During influenza A virus infection, the viral RNA polymerase transcribes the viral negative-sense segmented RNA genome and replicates it in a two-step process via complementary RNA within viral ...ribonucleoprotein (vRNP) complexes. While numerous viral and host factors involved in vRNP functions have been identified, dissecting the roles of individual factors remains challenging due to the complex cellular environment in which vRNP activity has been studied. To overcome this challenge, we reconstituted viral transcription and a full cycle of replication in a test tube using vRNPs isolated from virions and recombinant factors essential for these processes. This novel system uncovers the minimal components required for influenza virus replication and also reveals new roles of regulatory factors in viral replication. Moreover, it sheds light on the molecular interplay underlying the temporal regulation of viral transcription and replication. Our highly robust in vitro system enables systematic functional analysis of factors modulating influenza virus vRNP activity and paves the way for imaging key steps of viral transcription and replication.
Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge
. Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by ...the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein complexes
. RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA inefficiently in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity
. Host-adaptive mutations, particularly a glutamic-acid-to-lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit, enable avian FluPolA to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here we report cryo-electron microscopy structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain of ANP32A. The C-terminal low-complexity acidic region of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, suggesting a mechanism for how the adaptive PB2(E627K) mutation enables the replication of viral RNA in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a viral ribonucleoprotein complex.
Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral ...RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.
Avian influenza A viruses (IAVs) pose a public health threat, as they are capable of triggering pandemics by crossing species barriers. Replication of avian IAVs in mammalian cells is hindered by ...species-specific variation in acidic nuclear phosphoprotein 32 (ANP32) proteins, which are essential for viral RNA genome replication. Adaptive mutations enable the IAV RNA polymerase (FluPolA) to surmount this barrier. Here, we present cryo-electron microscopy structures of monomeric and dimeric avian H5N1 FluPolA with human ANP32B. ANP32B interacts with the PA subunit of FluPolA in the monomeric form, at the site used for its docking onto the C-terminal domain of host RNA polymerase II during viral transcription. ANP32B acts as a chaperone, guiding FluPolA towards a ribonucleoprotein-associated FluPolA to form an asymmetric dimer-the replication platform for the viral genome. These findings offer insights into the molecular mechanisms governing IAV genome replication, while enhancing our understanding of the molecular processes underpinning mammalian adaptations in avian-origin FluPolA.
Eukaryotic organelles have developed elaborate protein quality control systems to ensure their normal activity, among which Deg/HtrA proteases play an essential role. Plant Deg2 protease is a ...homologue of prokaryotic DegQ/DegP proteases and is located in the chloroplast stroma, where its proteolytic activity is required to maintain the efficiency of photosynthetic machinery during stress. Here, we demonstrate that Deg2 exhibits dual protease-chaperone activities, and we present the hexameric structure of Deg2 complexed with co-purified peptides. The structure shows that Deg2 contains a unique second PDZ domain (PDZ2) following a conventional PDZ domain (PDZ1), with PDZ2 orchestrating the cage assembly of Deg2. We discovered a conserved internal ligand for PDZ2 that mediates hexamer formation and thus locks the protease in the resting state. These findings provide insight into the diverse modes of PDZ domain-mediated regulation of Deg proteases.
Background: The PDZ protease Deg2 is involved in chloroplast protein quality control through a yet unknown molecular mechanism.
Results: A novel PDZ domain with an internal ligand mediates hexamer formation and locks Deg2 into the resting state.
Conclusion: Formation of the resting hexamer may be a common strategy in a Deg protease subfamily.
Significance: We provide structural insights into the PDZ domain-mediated regulation of Deg proteases.
Objectives T-2307, a novel arylamidine synthesized at Toyama Chemical Co., Ltd, has in vitro and in vivo broad-spectrum activities against pathogenic fungi. T-2307 particularly exhibits potent in ...vitro and in vivo activity against Candida albicans, suggesting that its uptake might be mediated by a transport system. In this report, we studied the uptake of T-2307 in C. albicans. Methods C. albicans cells and rat hepatocytes were exposed to 0.02 µM 14CT-2307. After incubation, the reaction mixture was concentrated and layered on a silicon layer (mixture of silicon oil and liquid paraffin) inside a tube. The tube was then centrifuged to transfer cells into the bottom layer (sodium hydroxide) for solubilization. The bottom layer was neutralized and measured for radioactivity. Results T-2307 was concentrated from the extracellular medium by C. albicans cells in 10 mM phosphate buffer solution supplemented with 1% glucose by 3200- to 5100-fold. The accumulation was approximately two orders of magnitude greater than that achieved with a rat hepatocyte preparation. T-2307 uptake was sensitive to temperature and extracellular pH, and was reduced in the presence of inhibitors of mitochondrial respiration, oxidative phosphorylation and plasma membrane proton pump, and by an uncoupler. Furthermore, T-2307 uptake was concentration dependent and an Eadie–Hofstee plot suggested the involvement of two transport systems. Conclusions The considerably higher concentrations of T-2307 were selectively accumulated in C. albicans via transporter-mediated systems, as compared with the concentrations in rat hepatocytes. This transporter-mediated uptake of T-2307 contributes to its potent anticandidal activity.
