Irma Herrera-Camacho

Interferon lambda inhibits dengue virus replication in epithelial cells.

BackgroundIn viral disease, infection is controlled at the cellular level by type I interferon (IFN-I), but dengue virus (DENV) has the ability to inhibit this response. Type III interferon, also known as lambda IFN (IFN-III or IFN-λ), is a complementary pathway to the antiviral response by IFN-I. This work analyzed the IFN-λ (IFN-III) mediated antiviral response against DENV serotype 2 (DENV-2) infection.MethodsDengue fever patients were sampled to determine their IFN-λ levels by ELISA. To study the IFN-λ response during DENV infection we selected the epithelial cell line C33-A, and we demonstrated that it is permissive to DENV-2 infection. The effect of IFN-λ on virus replication was determined in these cells, in parallel to the expression of IFN-stimulated genes (ISGs), and Suppressor of Cytokine Signaling (SOCS), genes measured by RT-qPCR.ResultsWe found increased (~1.8 times) serological IFN-λ in dengue fever patients compared to healthy blood donors. IFN-λ inhibited DENV-2 replication in a dose-dependent manner in vitro. The reduction of viral titer corresponded with increased ISG mRNA levels (MX1 and OAS1), with the highest inhibition occurring at ISG’s peak expression. Presence of IFN-negative regulators, SOCS1 and SOCS3, during DENV-2 infection was associated with reduced IFN-λ1 expression.ConclusionsEvidence described here suggests that IFN-λ is a good candidate inhibitor of viral replication in dengue infection. Mechanisms for the cellular and organismal interplay between DENV and IFN- λ need to be further studied as they could provide insights into strategies to treat this disease. Furthermore, we report a novel epithelial model to study dengue infection in vitro.

Biochemical characterization and structural prediction of a novel cytosolic leucyl aminopeptidase of the M17 family from Schizosaccharomyces pombe

A new leucyl aminopeptidase activity has been identified in the fission yeast Schizosaccharomyces pombe. The enzyme, which has been purified and named leucyl aminopeptidase yspII (LAP yspII), had a molecular mass of 320 and 54 kDa by gel filtration and SDS/PAGE, respectively, suggesting a homohexameric structure. The enzyme cleaved synthetic aminoacyl‐4‐nitroanilides at an optimum of pH 8.5, and preferred leucine and methionine as N‐terminal amino acids. A clear dependence on Mn2+ concentration for activity was found, and an apparent association constant of 0.33 mm was calculated for the metal ion. Bestatin behaved as a competitive inhibitor of LAP yspII (Ki = 0.14 µm), while chelating agents such as chloroquine, EDTA and 1,10‐phenanthroline also reduced enzyme activity. A MALDI‐MS analysis, followed by sequencing of two of the resulting peptides, showed that LAP yspII undoubtedly corresponds to the putative aminopeptidase C13A11.05 identified in the S. pombe genome project. The protein exhibited nearly 40% sequence identity to fungal and mammalian aminopeptidases belonging to the M17 family of metallopeptidases. Catalytic residues (Lys292 and Arg366), as well as those involved in coordination with the cocatalytic metal ions (Lys280, Asp285, Asp303, Asp362 and Glu364) and those forming the hydrophobic pocket for substrate binding (Met300, Asn360, Ala363, Thr390, Leu391, Ala483 and Met486), were perfectly conserved among all known aminopeptidases. The S. pombe enzyme is predicted to be formed two clearly distinguished domains with a well conserved C‐terminal catalytic domain showing a characteristic topology of eight β‐sheets surrounded by α‐helical segments in the form of a saddle.

Aminopeptidase yscCo‐II: a new cobalt‐dependent aminopeptidase from yeast—purification and biochemical characterization

Saccharomyces cerevisiae aminopeptidase yscCo‐II (APCo‐II) was purified to apparent homogeneity by gel filtration, affinity chromatography and anion‐exchange chromatography. APCo‐II is an hexameric cobalt‐dependent metallo‐enzyme with an estimated native molecular mass of 290 kDa. Enzyme activity is only detected in the presence of cobalt ions at pH 7.0. Substrate specificity studies indicate that aminopeptidase yscCo‐II cleaves only basic N‐terminal residues. PMSF, Cu2+, 1,10‐phenanthroline and bestatin were found to be very strong inhibitors of aminopeptidase yscCo‐II activity. Kinetic studies indicated that the enzyme has a similar Km and KaCo (activation constant of cobalt) and, following extraction of cobalt from the enzyme, activity was recovered only after cobalt addition. Copyright

Structure-function analysis of two variants of mumps virus hemagglutinin-neuraminidase protein

