Peter Pak-Hang Cheung

Hemagglutinin–neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets

A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin–neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility.

Histone code pathway involving H3 S28 phosphorylation and K27 acetylation activates transcription and antagonizes polycomb silencing

Histone H3 phosphorylation is a critical step that couples signal transduction pathways to gene regulation. To specifically assess the transcriptional regulatory functions of H3 phosphorylation, we developed an in vivo targeting approach and found that the H3 kinase MSK1 is a direct and potent transcriptional activator. Targeting of this H3 kinase to the endogenous c-fos promoter is sufficient to activate its expression without the need of upstream signaling. Moreover, targeting MSK1 to the α-globin promoter induces H3 S28 phosphorylation and reactivates expression of this polycomb-silenced gene. Importantly, we discovered a mechanism whereby H3 S28 phosphorylation not only displaces binding of the polycomb-repressive complexes, but it also induces a methyl-acetylation switch of the adjacent K27 residue. Our findings show that signal transduction activation can directly regulate polycomb silencing through a specific histone code-mediated mechanism.

Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population

ABSTRACT We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was completely inhibited at 250 µM and 31.25 µM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2′-(4-methylumbelliferryl)-α-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay. IMPORTANCE The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants. We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir. Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection. The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants. We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir. Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection.

Generation and characterization of influenza A viruses with altered polymerase fidelity

Genetic diversity of influenza A viruses (IAV) acquired through the error-prone RNA-dependent RNA polymerase (RdRP) or genetic reassortment enables perpetuation of IAV in humans through epidemics or pandemics. Here, to assess the biological significance of genetic diversity acquired through RdRP, we characterize an IAV fidelity variant derived from passaging a seasonal H3N2 virus in the presence of ribavirin, a purine analog that increases guanosine-to-adenosine mutations. We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses. The H5N1 PB1-V43I recombinant virus replicates to comparable titres as the wild-type virus in vitro or in the mouse lungs. However, a decrease in viral population diversity at day 3 post-inoculation is associated with a 10-fold reduced lethality and neurotropism in mice. Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

Herpes Simplex Virus ICP27 Induces Cytoplasmic Accumulation of Unspliced Polyadenylated α-Globin Pre-mRNA in Infected HeLa Cells

ABSTRACT Transcripts of most intron-bearing cellular genes must be processed by the splicing machinery in order to efficiently accumulate and gain access to the cytoplasm. However, we found that herpes simplex virus induces cytoplasmic accumulation of both spliced and unspliced polyadenylated α-globin RNAs in infected HeLa cells. Accumulation of the unspliced RNA required the immediate-early protein ICP27, and ICP27 was sufficient (in combination with ICP4) to produce this effect in a transient-transfection assay. However, expression of ICP27 did not markedly alter the levels of fully spliced α-globin transcripts in infected cells. These data demonstrate that the previously documented effects of ICP27 on the cellular splicing apparatus do not greatly inhibit splicing of α-globin RNA and argue that ICP27 induces a splicing-independent pathway for α-globin RNA accumulation and nuclear export.

Comparable Fitness and Transmissibility between Oseltamivir-Resistant Pandemic 2009 and Seasonal H1N1 Influenza Viruses with the H275Y Neuraminidase Mutation

ABSTRACT Limited antiviral compounds are available for the control of influenza, and the emergence of resistant variants would further narrow the options for defense. The H275Y neuraminidase (NA) mutation, which confers resistance to oseltamivir carboxylate, has been identified among the seasonal H1N1 and 2009 pandemic influenza viruses; however, those H275Y resistant variants demonstrated distinct epidemiological outcomes in humans. Specifically, dominance of the H275Y variant over the oseltamivir-sensitive viruses was only reported for a seasonal H1N1 variant during 2008-2009. Here, we systematically analyze the effect of the H275Y NA mutation on viral fitness and transmissibility of A(H1N1)pdm09 and seasonal H1N1 influenza viruses. The NA genes from A(H1N1)pdm09 A/California/04/09 (CA04), seasonal H1N1 A/New Caledonia/20/1999 (NewCal), and A/Brisbane/59/2007 (Brisbane) were individually introduced into the genetic background of CA04. The H275Y mutation led to reduced NA enzyme activity, an increased Km for 3′-sialylactose or 6′-sialylactose, and decreased infectivity in mucin-secreting human airway epithelial cells compared to the oseltamivir-sensitive wild-type counterparts. Attenuated pathogenicity in both RG-CA04NA-H275Y and RG-CA04 × BrisbaneNA-H275Y viruses was observed in ferrets compared to RG-CA04 virus, although the transmissibility was minimally affected. In parallel experiments using recombinant Brisbane viruses differing by hemagglutinin and NA, comparable direct contact and respiratory droplet transmissibilities were observed among RG-NewCalHA,NA, RG-NewCalHA,NA-H275Y, RG-BrisbaneHA,NA-H275Y, and RG-NewCalHA × BrisbaneNA-H275Y viruses. Our results demonstrate that, despite the H275Y mutation leading to a minor reduction in viral fitness, the transmission potentials of three different antigenic strains carrying this mutation were comparable in the naïve ferret model.

Generation and characterization of antibodies directed against di-modified histones, and comments on antibody and epitope recognition.

