New nonacyclic duclauxin derivatives with potent anti-influenza activities from Antarctic fungus Penicillium sp. CPCC 401065.

The heterotrimeric RNA-dependent RNA polymerase of influenza viruses catalyzes RNA replication and transcription activities in infected cell nuclei. The nucleotide polymerization activity is common to both replication and transcription processes, with an additional cap-snatching function being employed during transcription to steal short 5'-capped RNA primers from host mRNAs. Cap-binding, endonuclease, and polymerase activities have long been studied biochemically, but structural studies on the polymerase and its subunits have been hindered by difficulties in producing sufficient quantities of material. Recently, because of heightened effort and advances in expression and crystallization technologies, a series of high resolution structures of individual domains have been determined. These shed light on intrinsic activities of the polymerase, including cap snatching, subunit association, and nucleocytoplasmic transport, and open up the possibility of structure-guided development of new polymerase inhibitors. Furthermore, the activity of influenza polymerase is highly host- and cell type-specific, being dependent on the identity of a few key amino acid positions in the different subunits, especially in the C-terminal region of PB2. New structures demonstrate the surface exposure of these residues, consistent with ideas that they might modulate interactions with host-specific factors that enhance or restrict activity. Recent proteomic and genome-wide interactome and RNA interference screens have suggested the identities of some of these potential regulators of polymerase function.

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis. It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction.

Influenza virus RNA-dependent RNA polymerase is a heterotrimer composed of PA, PB1, and PB2 subunits. RNA-dependent RNA polymerase is required for both transcription and replication of influenza viral RNA taking place in the nucleus of infected cells. A "cap-snatching" mechanism is used to generate a 5'-capped primer for transcription in which the cap-binding domain of PB2 (PB2cap) captures the 5' cap of the host pre-mRNA. Our statistical analysis of PB2 sequences showed that residue Lys(339) located in the cap-binding pocket of H5N1 PB2cap was gradually replaced by Thr(339) over the past decade. To understand the role of this amino acid polymorphism, we solved the crystal structures of PB2cap with or without a pre-mRNA cap analog, m(7)GTP, in the presence of Lys(339) or Thr(339). The structures showed that Lys(339) contributes to binding the γ-phosphate group of m(7)GTP, and the replacement of Lys(339) by Thr eliminates this interaction. Isothermal titration calorimetry analysis showed that Thr(339) attenuated the PB2cap cap binding activity in vitro compared with Lys(339). Further functional studies confirmed that Thr(339)-PB2-containing ribonucleoprotein complex has a reduced influenza polymerase activity and RNA synthesis activity, and a reconstituted H5N1 virus containing the Thr(339) substitution exhibited a lower virulence to mice but more active replication in Madin-Darby canine kidney cells. The K339T substitution in the cap-binding pocket of PB2 modulates the polymerase activity and virulence by regulating the cap binding activity. It is informative to track variations in the cap-binding pocket of PB2 in surveillance of the evolution and spread of influenza virus.

In the influenza virus life cycle, viral RNA (vRNA) transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA), catalyzed by the viral RNA-dependent RNA polymerase in the host cell nucleus, are delicately controlled, and the levels of the three viral RNA species display very distinct synthesis dynamics. However, the underlying mechanisms remain elusive. Here, we demonstrate that in the context of virus infection with cycloheximide treatment, the expression of viral nonstructural protein 1 (NS1) can stimulate primary transcription, while the expression of viral NS2 inhibits primary transcription. It is known that the NS1 and NS2 proteins are expressed with different timings from unspliced and spliced mRNAs of the viral NS segment. We then simulated the synthesis dynamics of NS1 and NS2 proteins during infection by dose-dependent transfection experiments in ribonucleoprotein (RNP) reconstitution systems. We found that the early-expressed NS1 protein can stimulate viral mRNA synthesis, while the late-expressed NS2 protein can inhibit mRNA synthesis but can promote vRNA synthesis in a manner highly consistent with the dynamic changes in mRNA/vRNA in the virus life cycle. Furthermore, we observed that the coexistence of sufficient NS1 and NS2, close to the status of the NS1 and NS2 levels in the late stage of infection, could boost vRNA synthesis to the highest efficiency. We also identified key functional amino acids of NS1 and NS2 involved in these regulations. Together, we propose that the stoichiometric changes in the viral NS1 and NS2 proteins during infection are responsible for the fine regulation of viral RNA transcription and replication. IMPORTANCE In order to ensure efficient multiplication, influenza virus transcribes and replicates its segmented, negative-sense viral RNA genome in highly ordered dynamics across the virus life cycle. How the virus achieves such regulation remains poorly understood. Here, we demonstrate that the stoichiometric changes in the viral NS1 and NS2 proteins during infection could be responsible for the fine regulation of the distinct dynamics of viral RNA transcription and replication. We thus propose a fundamental mechanism exploited by influenza virus to dynamically regulate the synthesis of its viral RNA through the delicate control of viral NS1 and NS2 protein expression.