Rhizosphere microorganism is an important bio‐component for wastewater treatment in constructed wetlands (CWs). Microbial abundance and enzyme activities in the rhizospheres of nine plant species ...were investigated in an integrated vertical‐flow CW. The abundance of denitrifiers, as well as urease, acid, and alkaline phosphatase activities were positively correlated to plant root biomass. The abundance of bacteria, fungi, actinomycetes, ammonifiers, denitrifiers, and phosphorus decomposers, related to nutrient removal efficiencies in CWs, greatly varied among rhizospheres of different plant species (p < 0.05). Significant differences in rhizosphere enzyme activity among plant species were also observed (p < 0.05), with the exception of catalase activity. The principal component analysis using the data of microbial abundance and enzyme activity showed that Miscanthus floridulus, Acorus calamus, and Reineckia carnea were candidates to be used in CWs to effectively remove nitrogen and phosphorus from wastewater.
The results showed that Miscanthus floridulus, Acorus calamus, and Reineckia carnea were candidates to be used in constructed wetlands to effectively remove nitrogen and phosphorus from wastewater in regard to plant root biomass and rhizosphere microbial parameters.
Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans
. Influenza A viruses contain a segmented ...negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPol
) composed of PB1, PB2 and PA subunits
. Although the high-resolution crystal structure of FluPol
of bat influenza A virus has previously been reported
, there are no complete structures available for human and avian FluPol
. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase
-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPol
from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0-4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPol
forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPol
bound to the cRNA template reveals a binding site for the 3' cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPol
dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPol
dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPol
, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPol
that could be targeted in the development of antiviral drugs.
Abstract
SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. ...SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5′ capping of viral RNAs. The formation of the 5′ 7-methylguanosine (m7G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5′ triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5′ end of viral RNA via a 5′ to 5′ triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.
Influenza A virus (IAV) contains a segmented RNA genome that is transcribed and replicated by the viral RNA polymerase in the cell nucleus. Replicated RNA segments are assembled with viral polymerase ...and oligomeric nucleoprotein into viral ribonucleoprotein (vRNP) complexes which are exported from the nucleus and transported across the cytoplasm to be packaged into progeny virions. Host GTPase Rab11a associated with recycling endosomes is believed to contribute to this process by mediating the cytoplasmic transport of vRNPs. However, how vRNPs interact with Rab11a remains poorly understood. In this study, we utilized a combination of biochemical, proteomic, and biophysical approaches to characterize the interaction between the viral polymerase and Rab11a. Using pulldown assays, we showed that vRNPs but not complementary RNPs (cRNPs) from infected cell lysates bind to Rab11a. We also showed that the viral polymerase directly interacts with Rab11a and that the C-terminal two-thirds of the PB2 polymerase subunit (PB2-C) comprising the cap-binding, mid-link, 627, and nuclear localization signal (NLS) domains mediate this interaction. Small-angle X-ray scattering (SAXS) experiments confirmed that PB2-C associates with Rab11a in solution forming a compact folded complex with a 1:1 stoichiometry. Furthermore, we demonstrate that the switch I region of Rab11a, which has been shown to be important for binding Rab11 family-interacting proteins (Rab11-FIPs), is also important for PB2-C binding, suggesting that IAV polymerase and Rab11-FIPs compete for the same binding site. Our findings expand our understanding of the interaction between the IAV polymerase and Rab11a in the cytoplasmic transport of vRNPs.
The influenza virus RNA genome segments are replicated in the cell nucleus and are assembled into viral ribonucleoprotein (vRNP) complexes with viral RNA polymerase and nucleoprotein (NP). Replicated vRNPs need to be exported from the nucleus and trafficked across the cytoplasm to the cell membrane, where virion assembly takes place. The host GTPase Rab11a plays a role in vRNP trafficking. In this study, we showed that the viral polymerase directly interacts with Rab11a mediating the interaction between vRNPs and Rab11a. We mapped this interaction to the C-terminal domains of the PB2 polymerase subunit and the switch I region of Rab11a. Identifying the exact site of Rab11a binding on the viral polymerase could uncover a novel target site for the development of an influenza antiviral drug.
PDF
