The Extended C-Terminal Region of Influenza C Virus Nucleoprotein Is Important for Nuclear Import and Ribonucleoprotein Activity.

The influenza C virus (ICV) is a human-pathogenic agent, and the infections are frequently identified in children. Compared to influenza A and B viruses, the nucleoprotein of ICV (NPC) has an extended C-terminal region of which the functional significance is ill defined. We observed that the nuclear localization signals (NLSs) found on the nucleoproteins of influenza A and B virus subtypes are absent at corresponding positions on ICV. Instead, we found that a long bipartite nuclear localization signal resides at the extended C-terminal region, spanning from R513 to K549. Our experimental data determined that the KKMK motif within this region plays important roles in both nuclear import and polymerase activity. Similar to the influenza A viruses, NPC also binds to multiple human importin α isoforms. Taken together, our results enhance the understanding of the virus-host interaction of the influenza C virus.IMPORTANCE As a member of the Orthomyxoviridae family, the polymerase complex of the influenza C virus structurally resembles its influenza A and influenza B virus counterparts, but the nucleoprotein differs by possessing an extra C-terminal region. We have characterized this region in view of nuclear import and interaction with the importin α protein family. Our results demonstrate the functional significance of a previously uncharacterized region on Orthomyxoviridae nucleoprotein (NP). Based on this work, we propose that importin α binding to influenza C virus NP is regulated by a long bipartite nuclear localization signal. Since the sequence of influenza D virus NP shares high homology to that of the influenza C virus, this work will also shed light on how influenza D virus NP functions.

Structure and Function of Influenza Virus Ribonucleoprotein.

Influenza is a negative-sense single-stranded RNA virus with segmented genome. Each segment is encapsidated by a ribonucleoprotein (RNP) complex composed of RNA-dependent RNA polymerase (RdRP) and multiple copies of nucleoprotein (NP). The RNP complex plays a crucial role in viral life cycle, supporting and regulating transcription and replication of viral genome in infected cells. The structural characterization of RdRP and RNP in recent years has shed light on its functions and mechanism of action. In this review, we summarize current understanding on the structure of RNP complex, as well as the structure of each subunit. Crucial functions of RNP are also discussed.

Functional Importance of the Hydrophobic Residue 362 in Influenza A PB1 Subunit.

PB1, acting as the catalytic subunit of the influenza polymerase, has numerous sequentially and structurally conserved regions. It has been observed that the slight modification of residues in PB1 would greatly affect the polymerase activity and even host adaptation ability. Here, we identified a critical residue, 362M, on the polymerase activity and virus replication. By means of the minireplicon assay, we assured the importance of the hydrophobicity of PB1 362, and the possibility that the size and charge of the side chain might directly interfere with the polymerase function. We also proposed a hydrophobic core between the PA-arch and the PB1 ß-hairpin motifs and showed the importance of the core to the polymerase function.

Anti-influenza virus activities and mechanism of antrafenine analogs.

Antrafenine is a drug initially designed for anti-inflammation uses. In this work we have synthesized a library of its structural analogs and tested the anti-influenza activities. These analogs belong to a group of 2-(quinolin-4-yl)amino benzamides or 2-(quinolin-4-yl)amino benzoate derivatives. Best performers were identified, namely 12, 34, 41, with IC50 against A/WSN/33 (H1N1) of 5.53, 3.21 and 6.73 µM respectively. These chemicals were also effective against A/PR/8/34 (H1N1), A/HK/1/68 (H3N2) and B/Florida/04/2006 viruses. Time-of-addition study and minigenome luciferase reporter assay both supported that the compounds act on the ribonucleoprotein (RNP) components. Using 34 and 41 as representative compounds, we determined by microscale thermophoresis that this group of compounds bind to both PA C-terminal domain and the nucleoprotein (NP) which is the most abundant subunit of the RNP. Taken together, we have identified a new class of anti-influenza compounds with dual molecular targets and good potential to be further developed. IMPORTANCE: The influenza viruses, especially influenza A and B subtypes, cause many deaths each year. The high mutation rate of the virus renders available therapeutics less effective with time. In this work we identify a new class of compounds, structurally similar to the anti-inflammation drug antrafenine, with good potency against influenza A strains. The IC50 of the best performers are within low micromolar range and thus have good potential for further development.

