Development of peptide-conjugated morpholino oligomers as pan-arenavirus inhibitors.

Members of the Arenaviridae family are a threat to public health and can cause meningitis and hemorrhagic fever, and yet treatment options remain limited by a lack of effective antivirals. In this study, we found that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) complementary to viral genomic RNA were effective in reducing arenavirus replication in cell cultures and in vivo. PPMO complementary to the Junín virus genome were designed to interfere with viral RNA synthesis or translation or both. However, only PPMO designed to potentially interfere with translation were effective in reducing virus replication. PPMO complementary to sequences that are highly conserved across the arenaviruses and located at the 5' termini of both genomic segments were effective against Junín virus, Tacaribe virus, Pichinde virus, and lymphocytic choriomeningitis virus (LCMV)-infected cell cultures and suppressed viral titers in the livers of LCMV-infected mice. These results suggest that arenavirus 5' genomic termini represent promising targets for pan-arenavirus antiviral therapeutic development.

Antiviral effects of antisense morpholino oligomers in murine coronavirus infection models.

The recent emergence of novel pathogenic human and animal coronaviruses has highlighted the need for antiviral therapies that are effective against a spectrum of these viruses. We have used several strains of murine hepatitis virus (MHV) in cell culture and in vivo in mouse models to investigate the antiviral characteristics of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (P-PMOs). Ten P-PMOs directed against various target sites in the viral genome were tested in cell culture, and one of these (5TERM), which was complementary to the 5' terminus of the genomic RNA, was effective against six strains of MHV. Further studies were carried out with various arginine-rich peptides conjugated to the 5TERM PMO sequence in order to evaluate efficacy and toxicity and thereby select candidates for in vivo testing. In uninfected mice, prolonged P-PMO treatment did not result in weight loss or detectable histopathologic changes. 5TERM P-PMO treatment reduced viral titers in target organs and protected mice against virus-induced tissue damage. Prophylactic 5TERM P-PMO treatment decreased the amount of weight loss associated with infection under most experimental conditions. Treatment also prolonged survival in two lethal challenge models. In some cases of high-dose viral inoculation followed by delayed treatment, 5TERM P-PMO treatment was not protective and increased morbidity in the treated group, suggesting that P-PMO may cause toxic effects in diseased mice that were not apparent in the uninfected animals. However, the strong antiviral effect observed suggests that with further development, P-PMO may provide an effective therapeutic approach against a broad range of coronavirus infections.

Mapping the landscape of the lymphocytic choriomeningitis virus stable signal peptide reveals novel functional domains.

The stable signal peptide (SSP) of the lymphocytic choriomeningitis virus surface glycoprotein precursor has several unique characteristics. The SSP is unusually long, at 58 amino acids, and contains two hydrophobic domains, and its sequence is highly conserved among both Old and New World arenaviruses. To better understand the functions of the SSP, a panel of point and deletion mutants was created by in vitro mutagenesis to target the highly conserved elements within the SSP. We were also able to confirm critical residues required for separate SSP functions by trans-complementation. Using these approaches, it was possible to resolve functional domains of the SSP. In characterizing our SSP mutants, we discovered that the SSP is involved in several distinct functions within the viral life cycle, beyond translocation of the viral surface glycoprotein precursor into the endoplasmic reticulum lumen. The SSP is required for efficient glycoprotein expression, posttranslational maturation cleavage of GP1 and GP2 by SKI-1/S1P protease, glycoprotein transport to the cell surface plasma membrane, formation of infectious virus particles, and acid pH-dependent glycoprotein-mediated cell fusion.

Exacerbated pathology of viral encephalitis in mice with central nervous system-specific autoantibodies.

We examine here the outcome of viral encephalomyelitis [mouse hepatitis virus (MHV) A59, Theiler's encephalomyelitis virus, and Coxsackievirus B3] in mice with autoantibodies to a central nervous system (CNS)-specific antigen, myelin oligodendrocyte glycoprotein, that usually develop no clinical disease. Morbidity and mortality of the acute viral CNS disease was augmented by the presence of the autoantibodies in all three viral infections. Transfer of serum containing the autoantibodies at the time of infection with MHV was sufficient to reproduce the exacerbated disease. The presence of the autoantibodies was found to result in increased infiltration of mononuclear cells into the brain. Early demyelination was severely augmented in brains and spinal cords of MHV-infected mice with CNS-specific autoantibodies. The antibody-mediated exacerbation was shown to be independent of the complement system but to require expression of Fc receptors, because it was observed in C'-3-deficient but not in Fc receptor-deficient mice. Our study illustrates the possibility that infections can lead to much more profound immunopathology in the presence of an otherwise latent autoimmune condition.

HLA-A2-restricted protection against lethal lymphocytic choriomeningitis.

The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8(+) T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides-nucleoprotein NP(69-77), glycoprotein precursor GPC(10-18), GPC(447-455), and zinc-binding protein Z(49-58)-that displayed high-affinity binding (< or =275 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC(447-455)), after peptide immunization of HLA-A*0201 mice, induced CD8(+) T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV.