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.
"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. "
"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. "
[Show abstract][Hide abstract] ABSTRACT: Highlights
•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.
"The structure of the RNP complex were investigated using cryogenic electron microscopy [4,5], however, there have been no structural analyses of the entire RNP complex or the RNA polymerase complex at the atomic level. Partial domain structures of the RNA polymerase subunit have been reported [6-11]. "
[Show abstract][Hide abstract] ABSTRACT: Influenza pandemics with human-to-human transmission of the virus are of great public concern. It is now recognized that a number of factors are necessary for human transmission and virulence, including several key mutations within the PB2 subunit of RNA-dependent RNA polymerase. The structure of the middle domain in PB2 has been revealed with or without m(7)GTP, thus the middle domain is considered to be novel target for structure-based drug design. Here we report the crystal structure of the middle domain of H1N1 PB2 with or without m(7)GTP at 1.9Å and 2.0Å resolution, respectively, which has two mutations (P453H, I471T) to increase electrostatic potential and solubility. Here we report the m(7)GTP has unique conformation differ from the reported structure. 7-methyl-guanine is fixed in the pocket, but particularly significant change is seen in ribose and triphosphate region: the buried 7-methyl-guanine indeed binds in the pocket forming by H357, F404, E361 and K376 but the triphosphate continues directly to the outer domain. The presented conformation of m(7)GTP may be a clue for the anti-influenza drug-design.
PLoS ONE 11/2013; 8(11):e82020. DOI:10.1371/journal.pone.0082020 · 3.23 Impact Factor
Peixiang Ma, Yi Xue, Nicolas Coquelle, Jens D Haller, Tairan Yuwen, Isabel Ayala, Oleg Mikhailovskii, Dieter Willbold, Jacques-Philippe Colletier, Nikolai R Skrynnikov, Paul Schanda
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.