Matthew S. Walters

Zanamivir Susceptibility Monitoring and Characterization of Influenza Virus Clinical Isolates Obtained during Phase II Clinical Efficacy Studies

ABSTRACT Zanamivir is a highly selective neuraminidase (NA) inhibitor with demonstrated clinical efficacy against influenza A and B virus infections. In phase II clinical efficacy trials (NAIB2005 and NAIB2008), virological substudies showed mean reductions in virus shedding after 24 h of treatment of 1.5 to 2.0 log1050% tissue culture infective doses compared to a placebo, with no reemergence of virus after the completion of therapy. Paired isolates (n = 41) obtained before and during therapy with zanamivir demonstrated no shifts in susceptibility to zanamivir when measured by NA assays, although for a few isolates NA activity was too low to evaluate. In plaque reduction assays in MDCK cells, the susceptibility of isolates to zanamivir was extremely variable even at baseline and did not correlate with the speed of resolution of virus shedding. Isolates with apparent limited susceptibility to zanamivir by plaque reduction proved highly susceptible in vivo in the ferret model. Further sequence analysis of paired isolates revealed no changes in the hemagglutinin and NA genes in the majority of isolates. The few changes observed were all natural variants. No amino acid changes that had previously been identified in vitro as being involved with reduced susceptibility to zanamivir were observed. These studies highlighted problems associated with monitoring susceptibility to NA inhibitors in the clinic, in that no reliable cell-based assay is available. At present the NA assay is the best available predictor of susceptibility to NA inhibitors in vivo, as measured in the validated ferret model of infection.

Cilia Dysfunction in Lung Disease

A characteristic feature of the human airway epithelium is the presence of ciliated cells bearing motile cilia, specialized cell surface projections containing axonemes composed of microtubules and dynein arms, which provide ATP-driven motility. In the airways, cilia function in concert with airway mucus to mediate the critical function of mucociliary clearance, cleansing the airways of inhaled particles and pathogens. The prototypical disorder of respiratory cilia is primary ciliary dyskinesia, an inherited disorder that leads to impaired mucociliary clearance, to repeated chest infections, and to the progressive destruction of lung architecture. Numerous acquired lung diseases are also marked by abnormalities in both cilia structure and function. In this review we summarize current knowledge regarding airway ciliated cells and cilia, how they function to maintain a healthy epithelium, and how disorders of cilia structure and function contribute to inherited and acquired lung disease.

Cigarette smoking induces small airway epithelial epigenetic changes with corresponding modulation of gene expression

The small airway epithelium (SAE), the first site of smoking-induced lung pathology, exhibits genome-wide changes in gene expression in response to cigarette smoking. Based on the increasing evidence that the epigenome can respond to external stimuli in a rapid manner, we assessed the SAE of smokers for genome-wide DNA methylation changes compared with nonsmokers, and whether changes in SAE DNA methylation were linked to the transcriptional output of these cells. Using genome-wide methylation analysis of SAE DNA of nonsmokers and smokers, the data identified 204 unique genes differentially methylated in SAE DNA of smokers compared with nonsmokers, with 67% of the regions with differential methylation occurring within 2 kb of the transcriptional start site. Among the genes with differential methylation were those related to metabolism, transcription, signal transduction and transport. For the differentially methylated genes, 35 exhibited a correlation with gene expression, 54% with an inverse correlation of DNA methylation with gene expression and 46% a direct correlation. These observations provide evidence that cigarette smoking alters the DNA methylation patterning of the SAE and that, for some genes, these changes are associated with the smoking-related changes in gene expression.

Herpesvirus Saimiri Open Reading Frame 50 (Rta) Protein Reactivates the Lytic Replication Cycle in a Persistently Infected A549 Cell Line

ABSTRACT Herpesviruses occur in two distinct forms of infection, lytic replication and latent persistence. In this study, we investigated the molecular mechanisms that govern the latent-lytic switch in the prototype gamma-2 herpesvirus, herpesvirus saimiri (HVS). We utilized a persistently HVS-infected A549 cell line, in which HVS DNA is stably maintained as nonintegrated circular episomes, to assess the role of the open reading frame 50 (ORF 50) (Rta) proteins in the latent-lytic switch. Northern blot analysis and virus recovery assays determined that the ORF 50a gene product, when expressed under the control of a constitutively active promoter, was sufficient to reactivate the entire lytic replication cycle, producing infectious virus particles. Furthermore, although the ORF 50 proteins of HVS strains A11 and C488 are structurally divergent, they were both capable of inducing the lytic replication cycle in this model of HVS latency.

