Differential recognition of Old World and New World arenavirus envelope glycoproteins by subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P).

The arenaviruses are an important family of emerging viruses that includes several causative agents of severe hemorrhagic fevers in humans that represent serious public health problems. A crucial step of the arenavirus life cycle is maturation of the envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). Comparison of the currently known sequences of arenavirus GPCs revealed the presence of a highly conserved aromatic residue at position P7 relative to the SKI-1/S1P cleavage side in Old World and clade C New World arenaviruses but not in New World viruses of clades A and B or cellular substrates of SKI-1/S1P. Using a combination of molecular modeling and structure-function analysis, we found that residue Y285 of SKI-1/S1P, distal from the catalytic triad, is implicated in the molecular recognition of the aromatic "signature residue" at P7 in the GPC of Old World Lassa virus. Using a quantitative biochemical approach, we show that Y285 of SKI-1/S1P is crucial for the efficient processing of peptides derived from Old World and clade C New World arenavirus GPCs but not of those from clade A and B New World arenavirus GPCs. The data suggest that during coevolution with their mammalian hosts, GPCs of Old World and clade C New World viruses expanded the molecular contacts with SKI-1/S1P beyond the classical four-amino-acid recognition sequences and currently occupy an extended binding pocket.

Evaluation of the anti-arenaviral activity of the subtilisin kexin isozyme-1/site-1 protease inhibitor PF-429242.

The cellular protease subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in the proteolytic processing of the viral envelope glycoprotein precursor (GPC) of arenaviruses, a step strictly required for production of infectious progeny. The small molecule SKI-1/S1P inhibitor PF-429242 was shown to have anti-viral activity against Old World arenaviruses. Here we extended these studies and show that PF-429242 also inhibits GPC processing and productive infection of New World arenaviruses, making PF-429242 a broadly active anti-arenaviral drug. In combination therapy, PF-429242 potentiated the anti-viral activity of ribavirin, indicating a synergism between the two drugs. A hallmark of arenaviruses is their ability to establish persistent infection in vitro and in vivo. Notably, PF-429242 was able to efficiently and rapidly clear persistent infection by arenaviruses. Interruption of drug treatment did not result in re-emergence of infection, indicating that PF-429242 treatment leads to virus extinction.

Mouse granzyme K has pro-inflammatory potential.

Granzymes (gzms) are key components of T-killer (Tc) cells believed to mediate pro-apoptotic activities. Recent evidence suggests that gzms also possess non-cytotoxic activities that contribute to host defense. In this study, we show that Tc cells from lymphocytic choriomeningitis virus (LCMV)-infected wild-type (wt) and gzm A/B-deficient mice express similar levels of gzmK protein, with both mouse strains efficiently controlling infection. GzmK, in recombinant form or secreted by ex vivo-derived LCMV-immune gzmAxB(-/-) Tc cells, lacks pro-apoptotic activity. Instead, gzmK induces primary mouse macrophages to process and secrete interleukin-1ß, independent of the ATP receptor P2X(7). Together with the finding that IL-1Ra (Anakinra) treatment inhibits virus elimination but not generation of cytotoxic Tc cells in wt mice, the data suggest that Tc cells control LCMV through non-cytotoxic processes that involve gzmK.

Targeting inside-out phosphatidylserine as a therapeutic strategy for viral diseases.

There is a pressing need for antiviral agents that are effective against multiple classes of viruses. Broad specificity might be achieved by targeting phospholipids that are widely expressed on infected host cells or viral envelopes. We reasoned that events occurring during virus replication (for example, cell activation or preapoptotic changes) would trigger the exposure of normally intracellular anionic phospholipids on the outer surface of virus-infected cells. A chimeric antibody, bavituximab, was used to identify and target the exposed anionic phospholipids. Infection of cells with Pichinde virus (a model for Lassa fever virus, a potential bioterrorism agent) led to the exposure of anionic phospholipids. Bavituximab treatment cured overt disease in guinea pigs lethally infected with Pichinde virus. Direct clearance of infectious virus from the blood and antibody-dependent cellular cytotoxicity of virus-infected cells seemed to be the major antiviral mechanisms. Combination therapy with bavituximab and ribavirin was more effective than either drug alone. Bavituximab also bound to cells infected with multiple other viruses and rescued mice with lethal mouse cytomegalovirus infections. Targeting exposed anionic phospholipids with bavituximab seems to be safe and effective. Our study demonstrates that anionic phospholipids on infected host cells and virions may provide a new target for the generation of antiviral agents.

