Oropouche orthobunyavirus infection is mediated by the cellular host factor Lrp1.

Oropouche orthobunyavirus (OROV; Peribunyaviridae) is a mosquito-transmitted virus that causes widespread human febrile illness in South America, with occasional progression to neurologic effects. Host factors mediating the cellular entry of OROV are undefined. Here, we show that OROV uses the host protein low-density lipoprotein-related protein 1 (Lrp1) for efficient cellular infection. Cells from evolutionarily distinct species lacking Lrp1 were less permissive to OROV infection than cells with Lrp1. Treatment of cells with either the high-affinity Lrp1 ligand receptor-associated protein (RAP) or recombinant ectodomain truncations of Lrp1 significantly reduced OROV infection. In addition, chimeric vesicular stomatitis virus (VSV) expressing OROV glycoproteins (VSV-OROV) bound to the Lrp1 ectodomain in vitro. Furthermore, we demonstrate the biological relevance of the OROV-Lrp1 interaction in a proof-of-concept mouse study in which treatment of mice with RAP at the time of infection reduced tissue viral load and promoted survival from an otherwise lethal infection. These results with OROV, along with the recent finding of Lrp1 as an entry factor for Rift Valley fever virus, highlight the broader significance of Lrp1 in cellular infection by diverse bunyaviruses. Shared strategies for entry, such as the critical function of Lrp1 defined here, provide a foundation for the development of pan-bunyaviral therapeutics.

Structural characterization of peptidyl-tRNA hydrolase from Mycobacterium smegmatis by NMR spectroscopy.

BACKGROUND: Accumulation of toxic peptidyl-tRNAs in the bacterial cytoplasm is averted by the action of peptidyl-tRNA hydrolase (Pth), which cleaves peptidyl-tRNA into free tRNA and peptide. NMR studies are needed for a protein homolog with a complete crystal structure, for comparison with the NMR structure of Mycobacterium tuberculosis Pth. METHODS: The structure and dynamics of Mycobacterium smegmatis Pth (MsPth) were characterized by NMR spectroscopy and MD simulations. The thermal stability of MsPth was characterized by DSC. RESULTS: MsPth NMR structure has a central mixed seven stranded ß-sheet that is enclosed by six α-helices. NMR relaxation and MD simulations studies show that most of the ordered regions are rigid. Of the substrate binding segments, the gate loop is rigid, the base loop displays slow motions, while the lid loop displays fast timescale motions. MsPth displays high thermal stability characterized by a melting temperature of 61.71°C. CONCLUSION: The NMR structure of MsPth shares the canonical Pth fold with the NMR structure of MtPth. The motional characteristics for the lid region, the tip of helix α3, and the gate region, as indicated by MD simulations and NMR data, are similar for MsPth and MtPth. However, MsPth has relatively less rigid base loop and more compactly packed helices α5 and α6. The packing and the dynamic differences appear to be an important contributing factor to the thermal stability of MsPth, which is significantly higher than that of MtPth. SIGNIFICANCE: MsPth structure consolidates our understanding of the structure and dynamics of bacterial Pth proteins.

Immunological characterization of chimeras of high specificity antigens from Mycobacterium tuberculosis H37Rv.

Tuberculosis remains a serious global health problem. BCG is the only prophylactic TB vaccine and it shows variable protective efficacy. Chimeric protein subunit vaccines hold great potential as stand-alone vaccines or heterologous BCG prime boosters. We have designed a protein chimera, PP31, by combining Mtb ESAT-6 family antigen Rv1198 and MoCo biosynthesis family antigen Rv3111. Further, PP31 was extended by addition of latency antigen Rv1813c to yield PP43. Immunization of BALB/c mice with PP31 or PP43 with FIA adjuvant elicited strong humoral immune response. Restimulation of splenocytes of the immunized mice lead to significant proliferation of lymphocytes, secretion of cytokines IFN-γ, TNF, IL-2 of the Th1 class, IL-17A of the Th17 class, and IL-6. PP31 and PP43 also induced intracellular cytokine expression (IFN-γ, TNF, and IL-2) from both CD4+-CD44high and CD8+-CD44high T-cells. Antigen-specific IFN-γ+/IL-2+ double positive CD4+ T-cells were significantly higher in case of PP43 than PP31-immunized mice and control group. PP43 showed protection equivalent to heat-inactivated BCG in response to challenge of the immunized mice with Mtb H37Ra. Based on its immunogenicity and protective efficacy, PP43 appears to be a potential candidate for further development as a subunit vaccine against TB.

Biophysical and immunological characterization of the ESX-4 system ESAT-6 family proteins Rv3444c and Rv3445c from Mycobacterium tuberculosis H37Rv.

The ESAT-6 family proteins of Mycobacterium tuberculosis are regarded as the key mediators in mycobacterial virulence and are largely considered as antigens that can improve TB vaccines and diagnostics. We have characterized Rv3444c and Rv3445c proteins of the ESX-4 system of ESAT-6 family of M. tuberculosis H37Rv, and have experimentally established that these two proteins interact to form a heterodimeric complex. Complex formation resulted in induction of α-helical conformation and stability against chemical denaturation. To evaluate the immunogenic potential, we have immunized mice with Rv3444c or Rv3445c along with Freund's incomplete adjuvant (FIA). Immunization with Rv3444c-FIA or Rv3445c-FIA resulted in long term humoral responses. Re-stimulation of splenocytes from immunized mice resulted in significant lymphocyte proliferation with induction of TNF-α and IL-6. Further, the humoral responses to Rv3444c and Rv3445c antigens in Indian patients with active pulmonary TB (n = 44), and healthy individuals (n = 20), were investigated. Compared to healthy individuals, high levels of IgG against Rv3444c and Rv3445c were observed in TB patient's sera, indicating that these proteins are actively produced during the active phase of TB. Cellular immune responses to these proteins in active pulmonary TB patients (n = 5) were also investigated using peripheral blood mononuclear cells (PBMCs). Both the proteins induce significant lymphocyte proliferation and up-regulate the induction of TNF-α and IL-6 in TB patients.