Hyperphosphorylation of Histone Deacetylase 2 by Alphaherpesvirus US3 Kinases

ABSTRACT A serine/threonine (S/T) kinase encoded by the US3 gene of herpes simplex virus type 1 (HSV-1) is conserved in varicella-zoster virus (VZV) and pseudorabies virus (PRV). Expression of US3 kinase in cells transformed with US3 expression plasmids or infected with each virus results in hyperphosphorylation of histone deacetylase 2 (HDAC2). Mapping studies revealed that each US3 kinase phosphorylates HDAC2 at the same unique conserved Ser residue in its C terminus. HDAC2 was also hyperphosphorylated in cells infected with PRV lacking US3 kinase, indicating that hyperphosphorylation of HDAC2 by PRV occurs in a US3-independent manner. Specific chemical inhibition of class I HDAC activity increases the plaquing efficiency of VZV and PRV lacking US3 or its enzymatic activity, whereas only minimal effects are observed with wild-type viruses, suggesting that VZV and PRV US3 kinase activities target HDACs to reduce viral genome silencing and allow efficient viral replication. However, no effect was observed for wild-type or US3 null HSV-1. Thus, we have demonstrated that while HDAC2 is a conserved target of alphaherpesvirus US3 kinases, the functional significance of these events is virus specific.

RFX3 Modulation of FOXJ1 regulation of cilia genes in the human airway epithelium

BackgroundCiliated cells play a central role in cleansing the airways of inhaled contaminants. They are derived from basal cells that include the airway stem/progenitor cells. In animal models, the transcription factor FOXJ1 has been shown to induce differentiation to the ciliated cell lineage, and the RFX transcription factor-family has been shown to be necessary for, but not sufficient to induce, correct cilia development.MethodsTo test the hypothesis that FOXJ1 and RFX3 cooperatively induce expression of ciliated genes in the differentiation process of basal progenitor cells toward a ciliated cell linage in the human airway epithelium, primary human airway basal cells were assessed under conditions of in vitro differentiation induced by plasmid-mediated gene transfer of FOXJ1 and/or RFX3. TaqMan PCR was used to quantify mRNA levels of basal, secretory, and cilia-associated genes.ResultsBasal cells, when cultured in air-liquid interface, differentiated into a ciliated epithelium, expressing FOXJ1 and RFX3. Transfection of FOXJ1 into resting basal cells activated promoters and induced expression of ciliated cell genes as well as both FOXJ1 and RFX3, but not basal cell genes. Transfection of RFX3 induced expression of RFX3 but not FOXJ1, nor the expression of cilia-related genes. The combination of FOXJ1 + RFX3 enhanced ciliated gene promoter activity and mRNA expression beyond that due to FOXJ1 alone. Corroborating immunoprecipitation studies demonstrated an interaction between FOXJ1 and RFX3.ConclusionFOXJ1 is an important regulator of cilia gene expression during ciliated cell differentiation, with RFX3 as a transcriptional co-activator to FOXJ1, helping to induce the expression of cilia genes in the process of ciliated cell differentiation of basal/progenitor cells.