The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.

Structural studies of influenza virus RNPs by electron microscopy indicate molecular contortions within NP supra-structures

Schmallenberg virus (SBV), a newly emerged orthobunyavirus (family Bunyaviridae), has spread rapidly across Europe and has caused congenital abnormalities in the offspring of cattle, sheep, and goats. Like other orthobunyaviruses, SBV contains a tripartite negative-sense RNA genome that encodes four structural and two nonstructural proteins. The nucleoprotein (N) encapsidates the three viral genomic RNA segments and plays a crucial role in viral RNA transcription and replication. Here we report the crystal structure of the bacterially expressed SBV nucleoprotein to a 3.06-Å resolution. The protomer is composed of two domains (N-terminal and C-terminal domains) with flexible N-terminal and C-terminal arms. The N protein has a novel fold and forms a central positively charged cleft for genomic RNA binding. The nucleoprotein purified under native conditions forms a tetramer, while the nucleoprotein obtained following denaturation and refolding forms a hexamer. Our structural and functional analyses demonstrate that both N-terminal and C-terminal arms are involved in N-N interaction and oligomerization and play an essential role in viral RNA synthesis, suggesting a novel mechanism for viral RNA encapsidation and transcription.

The influenza virus RNA polymerase is a heterotrimer comprising the PB1, PB2 and PA subunits. PB1 is the core of the complex and accounts for the polymerase activity. We have studied the interaction of PB1 with model cRNA template by in vitro binding and Northwestern analyses. The binding to model cRNA was specific and showed an apparent Kd of approximately 7x10(-8) M. In contrast to the interaction with vRNA, PB1 was able to bind equally the 5' and 3' arm of the cRNA panhandle. The N-terminal 139 amino acids of PB1 and sequences between positions 267 and 493 proved positive for binding to cRNA, whereas the interaction with vRNA template previously was mapped to the N- and C-terminal regions. Competition experiments using the 5' and 3' arms of either the vRNA or cRNA panhandle indicated that the N-terminal binding site is shared by both templates. The data indicate that the PB1 RNA-binding sites are constituted by: (i) residues located at the N-terminus (probably common for vRNA and cRNA binding) and, either (ii) residues from the central part of PB1 (for cRNA) or (iii) residues from the C-terminal region of PB1 (for vRNA), and suggest that PB1 undergoes a conformational change upon binding to cRNA versus vRNA templates.

Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5' untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target.IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions, namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc finger functions, effectively repressing IAV replication. Based on our findings, we demonstrate that ZBTB25 regulates IAV RNA transcription and replication and serves as a promising antiviral target for IAV treatment.

Influenza viral RNA transcription in the infected cell is inhibited by alpha-amanitin, a specific inhibitor of the host nuclear RNA polymerase II. Because viral RNA transcription in vitro catalysed by the virion-associated transcriptase is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by RNA polymerase II. In addition, because we did not detect any capping and methylating enzymes in virions, we have proposed that the 5' terminal methylated cap found on in-vivo viral messenger RNA (mRNA) is derived from the putative primer RNAs. Our recent experiments have proved these two hypotheses. Purified globin mRNAs were shown to stimulate viral RNA transcription in vitro very effectively. The resulting transcripts directed the synthesis of all the non-glycosylated virus-specific proteins in cell-free systems. Other eukaryotic mRNAs were also active as primers. The presence of a 5' terminal methylated cap structure in the priming mRNA was required for its priming activity. Thus, with globin mRNA, removal of the cap eliminated essentially all of its priming activity, and much of this activity could be restored by enzymically recapping the globin mRNA. Using globin mRNA containing 32P only in its cap, we demonstrated that the 5' cap of the globin mRNA primer was physically transferred to the viral RNA transcripts during transcription. Gel electrophoretic analysis suggested that, in addition to the cap, about 10-15 other nucleotides were also transferred from the globin mRNA to the viral RNA transcripts. A mechanism for the priming of influenza viral RNA transcription by globin mRNA is proposed. Initial experiments strongly suggest that priming by capped host mRNAs also occurs during the synthesis of viral mRNA in vivo.