A point mutation from guanine (G) to adenine (A) at nucleotide position 1081 in the hemagglutinin-neuraminidase (HN) gene has been associated with neurovirulence of Urabe AM9 mumps virus vaccine. This mutation corresponds to a glutamic acid (E) to lysine (K) change at position 335 in the HN glycoprotein. We have experimentally demonstrated that two variants of Urabe AM9 strain (HN-A1081 and HN-G1081) differ in neurotropism, sialic acidbinding affinity and neuraminidase activity. In the present study, we performed a structure-function analysis of that amino acid substitution; the structures of HN protein of both Urabe AM9 strain variants were predicted. Based on our analysis, the E/K mutation changes the protein surface properties and to a lesser extent their conformations, which in turn reflects in activity changes. Our modeling results suggest that this E/K interchange does not affect the structure of the sialic acid binding motif; however, the electrostatic surface differs drastically due to an exposed short alpha helix. Consequently, this mutation may affect the accessibility of HN to substrates and membrane receptors of the host cells. Our findings appear to explain the observed differences in neurotropism of these vaccine strains.

Interaction of mumps virus V protein variants with STAT1-STAT2 heterodimer: experimental and theoretical studies

BackgroundMumps virus V protein has the ability to inhibit the interferon-mediated antiviral response by inducing degradation of STAT proteins. Two virus variants purified from Urabe AM9 mumps virus vaccine differ in their replication and transcription efficiency in cells primed with interferon. Virus susceptibility to IFN was associated with insertion of a non-coded glycine at position 156 in the V protein (VGly) of one virus variant, whereas resistance to IFN was associated with preservation of wild-type phenotype in the V protein (VWT) of the other variant.ResultsVWT and VGly variants of mumps virus were cloned and sequenced from Urabe AM9 vaccine strain. VGly differs from VWT protein because it possesses an amino acid change Gln103Pro (Pro103) and the Gly156 insertion. The effect of V protein variants on components of the interferon-stimulated gene factor 3 (ISGF3), STAT1 and STAT2 proteins were experimentally tested in cervical carcinoma cell lines. Expression of VWT protein decreased STAT1 phosphorylation, whereas VGly had no inhibitory effect on either STAT1 or STAT2 phosphorylation. For theoretical analysis of the interaction between V proteins and STAT proteins, 3D structural models of VWT and VGly were predicted by comparing with simian virus 5 (SV5) V protein structure in complex with STAT1-STAT2 heterodimer. In silico analysis showed that VWT-STAT1-STAT2 complex occurs through the V protein Trp-motif (W174, W178, W189) and Glu95 residue close to the Arg409 and Lys415 of the nuclear localization signal (NLS) of STAT2, leaving exposed STAT1 Lys residues (K85, K87, K296, K413, K525, K679, K685), which are susceptible to proteasome degradation. In contrast, the interaction between VGly and STAT1-STAT2 heterodimer occurs in a region far from the NLS of STAT2 without blocking of Lys residues in both STAT1 and STAT2.ConclusionsOur results suggest that VWT protein of Urabe AM9 strain of mumps virus may be more efficient than VGly to inactivate both the IFN signaling pathway and antiviral response due to differences in their finest molecular interaction with STAT proteins.

Modulation of apoptosis by V protein mumps virus

BackgroundThe Urabe AM9 vaccine strain of mumps virus contains two variants of V protein: VWT (of HN-A1081 viral population) and VGly (of HN-G1081). The V protein is a promoting factor of viral replication by blocking the IFN antiviral pathway.FindingsWe studied the relationship between V protein variants and IFN-α2b-induced apoptosis. V proteins decrease activation of the extrinsic IFN-α2b-induced apoptotic pathway monitored by the caspase 8 activity, being the effect greater with the VWT protein. Both V proteins decrease the activity of caspase 9 of the intrinsic apoptotic pathway. In a system without IFN, the VWT and VGly proteins expression promotes activation of caspases 3 and 7. However, when the cellular system was stimulated with IFN-α, this activity decreased partially. TUNEL assay shows that for treatment with IFN-α and ibuprofen of cervical adenocarcinoma cells there is nuclear DNA fragmentation but the V protein expression reduces this process.ConclusionsThe reduction in the levels of caspases and DNA fragmentation, suggesting that V protein, particularly VWT protein of Urabe AM9 vaccine strain, modulates apoptosis. In addition, the VWT protein shows a protective role for cell proliferation in the presence of antiproliferative signals.

Production of an enzymatically active and immunogenic form of ectodomain of Porcine rubulavirus hemagglutinin-neuraminidase in the yeast Pichia pastoris.