Anti-influenza virus phytochemicals from Radix Paeoniae Alba and characterization of their neuraminidase inhibitory activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Bai Shao (Radix Paeoniae Alba, BS), the root of Paeonia lactiflora Pall., in ancient China was used for Wen Bing (Warm Disease) treatment. Wen Bing has the symptoms of influenza. Ethanol extract of the root has recently been shown to possess anti-influenza activity. However, the active compounds have not yet been identified. AIM: We showed that BS aqueous extract was potent in inhibiting influenza A virus in infected cells. We aimed to isolate the bioactive compounds and characterize the anti-influenza mechanism. MATERIALS AND METHODS: Plaque reduction assay was performed for fractions isolated from BS. Hemagglutination inhibition assay and neuraminidase inhibition assay were performed to find the target protein. Molecular docking and reverse genetics were used to confirm the action site of gallic acid on the neuraminidase protein. RESULTS: We identified three tannin compounds gallic acid (GA), methyl gallate (MG) and pentagalloylglucose (PGG) in BS aqueous extract that could inhibit the replication of influenza A virus in MDCK cells. While only PGG was found to inhibit the influenza virus-induced hemagglutination of chicken erythrocytes, all three compounds significantly reduced the activity of the neuraminidase. The results from molecular docking and reverse genetics showed that GA interacted with Arg152 of neuraminidase protein. CONCLUSION: Three compounds GA, MG and PGG isolated from BS were found to inhibit influenza A virus in MDCK cells. GA interacts with amino acid Arg152 of the viral neuraminidase. Our study identified anti-influenza compounds of BS and demonstrated their antiviral mechanism, thus providing scientific evidence for using this herb for clinical treatment.

Amino acid substitutions affecting aspartic acid 605 and valine 606 decrease the interaction strength between the influenza virus RNA polymerase PB2 '627' domain and the viral nucleoprotein.

The influenza virus RNA genome is transcribed and replicated in the context of the viral ribonucleoprotein (vRNP) complex by the viral RNA polymerase. The nucleoprotein (NP) is the structural component of the vRNP providing a scaffold for the viral RNA. In the vRNP as well as during transcription and replication the viral polymerase interacts with NP but it is unclear which parts of the polymerase and NP mediate these interactions. Previously the C-terminal '627' domain (amino acids 538-693) of PB2 was shown to interact with NP. Here we report that a fragment encompassing amino acids 146-185 of NP is sufficient to mediate this interaction. Using NMR chemical shift perturbation assays we show that amino acid region 601 to 607 of the PB2 '627' domain interacts with this fragment of NP. Substitutions of these PB2 amino acids resulted in diminished RNP activity and surface plasmon resonance assays showed that amino acids D605 was essential for the interaction with NP and V606 may also play a partial role in the interaction. Collectively these results reveal a possible interaction surface between NP and the PB2 subunit of the RNA polymerase complex.

Identification of influenza polymerase inhibitors targeting C-terminal domain of PA through surface plasmon resonance screening.

Currently, many strains of influenza A virus have developed resistance against anti-influenza drugs, and it is essential to find new chemicals to combat this virus. The influenza polymerase with three proteins, PA, PB1 and PB2, is a crucial component of the viral ribonucleoprotein (RNP) complex. Here, we report the identification of a hit compound 221 by surface plasmon resonance (SPR) direct binding screening on the C-terminal of PA (PAC). Compound 221 can subdue influenza RNP activities and attenuate influenza virus replication. Its analogs were subsequently investigated and twelve of them could attenuate RNP activities. One of the analogs, compound 312, impeded influenza A virus replication in Madin-Darby canine kidney cells with IC50 of 27.0 ± 16.8 µM. In vitro interaction assays showed that compound 312 bound directly to PAC with Kd of about 40 µM. Overall, the identification of novel PAC-targeting compounds provides new ground for drug design against influenza virus in the future.

Identification of influenza polymerase inhibitors targeting polymerase PB2 cap-binding domain through virtual screening.

Influenza A virus is the major cause of epidemics and pandemics worldwide. In this study, virtual screening was used to identify compounds interacting with influenza A polymerase PB2 cap-binding domain (CBD). With a database of 21,351 small molecules, 28 candidate compounds were tested and one compound (225) was identified as hit compound. Compound 225 and three of its analogs (225D1, 426 and 426Br) were found to bind directly to PB2 CBD by surface plasmon resonance (SPR). The evaluation of compounds 426Br and 225 indicated that they could bind to PB2 CBD and inhibit influenza virus at low micromolar concentration. They were predicted to bind the cap binding site of the protein by molecular modeling and were confirmed by SPR assay using PB2 CBD mutants. These two compounds have novel scaffolds and could be further developed into lead compound for influenza virus inhibition.