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Viral determinants in H5N1 influenza A virus enable productive infection of HeLa cells

ABSTRACT: Influenza A virus (IAV) is a human respiratory pathogen that causes yearly global epidemics, and sporadic pandemics due to human adaptation of pathogenic strains. Efficient replication of IAV in different species is, in part, dictated by its ability to exploit the genetic environment of the host cell. To investigate IAV tropism in human cells, we evaluated the replication of IAV strains in a diverse subset of epithelial cell lines. HeLa cells were refractory to growth of human H1N1 and H3N2, and low pathogenic avian influenza (LPAIs) viruses. Interestingly, a human isolate of the highly pathogenic avian influenza (HPAI) virus H5N1 successfully propagated in HeLa cells to levels comparable to a human lung cell line. Heterokaryon cells generated by fusion of HeLa and permissive cells supported H1N1 growth, suggesting the absence of a host factor(s) required for replication of H1N1, but not H5N1, in HeLa cells. The absence of this factor(s) was mapped to reduced nuclear import, replication, and translation, and deficient viral budding. Using reassortant H1N1:H5N1 viruses, we found that the combined introduction of nucleoprotein (NP) and hemagglutinin (HA) from H5N1 was necessary and sufficient to enable H1N1 growth. Overall, this study suggests the absence of one or more cellular factors in HeLa cells that results in abortive replication of H1N1, H3N2, and LPAI viruses, but can be circumvented upon introduction of H5N1 NP and HA. Further understanding of the molecular basis of this restriction will provide important insights into virus-host interactions that underlie IAV pathogenesis and tropism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE140759 | GEO | 2019/11/21

REPOSITORIES: GEO

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Project description:Background: Influenza A virus (IAV) is a segmented negative-stranded RNA virus that brings a potentially serious threat to public health and animal husbandry. Mast cells play an important role in both the inherent and adaptive immune response. Previous studies have indicated that mast cells support the productive replication of H1N1, H5N1, and H7N2. To date, the distinct molecular mechanism behind the pathogenesis in mast cells among the three different viruses has been poorly understood. Methods: We investigated the genomic profiles in detail and the dynamic change of genomes regulated by different subtypes of IAV in mouse mast cells using microassays in order to the distinct molecular mechanism behind the pathogenesis in mast cells among the three different viruses. Mouse mast cells (P815) were infected at a multiplicity of infection of 1 (infectious viruses/cell) and incubated for 12 hr before collection of total RNA and microarray anlaysis using the Affymetrix platforms. Any two of the three IAV-infected groups were compared to detect genomic distinct. Based on the results of microassays, the further verification was performed by real-time PCR and western blotting. Here, the mRNA levels of 5-HT, PKG and HIF-1 in P815 cells following H1N1, H5N1 and H7N2 infection were determined by real-time PCR. The protein levels of exosomes in P815 cells following H1N1, H5N1 and H7N2 infection were detected by western blotting. Results: Compared with any two of the three IAV-infected groups, much more diﬀerentially expressed genes (DEGs), cellular functions and signaling pathways were confirmd in H1N1 or H7N2 group, and H7N2 group showed the highest levels. However, few DEGs were detected as well as various cellular functions and signaling pathways were dramatically suppressed in H5N1 group. With an in-depth study on H1N1 and H7N2 group, we demonstrated the 5-HT signaling pathway and cGMP/PKG signaling pathway terms were enriched in P815 cells infected by H1N1 while HIF-1 signaling pathway terms were enriched in P815 cells infected by H7N2 virus. Furthermore, RT-qPCR results also showed significantly increased mRNA of 5-HT and PKG in H1N1-infected P815 cells as well as HIF-1 in H7N2-infected P815 cells. Besides, exosomes were highly secreted from H1N1-infected or H7N2-infected P815 cells. Conclusions: H1N1 and H7N2 viruses could result in a variety of DEGs in mast cells and activate various cellular functions and signaling pathways while H5N1 virus cause the few DEGs in mast cells as well as suppression of cellular functions and signaling pathways. Furthermore, 5-HT signaling pathway and cGMP/PKG signaling pathway were preferentially activated in P815 cells infected by H1N1 while HIF-1 signaling pathway were preferentially activated in P815 cells infected by H7N2 virus. In addition, exosomes were also preferentially secreted from H1N1-infected or H7N2-infected P815 cells that are potentially pivotal in innate immunity to fight IAV infection. The study provides novel information and insight into the distinct of molecular mechanism of H1N1, H5N1 and H7N2 viruses in mast cells from the perspective of genomic profiles.

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Viral determinants in H5N1 influenza A virus enable productive infection of HeLa cells

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