Protein kinase C-theta critically regulates the proliferation and survival of pathogen-specific T cells in murine listeriosis.

Protein kinase C-theta (PKC-theta) is essential for the activation of T cells in autoimmune disorders, but not in viral infections. To study the role of PKC-theta in bacterial infections, PKC-theta(-/-) and wild-type mice were infected with Listeria monocytogenes (LM). In primary and secondary listeriosis, the numbers of LM-specific CD8 and CD4 T cells were drastically reduced in PKC-theta(-/-) mice, resulting in increased CFUs in spleen and liver of both PKC-theta(-/-) C57BL/6 and BALB/c mice. Furthermore, immunization with peptide-loaded wild-type dendritic cells induced LM-specific CD4 and CD8 T cells in wild-type but not in PKC-theta(-/-) mice. In listeriosis, transfer of wild-type T cells into PKC-theta(-/-) mice resulted in a normal control of Listeria, and, additionally, a selective expression of PKC-theta in LM-specific T cells was sufficient to drive a normal proliferation and survival of these T cells in LM-infected PKC-theta(-/-) recipients, illustrating a cell-autonomous function of PKC-theta in LM-specific T cells. Conversely, adoptively transferred PKC-theta(-/-) T cells were partially rescued from cell death and proliferated in LM-infected wild-type recipients, demonstrating that a PKC-theta deficiency of LM-specific T cells can be partially compensated for by a wild-type environment. Additionally, in vitro experiments showed that only the addition of IL-2, but not an inhibition of caspase-3, induced proliferation and prevented death of PKC-theta(-/-) T cells stimulated with LM-infected wild-type dendritic cells, further demonstrating that the impaired proliferation and survival of PKC-theta(-/-) T cells in listeriosis is not intrinsically fixed and can be experimentally improved.

An altered T cell repertoire in MECL-1-deficient mice.

Immunoproteasome subunits low-molecular mass polypeptide (LMP)2 and LMP7 affect Ag presentation by MHC class I molecules. In the present study, we investigated the function of the third immunosubunit LMP10/multicatalytic endopeptidase complex-like (MECL)-1 (beta2i) in MECL-1 gene-targeted mice. The number of CD8+ splenocytes in MECL-1-/- mice was 20% lower than in wild-type mice. Infection with lymphocytic choriomeningitis virus (LCMV) elicited a markedly reduced cytotoxic T cell (CTL) response to the LCMV epitopes GP276-286/Db and NP205-212/Kb in MECL-1-/- mice. The weak CTL response to GP276-286/Db was not due to an impaired generation of this epitope but was attributed to a decreased precursor frequency of GP276-286/Db-specific T cells. The expansion of TCR-Vbeta10+ T cells, which contain GP276-286/Db-specific cells, was reduced in LCMV-infected MECL-1-/- mice. Taken together, our data reveal an in vivo function of MECL-1 in codetermining the T cell repertoire for an antiviral CTL response.

Immunoproteasomes largely replace constitutive proteasomes during an antiviral and antibacterial immune response in the liver.

The proteasome is critically involved in the production of MHC class I-restricted T cell epitopes. Proteasome activity and epitope production are altered by IFN-gamma treatment, which leads to a gradual replacement of constitutive proteasomes by immunoproteasomes in vitro. However, a quantitative analysis of changes in the steady state subunit composition of proteasomes during an immune response against viruses or bacteria in vivo has not been reported. Here we show that the infection of mice with lymphocytic choriomeningitis virus or Listeria monocytogenes leads to an almost complete replacement of constitutive proteasomes by immunoproteasomes in the liver within 7 days. Proteasome replacements were markedly reduced in IFN-gamma(-/-) mice, but were only slightly affected in IFN-alphaR(-/-) and perforin(-/-) mice. The proteasome regulator PA28alpha/beta was up-regulated, whereas PA28gamma was reduced in the liver of lymphocytic choriomeningitis virus-infected mice. Proteasome replacements in the liver strongly altered proteasome activity and were unexpected to this extent, since an in vivo half-life of 12 days had been previously assigned to constitutive proteasomes in the liver. Our results suggest that during the peak phase of viral and bacterial elimination the antiviral cytotoxic T lymphocyte response is directed mainly to immunoproteasome-dependent T cell epitopes, which would be a novel parameter for the design of vaccines.