Two proteins were observed to be disassociated from 10 to 12hrs post infection, the last time point in the assay (data not shown). subcellular fractionation was done. Endogenous nucleolin expression in cytosolic and nuclear fractions was measured by western blotting using anti-nucleolin antibody and the viral NP expression using anti-viral NP antiserum. [a] Nucleolin and NP expression in two fractions of virus infected cells [b] Nucleolin expression in two fractions of mock infected cells. Marker proteins; -actin and c-Jun expression confirmed the purity of cytoplamic and nuclear fractions prepared from virus and mock infected cells.(TIF) pone.0164146.s002.tif (153K) GUID:?050F5688-FC79-43A2-8FE8-9E35B3D527E9 S3 Fig: Optimization of binding of recombinant viral NP and host nucleolin. BL-21 cells were transformed with the recombinant viral NP (pET29a+NP) or unrelated control protein and cell lysates were prepared. Either 100g of bacterial lysate incubated with different concentrations of Ni-NTA beads ranging from 12.5 to 100l or 100l of beads incubated with different concentrations of bacterial lysate ranging from 50 to 250g for 6hrs Mifepristone (Mifeprex) to immobilize the recombinant protein on Ni-NTA beads. Further, beads were washed and incubated with 1mg of A549 cell lysate. Next day, after washing the beads, bound protein complexes were eluted and subjected to SDS PAGE followed by immunoblotting with anti-nucleolin and anti-His antibodies. Cell lysates recovered after centrifugation following incubation with recombinant viral NP and control protein bound Ni-NTA beads were analyzed for endogenous nucleolin expression. [a] Binding of 110kDa nucleolin protein and the recombinant viral NP with the use of 100l beads [b] Dose dependent binding of nucleolin with viral NP [c] and [d] No visible Mifepristone (Mifeprex) binding of nucleolin with the control protein. Expression of recombinant viral NP, control protein and nucleolin was shown in the corresponding bacterial and A549 cell lysates.(TIF) pone.0164146.s003.tif (208K) GUID:?83993C5E-0828-47F0-993B-62CAB002E510 S4 Fig: Influenza A viral hemagglutination assay (HA assay). HA titer was measured in virus lysates harvested at 24hrs post contamination from A549 cells transfected with siRNA-NCL or siRNA-NT or pEGFP-NCL or pEGFP-C1. Viral lysates recovered from untransfected but virus infected and mock-infected cells at 24hrs post contamination were used as controls. Twofold serial dilutions of each sample was made in 1 PBS and incubated with guinea pig RBCs. Agglutination of RBCs was recorded for each sample. HA assay showing agglutination by virus lysate collected from siRNA-NCL cells up to 1 1:4 dilutions. No visible agglutination was observed by pEGFP-NCL cell lysate.(TIF) pone.0164146.s004.tif (378K) GUID:?826080CF-E5B0-4D20-83D2-284435BE42B2 S5 Fig: Titer of infectious viral progeny released from cells with depleted and overexpressed nucleolin. A549 cells were transfected with siRNA-NCL or siRNA-NT or pEGFP-NCL or pEGFP-C1 constructs followed by infections. Untransfected but virus or mock infected cells were included as controls. At 48hrs post contamination following 24hrs transfection, medium from infected cells was collected and the titer of the released infectious viral progeny in each sample was determined by TCID50 assay as described in Fig 7.(TIF) pone.0164146.s005.tif (71K) GUID:?335B49D2-3865-42DA-A07A-4314D443D0BF Data Availability StatementAll the relevant data are within the paper. Abstract Influenza A virus nucleoprotein, is Mifepristone (Mifeprex) usually a multifunctional RNA-binding protein, encoded by segment-5 of the unfavorable sense RNA genome. It serves as a key connector between the virus and the host during virus replication. It constantly shuttles between the cytoplasm and the nucleus interacting with various host cellular factors. In the current study, host proteins interacting with nucleoprotein of Influenza A virus of H1N1 2009 pandemic strain were identified by co-immunoprecipitation studies followed by MALDI-TOF/MS analysis. Here we report the host nucleolin, a major RNA-binding protein of the nucleolus as a novel interacting partner to influenza A virus nucleoprotein. We thus, explored the implications of this interaction in virus life cycle and our studies have shown that these two proteins interact early during contamination in the cytoplasm of infected cells. Depletion of nucleolin in A549 cells Goat polyclonal to IgG (H+L) by siRNA targeting endogenous nucleolin followed by influenza A virus contamination, disrupted its conversation with viral nucleoprotein, resulting in increased expression of gene transcripts Mifepristone (Mifeprex) encoding late viral proteins; matrix (M1) and hemagglutinin (HA) in infected cells. On the contrary, over expression of nucleolin in cells transiently transfected with pEGFP-NCL construct followed by virus infection significantly reduced the late viral gene transcripts, and consequently the viral titer. Altered expression of late viral genes and titers following manipulation of host cellular nucleolin, proposes the functional importance of its conversation with nucleoprotein during influenza A virus infection. Introduction Influenza A virus is usually a public health threat worldwide and contributes to a high-level of mortality during pandemics. Its segmented genetic composition allows re-assortment of gene segments between the strains of different host origins resulting in the emergence of a novel strain and an unpredictable pandemic. Eight unfavorable sense single stranded RNA gene segments of influenza A virus encode for 10 proteins. However, 7 more novel.