bα matrix  (Nonlinear Dynamics)

Journal: Virology

Article Title: Intravirion cohesion of matrix protein M1 with ribonucleocapsid is a prerequisite of influenza virus infectivity.

doi: 10.1016/j.virol.2016.02.021

Figure Lengend Snippet: Fig. 4. Preserving effect of Rimantadine against virus acidification. Virus A/HK/68 was grown in MDCK-H cells, treated with trypsin to cleave HA0 into HA1þHA2, and pelleted through 20% glycerol to remove virus-free RNPs. The virus was pre- treated with 200 mM of Rim for 20 min and acidified for 10 and 35 min (Acid), respectively, in the presense of Rim. Next, virus samples were either directly fractionated by uncoating centrifugation through NP40-containing glycerol (A) or treated with NP-40 in 15% glycerol at neutral pH and fractionated by differential centrifugation to obtain nucleocapsid pellets (NC) and soluble supernatant (Sol) fractions (B,C). NP and M1 polypeptides in the obtained fractions were analyzed by PAGE-WB using anti-NP and -M1 antibodies and ECL detection protocol. (A) Pellet fractions of viruses, which were pretreated for 20 min with and without Rim and then were acidified or not for 10 min. (B, C) NC and Sol fractions of viruses, which were pretreated with and without Rim and then were acidified either for 10 (B) or 35 min (C), respectively.

Article Snippet: After washing with PBS, membranes were incubated for 3 h at 20 °C in PBS containing 1% BSA, and either antisera specific to influenza H3 hemagglutinin (collection of the Institute of Virology, Marburg, Germany) and mouse monoclonal antibodies against influenza virus NP (clone A3; CDC, Atlanta), or influenza matrix M1 (Serotec, Germany).

Techniques: Preserving, Virus, Centrifugation

Journal: Virology

Article Title: Intravirion cohesion of matrix protein M1 with ribonucleocapsid is a prerequisite of influenza virus infectivity.

doi: 10.1016/j.virol.2016.02.021

Figure Lengend Snippet: Fig. 3. M1 and NP profiles of nucleocapsid fraction isolated from acidified influenza A virus. Influenza virus A/Aichi/2/68 (H3N2) virus was grown in MDCK-H cells (samples 1, 2) and treated with trypsin in vitro (3, 4). To separate contaminating virus-free RNPs, the virus was pelleted through 30% glycerol before acidification for 30 min at pH 4.5 (2, 4), followed by neutralization. The obtained virus samples were centrifuged through 2.5 ml of 30% glycerol containing 0.3% of NP-40, 0.2 M NaCl, 20 mM Tris–HCl (pH 8.0), at 27,000 rpm for 3 h in SW 55.1 rotor (Optima L-80XP). Pellets (nucleocapsid fraction) were analyzed by PAGE-WB using anti- HA, NP, and M1 antibodies. Lane V – intact non-treated A/Aichi/68 virus.

Article Snippet: After washing with PBS, membranes were incubated for 3 h at 20 °C in PBS containing 1% BSA, and either antisera specific to influenza H3 hemagglutinin (collection of the Institute of Virology, Marburg, Germany) and mouse monoclonal antibodies against influenza virus NP (clone A3; CDC, Atlanta), or influenza matrix M1 (Serotec, Germany).

Techniques: Isolation, Virus, In Vitro, Neutralization

Journal: Virology

Article Title: Intravirion cohesion of matrix protein M1 with ribonucleocapsid is a prerequisite of influenza virus infectivity.

doi: 10.1016/j.virol.2016.02.021

Figure Lengend Snippet: Fig. 7. Asymmetric structure of influenza A virus and its role in RNP release under acidification. Influenza A virus has two poles. RNP segments seem to locate at one end as a bunch-like structure (the head of the virion) (Noda et al. 2012; Vijayakrishnan et al. 2013) and interact with matrix M1 via the RNP polymerase promoter region (panels A, D). Such RNP orientation can provide (i) intersegment interaction via budding packaging signal located in the polymerase promoter region (Sugita et al., 2013; Fournier et al., 2012) and (ii) interaction of the RNP promoter region with the M1, which suppresses RNP polymerase complex activity inside the virion, according to data in Fig. 5 (Zhirnov, 1992). The opposite virion end is characterized by depleted amounts of the matrix M1 and the surface glycoproteins HA and NA and is enriched with M2 proteins (the virion tail) (panel A) (Nayak et al., 2009). The viral tail corresponds to the pinching off (scission) site of budding virus at the host plasma membrane (Barman and Nayak, 2007; Rossman et al., 2010b; Rossman and Lamb, 2011; Roberts et al., 2013). In acidic medium, the scission focus on virions with cleaved HA1þHA2 is transformed into a bleb where the M2 is concentrated and conducts proton influx and likely exchanges Kþ ions for protons inside the virion (Stauffer et al., 2014) (panel B). The M2 ion channels, in virus with uncleaved HA0, are locked in the closed state, despite external acidification (panels D and E). During infectious virus entry, RNP segments are moved from the head to the tail site (bleb domain), possibly by the M1 in cooperation with NEP (Yasuda et al., 1993), and released from M1 to be then translocated into cytosol where they can interact with cellular cargo machinery transporting them to the nucleus (panels F-H). Under artificial in vitro acidification, RNP segments in the HA1/2 virus prematurely lose M1 contact, activating the RNA polymerase function of the RNPs but destroying their tail-directed translocation inside the virion (panels A-C). In vitro acidified virions become unable to translocate RNPs from the virion cavity and to target them to the host nucleus. Thus, such preacidified virions fail to initiate the infection process when they interact with the target cell according to data in Fig. 6 (panel C). Images F, G, and H show the process that is believed to occur during productive infection displaying gradient increase of acidification on the pathway from early to late endosome and final RNP release into the host cell cytoplasm. Grey phone inside virions reflects the intensity of acidification.

Article Snippet: After washing with PBS, membranes were incubated for 3 h at 20 °C in PBS containing 1% BSA, and either antisera specific to influenza H3 hemagglutinin (collection of the Institute of Virology, Marburg, Germany) and mouse monoclonal antibodies against influenza virus NP (clone A3; CDC, Atlanta), or influenza matrix M1 (Serotec, Germany).

Techniques: Virus, Activity Assay, Clinical Proteomics, Membrane, Transformation Assay, In Vitro, Translocation Assay, Infection