Publisher Summary This chapter describes the approach and methods used in generating and purifying the polyclonal Phos Ser10/Ac Lys14 di-modified H3 antibody (hereafter Phos/Ac H3 Ab) from rabbit serum. While this particular affinity purification scheme may not be necessary for all antisera, the overall protocol can serve as a general guide for the generation and purification of antibodies against modified histones. The recent advances in the understanding of histone modifications are in part facilitated by the development and widespread uses of site- and modification-specific histone antibodies. Early efforts were focused on generating acetyl histone–specific antibodies to study the links between histone acetylation and a variety of nuclear processes. For example, by immunoblotting analyses, histones acetylated at specific sites are linked to histone deposition, transcription activation, and cell cycle progression. In addition, antibodies to di-modified H3 molecules are also developed to examine the significance of specific combinations of histone modifications. To date, two separate di-modified H3 antibodies have been generated, including one that specifically recognizes H3 phosphorylated at serine 10 (Ser10) and acetylated at lysine 14 (Lys14), the other that specifically recognizes H3 acetylated at Lys9 and phosphorylated at Ser10. In the case of this dimodified H3 antibody, previous studies have shown that H3 phosphorylation in mammalian cells is induced upon epidermal growth factor (EGF) stimulation and correlates with transcription activation of the immediate early genes. In addition to testing the purified antibody by enzyme-linked immunosorbent assay (ELISA), the specificity of histone modification antibodies should also be tested by western blot analyses coupled with peptide competition assays.

Highly pathogenic avian influenza A H5N1 and pandemic H1N1 virus infections have different phenotypes in Toll-like receptor 3 knockout mice.

Toll-like receptors (TLRs) play an important role in innate immunity to virus infections. We investigated the role of TLR3 in the pathogenesis of H5N1 and pandemic H1N1 (pH1N1) influenza virus infections in mice. Wild-type mice and those defective in TLR3 were infected with influenza A/HK/486/97 (H5N1) or A/HK/415742/09 (pH1N1) virus. For comparison, mice defective in the gene for myeloid differential factor 88 (MyD88) were also infected with the viruses, because MyD88 signals through a TLR pathway different from TLR3. Survival and body weight loss were monitored for 14 days, and lung pathology, the lung immune-cell profile, viral load and cytokine responses were studied. H5N1-infected TLR3(-/-) mice had better survival than H5N1-infected WT mice, evident by significantly faster regain of body weight, lower viral titre in the lung and fewer pathological changes in the lung. However, this improved survival was not seen upon pH1N1 infection of TLR3(-/-) mice. In contrast, MyD88(-/-) mice had an increased viral titre and decreased leukocyte infiltration in the lungs after infection with H5N1 virus and poorer survival after pH1N1 infection. In conclusion, TLR3 worsens the pathogenesis of H5N1 infection but not of pH1N1 infection, highlighting the differences in the pathogenesis of these two viruses and the different roles of TLR3 in their pathogenesis.

Elucidation of the Dynamics of Transcription Elongation by RNA Polymerase II using Kinetic Network Models.

RNA polymerase II (Pol II) is an essential enzyme that catalyzes transcription with high efficiency and fidelity in eukaryotic cells. During transcription elongation, Pol II catalyzes the nucleotide addition cycle (NAC) to synthesize mRNA using DNA as the template. The transitions between the states of the NAC require conformational changes of both the protein and nucleotides. Although X-ray structures are available for most of these states, the dynamics of the transitions between states are largely unknown. Molecular dynamics (MD) simulations can predict structure-based molecular details and shed light on the mechanisms of these dynamic transitions. However, the employment of MD simulations on a macromolecule (tens to hundreds of nanoseconds) such as Pol II is challenging due to the difficulty of reaching biologically relevant timescales (tens of microseconds or even longer). For this challenge to be overcome, kinetic network models (KNMs), such as Markov State Models (MSMs), have become a popular approach to access long-timescale conformational changes using many short MD simulations. We describe here our application of KNMs to characterize the molecular mechanisms of the NAC of Pol II. First, we introduce the general background of MSMs and further explain procedures for the construction and validation of MSMs by providing some technical details. Next, we review our previous studies in which we applied MSMs to investigate the individual steps of the NAC, including translocation and pyrophosphate ion release. In particular, we describe in detail how we prepared the initial conformations of Pol II elongation complex, performed MD simulations, extracted MD conformations to construct MSMs, and further validated them. We also summarize our major findings on molecular mechanisms of Pol II elongation based on these MSMs. In addition, we have included discussions regarding various key points and challenges for applications of MSMs to systems as large as the Pol II elongation complex. Finally, to study the overall NAC, we combine the individual steps of the NAC into a five-state KNM based on a nonbranched Brownian ratchet scheme to explain the single-molecule optical tweezers experimental data. The studies complement experimental observations and provide molecular mechanisms for the transcription elongation cycle. In the long term, incorporation of sequence-dependent kinetic parameters into KNMs has great potential for identifying error-prone sequences and predicting transcription dynamics in genome-wide transcriptomes.

Real-time Monitoring of Hydrophobic Aggregation Reveals a Critical Role of Cooperativity in Hydrophobic Effect

The hydrophobic interaction drives nonpolar solutes to aggregate in aqueous solution, and hence plays a critical role in many fundamental processes in nature. An important property intrinsic to hydrophobic interaction is its cooperative nature, which is originated from the collective motions of water hydrogen bond networks surrounding hydrophobic solutes. This property is widely believed to enhance the formation of hydrophobic core in proteins. However, cooperativity in hydrophobic interactions has not been successfully characterized by experiments. Here, we quantify cooperativity in hydrophobic interactions by real-time monitoring the aggregation of hydrophobic solute (hexaphenylsilole, HPS) in a microfluidic mixer. We show that association of a HPS molecule to its aggregate in water occurs at sub-microsecond, and the free energy change is −5.8 to −13.6 kcal mol−1. Most strikingly, we discover that cooperativity constitutes up to 40% of this free energy. Our results provide quantitative evidence for the critical role of cooperativity in hydrophobic interactions.