Entry of influenza A Virus with a α2,6-linked sialic acid binding preference requires host fibronectin.

The receptor binding specificity of influenza A virus is one of the major determinants of viral tropism and host specificity. In general, avian viral hemagglutinin prefers to bind to α2,3-linked sialic acid, whereas the human viral hemagglutinin prefers to bind to α2,6-linked sialic acid. Here, we demonstrate that host fibronectin protein plays an important role in the life cycle of some influenza A viruses. Treating cells with anti-fibronectin antibodies or fibronectin-specific small interfering RNA can inhibit the virus replication of human H1N1 influenza A viruses. Strikingly, these inhibitory effects cannot be observed in cells infected with H5N1 viruses. By using reverse genetics techniques, we observed that the receptor binding specificity, but not the origin of the hemagglutinin subtype, is responsible for this differential inhibitory effect. Changing the binding preference of hemagglutinin from α2,6-linked sialic acid to α2,3-linked sialic acid can make the virus resistant to the anti-fibronectin antibody treatment and vice versa. Our further characterizations indicate that anti-fibronectin antibody acts on the early phase of viral replication cycle, but it has no effect on the initial binding of influenza A virus to cell surface. Our subsequent investigations further show that anti-fibronectin antibody can block the postattachment entry of influenza virus. Overall, these results indicate that the sialic acid binding preference of influenza viral hemagglutinin can modulate the preferences of viral entry pathways, suggesting that there are subtle differences between the virus entries of human and avian influenza viruses.

Reverse phase protein array identifies novel anti-invasion mechanisms of YC-1.

YC-1 has recently been demonstrated to have potent anti-invasion and anti-metastatic activity in several cancer models, in addition to its anti-proliferation activity. However, the mechanism underlying its anti-invasion/anti-metastatic activity is largely unknown. Nasopharyngeal carcinoma (NPC) is a highly metastatic head and neck cancer in Southeast Asia. Here, we demonstrated that YC-1 inhibited invasiveness and proliferation of NPC cells, with the latter being accompanied by PARP cleavage, S-phase arrest and activation of Chk1/Chk2. We aimed at identifying novel anti-invasion mechanisms of YC-1 in NPC by a functional proteomic platform, the reverse phase protein array (RPPA). Our study revealed for the first time that multiple invasion-related signaling proteins (beta-catenin, caveolin, Src and EGFR), as well as several growth-related proteins (AMPKalpha, phospho-acetyl-CoA carboxylase (p-ACC), HER-2 and mTOR), which were previously un-described signaling proteins altered by YC-1, were found to be down-modulated by YC-1 in NPC cells. We hypothesized that YC-1-mediated downregulation of these invasion proteins contributed to its anti-invasion activity in NPC cells. Overexpression of EGFR, activated Src or caveolin, but not beta-catenin reversed the inhibitory effects of YC-1 on NPC cell invasion, with EGFR and activated Src having additional effects on rescuing NPC cells from YC-1-mediated growth inhibition. In summary, we have identified several novel anti-invasion mechanisms of YC-1 that could impact NPC, and possibly other cancers as well.

Evaluation of novel H1N1-specific primer-probe sets using commercial RT-PCR mixtures and a premixed reaction stored in a lyophilized format.

The recent emergence of a novel H1N1 influenza A virus in humans caused the first influenza pandemic of this century. Many clinical diagnostic laboratories are overwhelmed by the testing demands related to the infection. Three novel H1N1-specific primer-probe sets reported during the early phase of the pandemic were tested in three commercial real-time RT-PCR mixtures. The amplification efficiencies and detection limits of these assays were determined. A ready-to-use premixed RT-PCR stored in a lyophilized format was developed. The detection limits of the studied assays were highly variable, ranging from 1.68E-01 to 1.68E-05 TCID(50) per reaction. The detection limit of the lyophilized reaction mixture was found to be 1.68E-05 TCID(50) per reaction, but the amplification efficiency of the assay was lower than those deduced from the other assays. All respiratory samples from infected patients and all control nasopharyngeal aspirates were positive and negative, respectively, in the newly developed assay. The results highlighted that, to enhance the sensitivity of an assay, it is essential to evaluate a primer-probe set with different commercial RT-PCR assays. This study also demonstrated the feasibility of using lyophilized reaction mixtures for the molecular diagnosis of novel H1N1.