The mitochondrial carrier homolog 2 is involved in down-regulation of influenza A virus replication.

BACKGROUND: The mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein regulating mitochondrial metabolism and functions in lipid homeostasis and apoptosis. Experimental data on the interaction of MTCH2 with viral proteins in virus-infected cells are very limited. Here, the interaction of MTCH2 with PA subunit of influenza A virus RdRp and its effects on viral replication was investigated. METHODS: The human MTCH2 protein was identified as the influenza A virus PA-related cellular factor with the Y2H assay. The interaction between GST.MTCH2 and PA protein co-expressed in transfected HEK293 cells was evaluated by GST-pull down. The effect of MTCH2 on virus replication was determined by quantification of viral transcript and/or viral proteins in the cells transfected with MTCH2-encoding plasmid or MTCH2-siRNA. An interaction model of MTCH2 and PA was predicted with protein modeling/docking algorithms. RESULTS: It was observed that PA and GST.MTCH2 proteins expressed in HEK293 cells were co-precipitated by glutathione-agarose beads. The influenza A virus replication was stimulated in HeLa cells whose MTCH2 expression was suppressed with specific siRNA, whereas the increase of MTCH2 in transiently transfected HEK293 cells inhibited viral RdRp activity. The results of a Y2H assay and protein-protein docking analysis suggested that the amino terminal part of the viral PA (nPA) can bind to the cytoplasmic domain comprising amino acid residues 253 to 282 of the MTCH2. CONCLUSION: It is suggested that the host mitochondrial MTCH2 protein is probably involved in the interaction with the viral polymerase protein PA to cause negative regulatory effect on influenza A virus replication in infected cells.

An in silico prediction of interaction models of influenza A virus PA and human C14orf166 protein from yeast-two-hybrid screening data.

The human C14orf166 protein, also known as RNA transcription, translation, and transport factor, shows positive modulatory activity on the cellular RNA polymerase II enzyme. This protein is a component of the tRNA-splicing ligase complex and is involved in RNA metabolism. It also functions in the nucleo-cytoplasmic transport of RNA molecules. The C14orf166 protein has been reported to be associated with some types of cancer. It has been shown that the C14orf166 protein binds to the influenza A virus RNA polymerase PA subunit and has a stimulating effect on viral replication. In this study, candidate interactor proteins for influenza A virus PA protein were screened with a Y2H assay using HEK293 Matchmaker cDNA. The C14orf166 protein fragments in different sizes were found to interact with the PA. The three-dimensional structures of the viral PA and C14orf166 proteins interacting with the PA were generated using the I-TASSER algorithm. The interaction models between these proteins were predicted with the ClusPro protein docking algorithm and analyzed with PyMol software. The results revealed that the carboxy-terminal end of the C14orf166 protein is involved in this interaction, and it is highly possible that it binds to the carboxy-terminal of the PA protein. Although amino acid residues in the interaction area of the PA protein with the C14orf166 showed distribution from 450th to 700th position, the intense interaction region was revealed to be at amino acid positions 610-630.

Stenotrophomonas maltophilia outbreak with a commercial blood gas injector as the culprit and interventions for source and prevention: A possible passage between patient and ECMO water heater device.

BACKGROUND: Despite low virulence of Stenotrophomonas maltophilia, it represents one of the leading drug-resistant bacteria. We report a large outbreak of S. maltophilia infection associated with an unexpected source, which turned out to be a commercial needleless blood gas injector. METHODS: Over a period from January 1 to December10, 2021, 113 patients were identified to have S. maltophilia infection as documented by positive cultures from the clinical samples, extracorporeal membrane oxygenation (ECMO) water heater devices and commercial needleless blood gas injectors. RESULTS: Sixty-seven isolates (59 clinical, 4 ECMO, 4 blood gas injectors) were sent for molecular analysis. Both arbitrarily primed polymerase chain reaction and pulsed-field gel electrophoresis analyses showed 12 distinct genotypes. Of 67 isolates, 58 were clonally related (86.6%), with 52 indistinguishable strains from 4 blood gas needleless injectors, 46 patients' samples (78%), and 2 ECMO samples (50%). Two ECMO samples and 1 clinical sample were clonally identical. CONCLUSIONS: In the event that eradication of infections would not be possible despite taking all environmental disinfection measures including the ECMO devices, unexpected sources, such as a commercial needleless blood gas injector, should not be omitted from the list for surveillance. In addition, obtaining surveillance cultures of ECMO water reservoirs should be placed in the routine clinical practice.

Effects of Some Interferon-Related Proteins on Influenza A Viruse RNA Polymerase Activity.

