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 glycoproteinmediated cell fusion.The Arenaviridae comprise a group of enveloped RNA viruses that include several causative agents of hemorrhagic fevers in the New World and Africa. Among these are Lassa fever virus (LASV) and Junín virus (JUNV). The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), has a bisegmented, single-stranded, negative-sense RNA genome. Each of the two segments uses an ambisense coding strategy to direct the synthesis of two polypeptides. The large (L) segment (7.2 kb) encodes a small RING finger protein, Z, and the RNA-dependent RNA polymerase, the L protein, while the small (S) segment (3.4 kb) encodes the nucleoprotein, NP, and the glycoprotein (GP) precursor pGPC (5). The 498-aminoacid pGPC of LCMV consists of three domains (Fig. 1A) that are produced as independent polypeptides by posttranslational processing: residues 1 to 58 comprise the stable signal peptide (SSP), which is cotranslationally cleaved by signal peptidase and followed by the GP precursor GPC (residues 59 to 498), which is further processed into GP1 (residues 59 to 265) and GP2 (residues 266 to 498) (5, 7, 26) by the cellular protease, SKI-1/S1P (3). GP1 is heavily glycosylated, contains the receptor binding site and antibody neutralization sites, and is noncovalently associated with GP2. GP2 contains a transmembrane region and anchors the GP complex in the lipid bilayer of the cell membrane and virus envelope (6). LCMV cell entry involves a fusion event that requires exposure to acidic pH (9) to trigger GP1 dissociation from GP2 and to induce irreversible conformational changes in GP2 that mediate membrane fusion (10).Signal peptides (SPs) mediate protein translocation into the lumen of the endoplasmic reticulum (ER), promoting proper folding and posttranslational modifications of these polypeptides. Following translocation, an SP is typically cleaved and r...

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.

Background. Acute kidney injury (AKI) represents a major clinical problem with high mortality and limited causal treatments. The use of cell therapy has been suggested as a potential modality to improve the course and outcome of AKI.Methods. We investigated the possible renoprotection of freshly isolated, uncultured adipose tissue-derived stem and regenerative cells (ADRCs) before and after cryopreservation in a rat ischemia–reperfusion (I–R) model of AKI.Results. We demonstrated that ADRC therapy drastically reduced mortality (survival 100% vs. 57%, ADRC vs. controls, respectively) and significantly reduced serum creatinine (sCr on Day 3: 3.03 ± 1.58 vs. 7.37 ± 2.32 mg/dL, ADRC vs. controls, respectively). Histological analysis further validated a significantly reduced intratubular cast formation, ameliorated acute tubular epithelial cell necrosis and mitigated macrophage infiltration. Furthermore, a reduced RNA expression of CXCL2 and IL-6 was found in the ADRC group which could explain the reduced macrophage recruitment. Use of cryopreserved ADRCs resulted in an equally high survival (90% vs. 33% in the control group) and similarly improved renal function (sCr on Day 3: 4.64 ± 2.43 vs. 7.24 ± 1.40 mg/dL in controls).Conclusions. Collectively, these results suggest a potential clinical role for ADRC therapy in patients with AKI. Importantly, cryopreservation of ADRCs could offer an autologous treatment strategy for patients who are at high risk for AKI during planned interventions.

With the development of modern chemistry and biology, non-proteinogenic amino acids (NPAAs) have become a powerful tool for developing peptide-based drug candidates. Drug-like properties of peptidic medicines, due to the smaller size and simpler structure compared to large proteins, can be changed fundamentally by introducing NPAAs in its sequence. While peptides composed of natural amino acids can be used as drug candidates, the majority have shown to be less stable in biological conditions. The impact of NPAA incorporation can be extremely beneficial in improving the stability, potency, permeability, and bioavailability of peptide-based therapies. Conversely, undesired effects such as toxicity or immunogenicity should also be considered. The impact of NPAAs in the development of peptide-based therapeutics is reviewed in this article. Further, numerous examples of peptides containing NPAAs are presented to highlight the ongoing development in peptide-based therapeutics.

Recovery from Lassa virus (LASV) infection usually precedes the appearance of neutralizing antibodies, indicating that cellular immunity plays a primary role in viral clearance. To date, the role of LASV-specific CD8 ؉ T cells has not been evaluated in humans. To facilitate such studies, we utilized a predictive algorithm to identify candidate HLA-A2 supertype epitopes from the LASV nucleoprotein and glycoprotein precursor (GPC) genes. We identified three peptides (GPC 42-50 , GLVGLVTFL; GPC 60-68 , SLYKGVYEL; and GPC 441-449 , YLISIFLHL) that displayed high-affinity binding (<98 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 LASV GPC in human HLA-A*0201-positive target cells. HLA-A*0201 mice immunized with either GPC 42-50 or GPC 60-68 were protected against challenge with a recombinant vaccinia virus that expressed LASV GPC. The epitopes identified in this study represent potential diagnostic reagents and candidates for inclusion in epitope-based vaccine constructs. Our approach is applicable to any pathogen with existing sequence data, does not require manipulation of the actual pathogen or access to immune human donors, and should therefore be generally applicable to category A through C agents and other emerging pathogens.