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
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.
Available from: sciencedirect.com
- "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  . NP plays regulatory roles in transcription, replication, and virus maturation , and it is one of the most conserved proteins among influenza A viruses with >90% protein sequence homology  . "
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ABSTRACT: Vaccination is at present the most efficient way of preventing influenza infections. Currently used inactivated influenza vaccines can induce virus-neutralizing antibodies that are protective against a particular influenza strain, but hamper the induction of cross-protective T-cell responses to later infections. Thus, influenza vaccines need to be updated annually in order to confer protection against circulating influenza strains. This study aims at developing an efficient vaccine that can induce broader protection against influenza. For this purpose, we have used the highly conserved nucleoprotein (NP) from an influenza A virus subtype H7N7 strain, and inserted it into a vaccine format that targets an antigen directly to relevant antigen presenting cells (APCs). The vaccine format consists of bivalent antigenic and targeting units, linked via an Ig-based dimerization unit. In this study, NP was linked to MIP-1α, a chemokine that targets the linked antigen to chemokine receptors 1, 3 and 5 expressed on various APCs. The vaccine protein was indirectly delivered by DNA. Mice were vaccinated intradermally with plasmids, in combination with electroporation to enhance cellular uptake of DNA. We found that a single DNA vaccination was sufficient for induction of both antibody and T cell responses in BALB/c mice. Targeting of nucleoprotein to chemokine receptors enhanced T cell responses but not antibody responses. Moreover, a single dose of MIP1α-NP conferred protection in BALB/c mice against a lethal challenge with an H1N1 influenza virus. The observed cross-protection was mediated by CD8(+) T cells.
Vaccine 09/2015; DOI:10.1016/j.vaccine.2015.08.094 · 3.62 Impact Factor
Available from: Sung Lee
- "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.
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Structure 01/2015; 23(3). DOI:10.1016/j.str.2014.12.013 · 5.62 Impact Factor
Available from: Michinori Kohara
- "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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•Type III IFNs are predominantly induced in human hepatocytes during HBV infection
•RIG-I senses the HBV genotype A, B, and C for the induction of type III IFNs
•The 5′-ε region of HBV pgRNA is a key element for the RIG-I-mediated recognition
•RIG-I counteracts the interaction of HBV P with pgRNA to suppress viral replication
Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5′-ε region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5′-ε region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this ε region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.
Immunity 12/2014; DOI:10.1016/j.immuni.2014.12.016 · 21.56 Impact Factor
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