Tropism and Infectivity of Pandemic Influenza A H1N1/09 Virus in the Human Placenta.

Influenza virus infection in pregnant women may put the fetus at higher risk; however, to date, there has been no detailed research about the expression of influenza virus receptors in the human placenta. We employed the lectin staining technique, which is a classic influenza virus receptor research method for studying the distribution of viral receptors in the human placenta. In addition, we examined the susceptibility of the human placenta to H1N1/09, by detecting viral proteins and RNA at different time points post-infection. We found that the human placenta expressed both avian and human influenza A virus receptors (α-2, 3-linked sialic acid and α-2, 6-linked sialic acid). In addition, H1N1/09 did not only infect the human placenta, but also replicated and was released into the culture media. We concluded that the human placenta is susceptible to the 2009 influenza A virus (H1N1/09) infection, and that particular attention should be paid to shielding pregnant women from infection during influenza season.

Influenza PB1-F2 Inhibits Avian MAVS Signaling.

RIG-I plays an essential role in the duck innate immune response to influenza infection. RIG-I engages the critical adaptor protein mitochondrial antiviral signaling (MAVS) to activate the downstream signaling pathway. The influenza A virus non-structural protein PB1-F2 interacts with MAVS in human cells to inhibit interferon production. As duck and human MAVS share only 28% amino acid similarity, it is not known whether the influenza virus can similarly inhibit MAVS signaling in avian cells. Using confocal microscopy we show that MAVS and the constitutively active N-terminal end of duck RIG-I (2CARD) co-localize in DF-1 cells, and duck MAVS is pulled down with GST-2CARD. We establish that either GST-2CARD, or duck MAVS can initiate innate signaling in chicken cells and their co-transfection augments interferon-beta promoter activity. Demonstrating the limits of cross-species interactions, duck RIG-I 2CARD initiates MAVS signaling in chicken cells, but works poorly in human cells. The D122A mutation of human 2CARD abrogates signaling by affecting MAVS engagement, and the reciprocal A120D mutation in duck 2CARD improves signaling in human cells. We show mitochondrial localization of PB1-F2 from influenza A virus strain A/Puerto Rico/8/1934 (H1N1; PR8), and its co-localization and co-immunoprecipitation with duck MAVS. PB1-F2 inhibits interferon-beta promoter activity induced by overexpression of either duck RIG-I 2CARD, full-length duck RIG-I, or duck MAVS. Finally, we show that the effect of PB1-F2 on mitochondria abrogates TRIM25-mediated ubiquitination of RIG-I CARD in both human and avian cells, while an NS1 variant from the PR8 influenza virus strain does not.

The core promoter controls basal and inducible expression of duck retinoic acid inducible gene-I (RIG-I).

Retinoic acid inducible gene-I (RIG-I) is a cytoplasmic RNA sensor for detecting a variety of RNA viruses including influenza A viruses. Detection ultimately produces Type I interferon (IFN), which stimulates expression of interferon stimulated genes (ISGs), including RIG-I itself in a positive feedback loop. The structure and function of RIG-I is conserved across phylogeny, despite significant protein sequence divergence, however, the promoter sequences do not show the expected phylogenetic relationships and it is not known whether they are similarly regulated. We previously cloned duck RIG-I and showed it is highly induced during influenza A infection consistent with induction by the interferon produced. Here, we identified the Pekin duck RIG-I promoter and constructed promoter reporter vectors, which we transfected into duck embryonic fibroblasts or chicken DF-1 cells and tested in dual luciferase assays. We showed that activation of the Mitochondrial Antiviral Signalling (MAVS) pathway using the constitutively active N-terminal region of RIG-I or polyinosinic-polycytidylic acid (poly I:C) led to stimulation of duck RIG-I promoter activity. Using deletion constructs we showed the core promoter lies in the proximal 250 basepairs, and we identified essential cis-regulatory elements, a GC-box and an interferon-sensitive response element (ISRE), responsible for basal and inducible expression, respectively. Using mCherry-tagged interferon regulatory factors (IRFs) cloned from chickens and ducks, we show overexpression of chIRF7 induced the duck RIG-I promoter, and this required the ISRE site. Finally, we also demonstrated that overexpressed chIRF7 translocated to the nucleus, which was augmented by MAVS activation using RIG-I 2CARD. Our findings demonstrate that RIG-I expression is induced by chIRF7, in a positive regulatory loop. These studies show that the duck RIG-I promoter is appropriately regulated in chicken cells, necessary for the potential generation of transgenic chickens expressing RIG-I.

Immunoglobulin G locus events in soft tissue sarcoma cell lines.

Recently immunoglobulins (Igs) have been found to be expressed by cells other than B lymphocytes, including various human carcinoma cells. Sarcomas are derived from mesenchyme, and the knowledge about the occurrence of Ig production in sarcoma cells is very limited. Here we investigated the phenomenon of immunoglobulin G (IgG) expression and its molecular basis in 3 sarcoma cell lines. The mRNA transcripts of IgG heavy chain and kappa light chain were detected by RT-PCR. In addition, the expression of IgG proteins was confirmed by Western blot and immunofluorescence. Immuno-electron microscopy localized IgG to the cell membrane and rough endoplasmic reticulum. The essential enzymes required for gene rearrangement and class switch recombination, and IgG germ-line transcripts were also identified in these sarcoma cells. Chromatin immunoprecipitation results demonstrated histone H3 acetylation of both the recombination activating gene and Ig heavy chain regulatory elements. Collectively, these results confirmed IgG expression in sarcoma cells, the mechanism of which is very similar to that regulating IgG expression in B lymphocytes.

Transcription factors E2A, FOXO1 and FOXP1 regulate recombination activating gene expression in cancer cells.

It has long been accepted that immunoglobulins (Igs) were produced by B lymphoid cells only. Recently Igs have been found to be expressed in various human cancer cells and promote tumor growth. Recombination activating gene 1 (RAG1) and RAG2, which are essential enzymes for initiating variable-diversity-joining segment recombination, have also been found to be expressed in cancer cells. However, the mechanism of RAG activation in these cancer cells has not been elucidated. Here, we investigated the regulatory mechanism of RAG expression in four human cancer cell lines by analyzing transcription factors that induce RAG activation in B cells. By RT-PCR, Western blot and immunofluorescence, we found that transcription factors E2A, FOXO1 and FOXP1 were expressed and localized to the nuclei of these cancer cells. Over-expression of E2A, FOXO1 or Foxp1 increased RAG expression, while RNA interference of E2A, FOXO1 or FOXP1 decreased RAG expression in the cancer cells. Chromatin immunoprecipitation experiments showed acetylation of RAG enhancer (Erag) and E2A, FOXO1 or FOXP1 were bound to Erag in vivo. These results indicate that in these cancer cells the transcription factors E2A, FOXO1 and FOXP1 regulate RAG expression, which initiates Ig gene rearrangement much in the way similar to B lymphocytes.