Blue-eye disease (BED) of swine is a viral disease endemic in Mexico. The etiological agent is a paramyxovirus classified as Porcine rubulavirus (PoRV-LPMV), which exhibits in its envelope the hemagglutinin-neuraminidase (HN) glycoprotein, the most immunogenic and a major target for vaccine development. We report in this study the obtaining of ectodomain of PoRV HN (eHN) through the Pichia pastoris expression system. The expression vector (pPICZαB-HN) was integrated by displacement into the yeast chromosome and resulted in a Mut(+) phenotype. Expressed eHN in the P. pastoris X33 strain was recovered from cell-free medium, featuring up to 67 nmol/min/mg after 6 days of expression. eHN was recognized by the serum of infected pigs with strains currently circulating in the Mexican Bajio region. eHN induces antibodies in mice after 28 days of immunization with specific recognition in ELISA test. These antibodies were able to inhibit >80% replication by viral neutralization assays in cell culture. These studies show the obtaining of a protein with similar characteristics to the native HN and which may be a candidate to propose a vaccine or to use the antigen in a serologic diagnostic test.

Transfection of influenza A virus nuclear export protein induces the expression of tumor necrosis factor alpha.

Influenza A virus genomic segments eight codes for non-structural 1 (NS1) protein that is involved in evasion of innate antiviral response, and nuclear export protein (NEP) that participates in the export of viral ribonucleoprotein (RNP) complexes, transcription and replication. Tumor necrosis factor alpha (TNF-α) is highly expressed during influenza virus infections and is considered an anti-infective cytokine. NS1 and NEP proteins were overexpressed and their role on TNF-α expression was evaluated. Both TNF-α mRNA and protein increased in cells transfected with NEP but not with NS1. We further investigate if NS1 or NEP regulates the activity of TNF-α promoter. In the presence of NEP the activity of TNF-α promoter increased significantly compared with the control (83.5±2.9 vs. 30.9±2.8, respectively; p=0.001). This effect decreased 15-fold when the TNF-α promoter distal region was deleted, suggesting the involvement of mitogen-activated protein kinases (MAPK) and NF-kB response elements. This was corroborated by testing the effect produced on TNF-α promoter by the treatment with Raf/MEK/ERK (U0126), NF-kB (Bay-11-7082) and PI3K (Ly294-002) cell signaling inhibitors. Treatment with U0126 and Bay-117082 reduced the activity of TNF-α promoter mediated by NEP (41.5±3.2, 70% inhibition; and 80.6±7.4, 35% inhibition, respectively) compared to mock-treated control. The results suggest a new role for NEP protein that participates in the transcriptional regulation of human TNF-α expression.

Transcription of interferon stimulated genes in response to porcine rubulavirus infection in vitro

Porcine rubulavirus (PoRV) is an emerging virus causing meningo-encephalitis and reproductive failures in pigs. Little is known about the pathogenesis and immune evasion of this virus; therefore research on the mechanisms underlying tissue damage during infection is essential. To explore these mechanisms, the effect of PoRV on the transcription of interferon (IFN) pathway members was analyzed in vitro by semi-quantitative RT-PCR. Ten TCID50 of PoRV stimulated transcription of IFNα, IFNβ, STAT1, STAT2, p48 and OAS genes in neuroblastoma cells, whereas infection with 100 TCID50 did not stimulate transcription levels more than non-infected cells. When the cells were primed with IFNα, infection with 1 TCDI50 of PoRV sufficed to stimulate the transcription of the same genes, but 10 and 100 TCID50 did not modify the transcription level of those genes as compared with non-infected and primed controls. MxA gene transcription was observed only when the cells were primed with IFNα and stimulated with 10 TCID50, whereas 100 TCID50 of PoRV did not modify the MxA transcription level as compared to non-infected and primed cells. Our results show that PoRV replication at low titers stimulates the expression of IFN-responsive genes in neuroblastoma cells, and suggest that replication of PoRV at higher titers inhibits the transcription of several members of the IFN pathway. These findings may contribute to the understanding of the pathogenesis of PoRV.

Study of Cellular Processes in Higher Eukaryotes Using the Yeast Schizosaccharomyces pombe as a Model

Schizosaccharomyces pombe (Sz. pombe), or fission yeast, is an ascomycete unicellular fungus that has been used as a model system for studying diverse biological processes of higher eukaryotic cells, such as the cell cycle and the maintenance of cell shape, apoptosis, and ageing. Sz. pombe is a rod-shaped cell that grows by apical extension; it divides along the long axis by medial fission and septation. The fission yeast has a doubling time of 2–4 hours, it is easy and inexpensive to grow in simple culture conditions, and can be maintained in the haploid or the diploid state. Sz. pombe can be genetically manipulated using methods such as mutagenesis or gene disruption by homologous recombination. Fission yeast was defined as a micro-mammal because it shares many molecular, genetic, and biochemical features with cells of higher eukaryotes in mRNA splicing, post-translational modifications as N-glycosylation protein, cell-cycle regulation, nutrient-sensing pathways as the target of rapamycin (TOR) network, cAMP-PKA pathway, and autophagy. This chapter uses Sz. pombe as a useful model for studying important cellular processes that support life such as autophagy, apoptosis, and the ageing process. Therefore, the molecular analysis of these processes in fission yeast has the potential to generate new knowledge that could be applied to higher eukaryotes.