Organization of the Influenza Virus Replication Machinery

National Resource for Automated Molecular Microscopy, Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Science (Impact Factor: 33.61). 11/2012; 338(6114). DOI: 10.1126/science.1227270
Source: PubMed


Influenza virus ribonucleoprotein complexes (RNPs) are central to the viral life cycle and in adaptation to new host species. RNPs are composed of the viral genome, viral polymerase, and many copies of the viral nucleoprotein. In vitro cell expression of all RNP protein components with four of the eight influenza virus gene segments enabled structural determination of native influenza virus RNPs by cryo-EM. The cryo-EM structure reveals the architecture and organization of the native RNP, thereby defining the attributes of its largely helical structure and how polymerase interacts with NP and the viral genome. Observations of branched-RNP structures in negative stain EM and their putative identification as replication intermediates suggest a mechanism for viral replication by a second polymerase on the RNP template.

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    • "NP is a major component of the ribonucleoprotein (RNP) complexes. The RNP consists of RNA, multiple copies of NP, and polymerase subunits (PB1, PB2 and PA), together forming a large loop that is twisted into a helical rod-like structure [19] [20]. NP plays regulatory roles in transcription, replication, and virus maturation [21], and it is one of the most conserved proteins among influenza A viruses with >90% protein sequence homology [22] [23]. "
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    Vaccine 09/2015; DOI:10.1016/j.vaccine.2015.08.094 · 3.62 Impact Factor
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    • "Sample dilution was adjusted to achieve a homogeneous separation of particles. Samples were stained as previously described (Moeller et al., 2012) (Tao et al., 2013) using a 2% uranyl formate solution. EM micrographs were acquired using a Tecnai F20 Twin transmission electron microscope operating at 200 kV, using a dose of $45 e À /A ˚ 2 and nominal underfocus ranging from 0.7 mm to 1.7 mm. "
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    ABSTRACT: ATP-binding cassette (ABC) exporters are ubiquitously found in all kingdoms of life and their members play significant roles in mediating drug pharmacokinetics and multidrug resistance in the clinic. Significant questions and controversies remain regarding the relevance of their conformations observed in X-ray structures, their structural dynamics, and mechanism of transport. Here, we used single particle electron microscopy (EM) to delineate the entire conformational spectrum of two homologous ABC exporters (bacterial MsbA and mammalian P-glycoprotein) and the influence of nucleotide and substrate binding. Newly developed amphiphiles in complex with lipids that support high protein stability and activity enabled EM visualization of individual complexes in a membrane-mimicking environment. The data provide a comprehensive view of the conformational flexibility of these ABC exporters under various states and demonstrate not only similarities but striking differences between their mechanistic and energetic regulation of conformational changes. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Structure 01/2015; 23(3). DOI:10.1016/j.str.2014.12.013 · 5.62 Impact Factor
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    • "Mechanistically, RIG-I was found to counteract the accessibility of HBV P protein to the 5 0 -ε stem-loop of pgRNA, which is an important process for the initiation of viral replication (Bartenschlager and Schaller, 1992). As is the case with this, several viral PAMPs known to be recognized by RIG-I, for example, the poly-U/UC tract in the 3 0 nontranslated region of HCV genome (Saito et al., 2008) and 5 0 terminal region of influenza virus genome (Baum et al., 2010) were previously reported to be directly or indirectly critical for viral replication (You and Rice, 2008; Huang et al., 2005; Moeller et al., 2012). In this respect, one could envisage that such an exquisite targeting by RIG-I would confer a unique machinery to ensure efficient antiviral activities of RIG-I. "
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    Immunity 12/2014; DOI:10.1016/j.immuni.2014.12.016 · 21.56 Impact Factor
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