The modular architecture of the nuclear pore complex (NPC) consists of a membrane‐embedded basic framework (the ‘spoke complex’) made up of eight multidomain spokes with two rings on each face (Figure 1A). The ring facing the cytoplasm is decorated with eight ∼50 nm fibrils extending into the cytosol, and the nuclear ring is capped with a basket‐like assembly of eight thin, 50–100 nm filaments joined distally by a 30–40 nm diameter terminal ring. The center of the basic framework harbors the ‘gated transport channel’, which is involved in signal‐mediated bidirectional transport of macromolecules and is plugged to the central ‘transporter’, whose ultimate structure and functional role in mediated nuclear transport remains to be established. N.Pante and U.Aebi (Basel) reported their approaches to the functional roles of the different NPC components and to dissection of the steps of nuclear import and export at the NPC by direct visualization of gold‐labeled proteins and RNAs moving in or out of the cell nucleus through the NPC (Figure 1B). Figure 1. ( A ) Current consensus model of the membrane‐bound NPC. Its major structural components include the basic framework, the central plug or gated channel, the cytoplasmic and nuclear rings, and the cytoplasmic fibrils and nuclear basket. ( B ) Visualization of the different steps of nuclear import of an NLS‐bearing protein through the NPC. At the bottom are selected examples of cross‐sectioned NPCs with associated nucleoplasmin–gold particles (8 nm diameter), depicted at different stages of transport; at the top is a schematic diagram of the distinct steps visualized by electron microscopy. In the first step of transport, the NLS protein to be imported associates with the NLS receptor (importins α and β) (1). This step takes place in the cytoplasm and does not require physical interaction with any NPC component. In a second step, this ‘targeting complex’ docks …

A Descriptive Study of Viral RNA Sequences Identified from Single-Cell Analysis of Acute Myeloid Leukemia

It is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus (IAV). As nucleoprotein (NP) and Matrix protein 1 (M1) are the most abundant proteins in the viral ribonucleoprotein (vRNP) complex and matrix layer of influenza A virus, and NS1 exerts most functions and interactions with host factor within IAV infection period as reports, these three proteins were widely developed for their hyper-phosphorylation state during IAV infectious course. The NP is a major component of the vRNP complex. During the replication of influenza virus, and each viral RNA segment is encapsulated by multiple copies of the NP. The vRNP complex undergoes a course of nuclear import, viral RNA replication and transcription, and nuclear export, during which NP serves as one of the determinants. Dozens of phosphorylation sites have been identified and determined to regulate the viral polymerase activity and nuclear-cytoplasmic shuttling of NP. Residue S165 have been demonstrated to mediate self-oligomerization of NP through phosphorylation by PKC, while Y10 and Y296 dynamically controlled the nuclear import and export respectively. M1, the most abundant protein in virions, also has multiple functions in the influenza A virus life cycle, including uncoating, transcription, nuclear export of vRNPs, assembly, and budding. Early studies indicated that M1 contains phosphoserine and phosphothreonine residues and has the potential to be phosphorylated by PKC and ERKs. Recently, several phosphorylation sites on M1 (including a phosphotyrosine) were reported. However, a majority of phosphorylated residues of M1 has not yet been identified, except for phosphorylated Y132 on M1 which has been confirmed to mediate the nuclear import of M1 via modulation of M1-importin interaction. The multi-functional NS1, which is encoded by the eighth RNA segment, is one of the IAV virulence factors. The multi-functional NS1, which is encoded by the eighth RNA segment, is one of the IAV virulence factors. Through binding to double-strand RNA and retinoic acid-induced gene 1 protein (RIG-I), NS1 antagonizes host innate/adaptive immune responses and inhibits interferon (IFN) production, and NS1 also binds to the NP of the vRNP complex to facilitate efficient virus replication. Several reports demonstrated that cellular host kinase such as PKC α , CDK, and ERK involved into the NS1 phosphorylation, causing phosphorylation occurred on multiple sites on NS1. These sites were proved to control IAV replication through mediating the association of NS1 with dsDNA, TRIM25, or RIG-I, being known as major sensors in RIG-like receptor signal pathway to production antiviral IFN. In addition, there exist phosphorylation occurring in other viral proteins, included PA, PB1, PB1-F2, NEP, and M2. In the present report, we majorly retrospectively reviewed the phosphorylation sites and their functions on these three proteins, and briefly summarized the phosphorylation researches on other viral proteins. We believed that this report provided a novel aspect of virus-host interaction study, and a theoretical basis for searching anti-flu target works in drug research and development area.

Influenza virus, an RNA virus, synthesizes its messenger RNA in the nucleus of infected cells

Influenza virus uses a unique mechanism to initiate viral transcription named cap-snatching. The PB2 subunit of the viral heterotrimeric RNA polymerase binds the cap structure of cellular pre-mRNA to promote its cleavage by the PA subunit. The resulting 11–13 capped oligomer is used by the PB1 polymerase subunit to initiate transcription of viral proteins. VX-787 is an inhibitor of the influenza A virus pre-mRNA cap-binding protein PB2. This clinical stage compound was shown to bind the minimal cap-binding domain of PB2 to inhibit the cap-snatching machinery. However, the binding of this molecule in the context of an extended form of the PB2 subunit has remained elusive. Here we generated a collection of PB2 truncations to identify a PB2 protein representative of its structure in the viral heterotrimeric protein. We present the crystal structure of VX-787 bound to a PB2 construct that recapitulates VX-787's biological antiviral activity in vitro. This co-structure reveals more extensive interactions than previously identified and provides insight into the observed resistance profile, affinity, binding kinetics, and conformational rearrangements induced by VX-787.