methodology, we have searched for new sites at the human adenovirus serotype 5 major capsid protein hexon for a vaccine antigen insertion. To this end, we utilized sites in the hexon hypervariable region (HVR) 7, 8 and 9 to display a 15-mer peptide containing the main neutralizing epitope of porcine reproductive and respiratory syndrome virus. However, we could not rescue the viruses with the insertions of the peptide into HVR 8 and 9, consistent with the viruses being unable to tolerate insertions at these sites. In contrast, the virus with the insertion of the peptide in HVR 7 was viable - growing well in cell culture and the inserted peptide was exposed on the virion surface.

Role of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) signalling pathways in porcine reproductive and respiratory syndrome virus (PRRSV) replication.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a positive sense, single-stranded RNA genome virus that has become a major infection in swine, exerting huge economic losses to the industry worldwide. Detailed knowledge concerning the molecular mechanisms by which the virus manipulates the host cell signals transduction machinery is not only critical to further our understanding of viral replication and pathogenesis, but also guides our efforts to design new and improved therapeutic strategies. The phosphatidylinositol-3-kinase (PI3K)-dependent Akt and the mammalian target of rapamycin (mTOR) (PI3K/Akt/mTOR) are major host cell signalling pathways that regulate protein synthesis, cell growth, proliferation, migration and survival. It is also established that many viruses exploit these signalling cascades for their own benefit, driving viral protein expression, replication, as well as the suppression of the host's antiviral activities. In this article, we will review the role of these signalling pathways during PRRSV replication, and discuss some of our recent findings implicating mTOR.

Potential role of porcine reproductive and respiratory syndrome virus structural protein GP2 in apoptosis inhibition.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious threat to the pork industry, and its pathogenesis needs further investigations. To study the role of two structural proteins of PRRSV in virus-host cells interactions, two stable cell lines (MARC-2a and MARC-N) expressing GP2 and N proteins, respectively, were established. We induced apoptosis in these cells by treating them with staurosporine and found a significant reduction in the number of apoptotic cells in MARC-2a as compared to MARC-N and MARC-145 cells. In addition, we found significantly higher activities of transcriptional factors (NF- κ B and AP-1) in both cell lines as compared to MARC-145 (parent cells). Overall, our data suggest that, although both stable cell lines activate NF- κ B and AP-1, GP2 triggers the antiapoptotic process through an intermediate step that needs to be further investigated.

Development of a DNA-launched replicon as a vaccine for porcine reproductive and respiratory syndrome virus.

Though a modified live attenuated vaccine (MLV) is available against porcine reproductive and respiratory syndrome virus (PRRSV), its limitations in protective efficacy, safety and few others warrant the development of newer vaccines. In this study, we have constructed a propagation-defective DNA-launched PRRSV replicon as a vaccine candidate and evaluated its immunogenicity and protective efficacy in a group of pigs along with MLV vaccinated group. Our data showed that prior to the intranasal challenge with a homologous strain of PRRSV, only MLV vaccinated pigs developed antibody response measured by ELISA and none of the pigs in any group developed PRRSV neutralizing antibodies in serum. The MLV vaccinated group also showed high PRRSV-specific INF-γ response, whereas the replicon-vaccinated pigs showed low but detectable INF-γ response. After 14 days post challenge, all groups showed similar PRRSV-specific serum neutralizing titers and were positive for PRRSV-specific ELISA antibody. In addition, the replicon-vaccinated group showed a significant reduction in viremia in comparison to the control group. In conclusion, vaccination with the PRRSV DNA-launched replicon decreased the viremia and viral load in bronchoalveolar lavage fluids of the PRRSV-challenged pigs and increased numbers of IFN-γ producing cells. Thus, the vaccine is partially protective and is a potential vaccine candidate for future with further improvement. The possible means of improvement is the expression of immunostimulatory genes by the replicon. We demonstrated the feasibility of this approach by expression of a foreign gene encoding firefly luciferase after transfection of cultured cells with the replicon plasmid DNA.

The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks.

Mucosal adenovirus-vectored vaccine for measles.

Several problems associated with the available anti-measles vaccine emphasize the need for a single shot anti-measles vaccine which is efficacious by mucosal route of administration and functional in the presence of anti-measles neutralizing antibodies. To achieve these goals, we constructed two recombinant human adenoviruses (collectively designated Ad-F/H) carrying genes for measles virus (MV) fusion (F) and haemagglutinin (H) proteins. Single intranasal or intramuscular vaccination of mice and cotton rats with Ad-F/H elicited high MV-specific serum neutralizing-antibody titers. Furthermore, bronchoalveolar lavage samples from mice vaccinated intranasally with Ad-F/H showed a 100-fold increase in MV-specific IgA titers compared with intramuscularly vaccinated mice. Moreover, Ad-F/H vaccine administered intranasally, but not intramuscularly, completely protected challenged cotton rats from MV replication in the lungs.

Immunogenicity of a receptor-binding domain of SARS coronavirus spike protein in mice: implications for a subunit vaccine.

We studied the immunogenicity of an anti-SARS subunit vaccine comprised of the fragment of the SARS coronavirus (SARS-CoV) spike protein amino acids 318-510 (S318-510) containing the receptor-binding domain. The S protein fragment was purified from the culture supernatant of stably transformed HEK293T cells secreting a tagged version of the protein. The vaccine was given subcutaneously to 129S6/SvEv mice in saline, with alum adjuvant or with alum plus CpG oligodeoxynucleotides (ODN). Mice immunized with the adjuvanted antigen elicited strong antibody and cellular immune responses; furthermore, adding the CpG ODN to the alum resulted in increased IgG2a antibody titers and a higher number of INF-gamma-secreting murine splenocytes. Mice vaccinated with S318-510 deglycosylated by PNGase F (dgS318-510) showed a lower neutralizing antibody response but had similar numbers of INF-gamma-producing cells in the spleen. This finding suggests that carbohydrate is important for the immunogenicity of the S318-510 protein fragment and provide useful information for designing an effective and safe SARS subunit vaccine.

Comparative evaluation of two severe acute respiratory syndrome (SARS) vaccine candidates in mice challenged with SARS coronavirus.

Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated by beta-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection.

Augmentation of immune responses to SARS coronavirus by a combination of DNA and whole killed virus vaccines.

We studied the immunogenicity of a DNA SARS-vaccine, a whole killed virus, or a whole killed and DNA vaccine combination. The DNA vaccine contained a plasmid encoding the SARS coronavirus (SARS-CoV) S protein under the control of the human CMV promoter and intron A. The whole killed virus vaccine was comprised of SARS-CoV, propagated in Vero-E6 cells, with subsequent beta-propilactone inactivation and formulated with aluminum hydroxide adjuvant. Mice immunized twice with the DNA vaccine and once with the whole killed virus elicited higher antibody responses than mice immunized three times with the DNA vaccine or once with the whole killed virus vaccine. Mice immunized twice with the whole killed virus vaccine elicited higher antibody responses than mice immunized three times with the DNA vaccine or once with the whole killed virus vaccine. However, a combination of the vaccines induced T-helper type 1 (Th1) immune responses while the whole killed virus vaccine induced T helper type 2 (Th2) immune response. These results demonstrate that combination of the DNA vaccine and the whole killed virus vaccine can be used to enhance the magnitude and change the bias of the immune responses to SARS-CoV.