Objectives: Interferons (IFNs) are one of the most important components of innate immunity against viruses, especially those carrying the RNA genomes such as influenza viruses. Upon viral infection, the IFNs are rapidly secreted, inducing the expression of several genes in the target cells and establishing an antiviral state. In this study, the effects of proteins encoded by some IFN-related genes on influenza A virus RNA-dependent RNA polymerase enzyme were investigated. We evaluated the importance of these proteins in the pathogenesis of different influenza A virus types. Materials and Methods: The IFN-related genes were amplified by polymerase chain reaction from the HEK293 cDNA library and cloned into pCHA expression vector. The expression of genes and subcellular localizations of the proteins were determined by Western blotting and immunofluorescence staining, respectively. The effects of IFNs-related proteins on virus RdRP enzyme were determined by influenza A virus mini-replicons. Results: The study revealed that the influenza A virus infections significantly altered the transcript level of the IFN-related CCL5, IFIT1, IFIT3, IFITM3, and OAS1 genes in HEK293 cells. It was determined that the alteration of the gene expression was also related to the virus type. The mini-replicon assays showed that the transient expression of CCL5, IFI27, OAS1, IFITM3, IFIT1, and IFIT3 have inhibitory effects on WSN and/or DkPen type virus RdRP enzymes. We observed that the proteins except OAS1 inhibited WSN type RdRP enzyme at a higher level than that of DkPen enzyme. Conclusion: It was concluded that influenza A virus infection significantly alters the IFN-related gene expression in the cells. Most of the proteins encoded from these genes showed an inhibitory effect on the virus RdRP enzymes in the HEK293 cells. The inhibition of the influenza virus RdRP with IFN-related proteins may be the result of direct or indirect interactions between the host proteins and the viral enzyme subunits.

Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication.

BACKGROUND: Replication of the influenza A viruses occurs in the cells through the viral RdRP consisting of PB1, PB2, and PA. Several cellular proteins are involved in these processes. This study aims to reveal the interaction between human SNX2 protein and the PA protein and the effects of the SNX2 on the virus replication. RESULTS: To identify potential host interacting proteins to the PA, yeast two-hybrid assay was carried out with HEK293 cell cDNA library and the PA as a bait. We focused on SNX2 protein, which interacts with the PA in the yeast cells. By using the co-immunoprecipitation assays, it has been demonstrated that the amino-terminal part of the PA was important for binding to the SNX2. Immunolocalization of the proteins in HeLa cells supported this interaction. Knockdown of the SNX2 with siRNA in the cells resulted in a significant increase in both viral transcripts and virus growth. However, the increase of SNX2 in transfected cells didn't cause a significant change in the viral RdRP activity in minireplicon assay. This may suggest that the negative effect of SNX2 on the virus replication could be saturated with its authentic intra-cellular amount. CONCLUSIONS: This study revealed that the SNX2 and PA protein interact with each other in both yeast and HEK293 cells, and the SNX2 has a negative regulatory function on the virus replication. However, more knowledge is required to elucidate the action mechanism of the SNX2 on the influenza A virus replication at the molecular level.

Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase.

Influenza A viruses frequently change their genetic characteristics, which leads to the emergence of new viruses. Consequently, elucidation of the relationship between influenza A virus and host cells has a great importance to cope with viral infections. In this study, it was aimed to determine expression profiles of interferon response genes in human embryonic kidney 293 (HEK293) cells infected with human (A/WSN-H1N1) and avian influenza A viruses (duck/Pennsylvania/10218/84/H5N2) or transfected with plasmids encoding viral RdRP subunits and, to obtain clues about the genes that may be important for the viral pathogenesis. The HEK293 cells cultured in a 12-well plate were infected with influenza A viruses or transfected with plasmids encoding viral polymerase. Total RNA extraction and cDNA preparation were carried out with commercial kits. Qiagen 96-well-RT2 Profiler PCR Array plates designated for interferons response genes were used for quantitation of the transcripts. The relative quantities of transcripts were normalized with STAT3 gen, and the results were evaluated. Quantitative RT-PCR results showed that there are substantial differences of the interferon response gene transcription in cells infected with viruses or transfected with plasmids. A higher number of interferon-related genes were found to be downregulated in the cells infected with DkPen compared to WSN. On the other hand, significant differences in the expression profiles of interferon response genes were observed in the cells expressing viral PA protein. In particular, avian influenza PA protein was found to cause more aggressive changes on the transcript levels. Human and avian influenza A viruses cause a substantial change in interferon response gene expression in HEK293 cells. However, a higher number of genes were downregulated in the cells infected with avian influenza DkPen compared to WSN. It has been also concluded that the viral PA protein is one of the important viral factors affecting the transcript level of host genes.