The TBSV viral RNA is recruited to the membrane from the soluble fraction with the help of TBSV replication proteins and host factors present in the yeast CFE. The in vitro RNA recruitment reaction was performed according to [20, 23] , except that 32 Plabeled DI-72 (+)repRNA were used and rCTP, rUTP, 32 P-labeled UTP, and Actinomycin D were omitted from the assay. As a negative control, p33 and p92 were omitted from the reaction to detect DI-72 binding nonselectively to host proteins present in the membrane. In vitro binding assay with purified recombinant eEF1Bc (Tef3). The TBSV (+)RNA templates were the four noncontiguous segments of the TBSV (+)RNA that are present in defective interfering RNAs, including DI-72 repRNA used in this study. RI(+) represents the 59-UTR, RII(+)-SL is an internal highly conserved sequence that binds to p33 replication protein, RIII(+) is ashort conserved sequence closed to the 39 end, andSL3-2-1(+), which contains the promoter region (SL1) for initiation and the replication silencer element (within SL3) that down-regulates initiation. The assay contained 32 P-labeled free ssRNA (as shown), plus 0.6 pmol purified recombinant eEF1Bc, respectively. The bound RNA-protein complexes were separated on nondenaturing 5% acrylamide gels. Quantification of the free (unshifted) RNA was done with ImageQuant. (B) RNA gel shift analysis shows SL3-2-1(+) RNA binds competitively to eEF1Bc. The RNA templates representing the 39 end of the TBSV RNA and the deleted nucleotides are shown schematically. The cold competitor was SL3-2-1(+) RNA, which represents a large portion of the 39-UTR ( Figure 4A ). The eEF1Bc -32 P-labeled ssRNA complex was visualized on nondenaturing 5% acrylamide gels. (EPS) Figure S2 eEF1Bcdoes not affect the template recruitment step in vitro. Purified recombinant p33/p92 and 32 P-labeled DI-72 (+)repRNA and eEF1Bc (affinity purified recombinant Tef3) were added to a whole cell extract (CFE), followed by centrifugation/ washing to remove the 32 P-labeled repRNA that is not bound to the membrane. Then the membrane-bound RNA was analyzed in a denaturing PAGE gel. Note that the repRNA binds to the cellular membrane fraction nonspecifically (,20% level) in the absence of the viral replication proteins. (EPS) Mannose-Binding Lectin Contributes to Deleterious Inflammatory Response in Pandemic H1N1 and Avian H9N2 Infection Background. Mannose-binding lectin (MBL) is a pattern-recognition molecule, which functions as a first line of host defense. Pandemic H1N1 (pdmH1N1) influenza A virus caused massive infection in 2009 and currently circulates worldwide. Avian influenza A H9N2 (H9N2/G1) virus has infected humans and has the potential to be the next pandemic virus. Antiviral function and immunomodulatory role of MBL in pdmH1N1 and H9N2/G1 virus infection have not been investigated. Methods. In this study, MBL wild-type (WT) and MBL knockout (KO) murine models were used to examine the role of MBL in pdmH1N1 and H9N2/G1 virus infection. Results. Our study demonstrated that in vitro, MBL binds to pdmH1N1 and H9N2/G1 viruses, likely via the carbohydrate recognition domain of MBL. Wild-type mice developed more severe disease, as evidenced by a greater weight loss than MBL KO mice during influenza virus infection. Furthermore, MBL WT mice had enhanced production of proinflammatory cytokines and chemokines compared with MBL KO mice, suggesting that MBL could upregulate inflammatory responses that may potentially worsen pdmH1N1 and H9N2/G1 virus infections. Conclusions. Our study provided the first in vivo evidence that MBL may be a risk factor during pdmH1N1 and H9N2/G1 infection by upregulating proinflammatory response. The pandemic H1N1 2009 (pdmH1N1) influenza A virus has spread globally since the outbreak first started in Mexico in 2009. As of August 2010, the virus had already caused more than 18 449 deaths in at least 214 countries worldwide [1] . The World Health Organization officially announced the step-down of pdmH1N1 to postpandemic phase on 10 August 2010, and the virus was expected to circulate as a seasonal virus in the human population thereafter [2] . Gene segments of the pdmH1N1 virus are derived from multiple lineages, including the Eurasian swine, the classical swine, and the triple reassortant swine lineages. Multiple genetic reassortment events of viral components have taken place and thus gave rise to this novel pandemic virus [3] . Clinical symptoms of pdmH1N1 infection are usually mild, possibly due to the cross-protection offered by memory cytotoxic T lymphocytes established from previous exposure to seasonal influenza [4] .