Smoking Dysregulates the Human Airway Basal Cell Transcriptome at COPD Risk Locus 19q13.2

Genome-wide association studies (GWAS) and candidate gene studies have identified a number of risk loci associated with the smoking-related disease COPD, a disorder that originates in the airway epithelium. Since airway basal cell (BC) stem/progenitor cells exhibit the earliest abnormalities associated with smoking (hyperplasia, squamous metaplasia), we hypothesized that smoker BC have a dysregulated transcriptome, enriched, in part, at known GWAS/candidate gene loci. Massive parallel RNA sequencing was used to compare the transcriptome of BC purified from the airway epithelium of healthy nonsmokers (n = 10) and healthy smokers (n = 7). The chromosomal location of the differentially expressed genes was compared to loci identified by GWAS to confer risk for COPD. Smoker BC have 676 genes differentially expressed compared to nonsmoker BC, dominated by smoking up-regulation. Strikingly, 166 (25%) of these genes are located on chromosome 19, with 13 localized to 19q13.2 (p<10−4 compared to chance), including 4 genes (NFKBIB, LTBP4, EGLN2 and TGFB1) associated with risk for COPD. These observations provide the first direct connection between known genetic risks for smoking-related lung disease and airway BC, the population of lung cells that undergo the earliest changes associated with smoking.

Histone Deacetylases 1 and 2 Are Phosphorylated at Novel Sites during Varicella-Zoster Virus Infection

ABSTRACT ORF66p, a virion-associated varicella-zoster virus (VZV) protein, is a member of a conserved Alphaherpesvirinae kinase family with homology to herpes simplex virus US3 kinase. Expression of ORF66p in cells infected with VZV or an adenovirus expressing only ORF66p results in hyperphosphorylation of histone deacetylase 1 (HDAC1) and HDAC2. Mapping studies reveal that phosphorylation is at a unique conserved Ser residue in the C terminus of both HDACs. This modification requires an active kinase domain in ORF66p, as neither protein is phosphorylated in cells infected with VZV lacking kinase activity. However, hyperphosphorylation appears to occur indirectly, as within the context of in vitro kinase reactions, purified ORF66p phosphorylates a peptide derived from ORF62p, a known substrate, but does not phosphorylate HDAC. These results support a model where ORF66p is necessary but not sufficient to effect hyperphosphorylation of HDAC1 and HDAC2.

Smoking accelerates aging of the small airway epithelium

BackgroundAging involves multiple biologically complex processes characterized by a decline in cellular homeostasis over time leading to a loss and impairment of physiological integrity and function. Specific cellular hallmarks of aging include abnormal gene expression patterns, shortened telomeres and associated biological dysfunction. Like all organs, the lung demonstrates both physiological and structural changes with age that result in a progressive decrease in lung function in healthy individuals. Cigarette smoking accelerates lung function decline over time, suggesting smoking accelerates aging of the lung. Based on this data, we hypothesized that cigarette smoking accelerates the aging of the small airway epithelium, the cells that take the initial brunt of inhaled toxins from the cigarette smoke and one of the primary sites of pathology associated with cigarette smoking.MethodsUsing the sensitive molecular parameters of aging-related gene expression and telomere length, the aging process of the small airway epithelium was assessed in age matched healthy nonsmokers and healthy smokers with no physical manifestation of lung disease or abnormalities in lung function.ResultsAnalysis of a 73 gene aging signature demonstrated that smoking significantly dysregulates 18 aging-related genes in the small airway epithelium. In an independent cohort of male subjects, smoking significantly reduced telomere length in the small airway epithelium of smokers by 14% compared to nonsmokers.ConclusionThese data provide biologic evidence that smoking accelerates aging of the small airway epithelium.

The RING Finger Domain of Varicella-Zoster Virus ORF61p Has E3 Ubiquitin Ligase Activity That Is Essential for Efficient Autoubiquitination and Dispersion of Sp100-Containing Nuclear Bodies

ABSTRACT Varicella zoster virus encodes an immediate-early (IE) protein termed ORF61p that is orthologous to the herpes simplex virus IE protein ICP0. Although these proteins share several functional properties, ORF61p does not fully substitute for ICP0. The greatest region of similarity between these proteins is a RING finger domain. We demonstrate that disruption of the ORF61p RING finger domain by amino acid substitution (Cys19Gly) alters ORF61p intranuclear distribution and abolishes ORF61p-mediated dispersion of Sp100-containing nuclear bodies. In addition, we demonstrate that an intact ORF61p RING finger domain is necessary for E3 ubiquitin ligase activity and is required for autoubiquitination and regulation of protein stability.