No abstract

Detection and elimination of virus-infected cells by cytotoxic T lymphocytes depends on recognition of virus-derived peptides

Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP 5 and M and the minor proteins GP 2a , E, GP 3 , and GP 4 . Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP 5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indicating that these proteins interact with each other and are assembled into virions as a multimeric complex. Independent evidence for such complexes was obtained by coexpression of the minor envelope proteins in BHK-21 cells using a Semliki Forest virus expression system. By analyzing the maturation of their N-linked oligosaccharides, we found that the glycoproteins were each retained in the endoplasmic reticulum unless expressed together, in which case they were collectively transported through the Golgi complex to the plasma membrane and were even detected in the extracellular medium. As the PRRSV particles lacking the minor envelope proteins are not infectious, we hypothesize that the virion surface structures formed by these proteins function in viral entry by mediating receptor binding and/or virus-cell fusion.Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family of Arteriviridae, which also comprises Equine arteritis virus (EAV), Lactate dehydrogenase-elevating virus (LDV), and Simian hemorrhagic fever virus (24,42). This family belongs to the order of Nidovirales, together with the Coronaviridae, Toroviridae, and Roniviridae (2, 3). Arteriviruses are enveloped RNA viruses that contain a positive-strand RNA genome and synthesize a 3Ј nested set of six or eight subgenomic RNAs (sgRNAs) that encode the structural proteins. Analyses of purified virions of PRRSV have indicated that they are composed of seven proteins, i.e., four envelope glycoproteins named GP 2a (encoded by open reading frame 2a [ORF2a]), GP 3 (ORF3), GP 4 (ORF4), and GP 5 (ORF5); a nonglycosylated membrane protein M (ORF6); the nucleocapsid protein N (ORF7); and a nonglycosylated envelope protein, E, that is expressed from a second ORF (ORF2b) entirely contained within ORF2 (22,25,35,48). The 29-to 30-kDa GP 2a and 31-to 35-kDa GP 4 proteins are both putative class I integral membrane proteins with an N-terminal signal sequence and a C-terminal membrane anchor, containing two and four predicted N-glycosylation sites, respectively (22). T...

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.

In mice, vaccines inducing antibodies to the extracellular domain of the M2 protein (M2e) can confer protection to influenza A virus infection. Unlike the surface glycoproteins, haemagglutinin and neuraminidase, this domain of M2 is highly conserved and is therefore a potential broadspectrum immunogen. In this study, the protection conferred by vaccines inducing antibodies to M2e was evaluated in a challenge model for swine influenza in pigs. A protein resulting from the fusion between M2e and the hepatitis B virus core protein (M2eHBc), with or without adjuvant, was evaluated. In addition, a DNA construct expressing a fusion protein between M2e and influenza virus nucleoprotein (M2eNP) was evaluated to see if the broad-spectrum protection conferred by antibodies could be further enhanced by T helper cells and cytotoxic T cells. All vaccines induced an antibody response against M2e, and the M2eNP DNA vaccine additionally induced an influenza virus-specific lymphoproliferation response. However, after challenge with a swine influenza virus (H1N1), no protection was observed in the vaccinated groups compared with the non-vaccinated control group. On the contrary, vaccinated pigs showed more severe clinical signs than the control pigs. The M2eNP DNA-vaccinated pigs showed the most severe clinical signs and three out of six pigs died on days 1 and 2 post-challenge. These results indicate that antibodies to M2e, especially in combination with cell-mediated immune responses, exacerbate disease. Thus, clinical signs after infection should be observed closely in further studies using M2e as an immunogen and caution should be exercised in using M2e in humans.

No abstract

Bovine herpesvirus 1 (BHV-1) interferes with peptide translocation by the transporter associated with antigen processing (TAP). Recently, the UL49.5 gene product of BHV-1 was identified as the protein responsible for the observed inhibition of TAP. In BHV-1-infected cells and virions, the UL49.5 protein forms a complex with glycoprotein M (gM). Hence, it was investigated whether UL49.5 can combine the interactions with gM and the TAP complex. In cell lines constitutively expressing both UL49.5 and gM, UL49.5 appears to be required for functional processing of gM. Immunofluorescence-confocal laser scanning microscopy demonstrated that both proteins are interdependent for their redistribution from the endoplasmic reticulum to the trans-Golgi network. Remarkably, expression of cloned gM results in the abrogation of the UL49.5-mediated inhibition of TAP and prevents the degradation of the transporter. However, in BHV-1-infected cells, differences in UL49.5 and gM expression kinetics were seen to create a window of opportunity at the early stages of infection, during which time the UL49.5 protein can act on TAP without gM interference. Moreover, in later periods, non-gM-associated UL49.5 can be detected in addition to the UL49.5/gM complex. Thus, it has been deduced that different functions of UL49.5, editing of gM processing and inhibition of TAP, can be combined during BHV-1 infection.

A marker vaccine elicits an antibody response in the host that can be distinguished from the antibody response induced by a wild-type strain. To obtain a bovine herpesvirus 1 (BHV-1) marker vaccine, we constructed a glycoprotein E (gE) deletion mutant. This was obtained by removing the complete gE coding region from the BHV-1 genome. To attenuate the gE deletion mutant further, we also introduced a small deletion in the thymidine kinase (TK) gene. We selected three mutants: the gE deletion mutant, a TK deletion mutant and a gE/TK double deletion mutant, and examined their virulence and immunogenicity in calves.After intranasal inoculation, the TK deletion mutant showed some residual virulence, whereas the gE and gE/TK deletion mutants were avirulent. The calves inoculated with the deletion mutants were protected against disease after challenge exposure and shed significantly less virus than control calves. Deleting the gE gene, therefore, has little effect on the immunogenicity of BHV-1, but is sufficient to reduce the virulence of BHV-1 in calves. These findings led us to conclude that the gE deletion mutant is a good candidate for a modified live BHV-1 marker vaccine.