HIV-1 Proteins gp120 and Tat Promote Epithelial-Mesenchymal Transition and Invasiveness of HPV-Positive and HPV-Negative Neoplastic Genital and Oral Epithelial Cells.

The incidence of human papillomavirus (HPV)-associated anogenital and oropharyngeal cancer in human immunodeficiency virus (HIV)-infected individuals is substantially higher than in HIV-uninfected individuals. HIV may also be a risk factor for the development of HPV-negative head and neck, liver, lung, and kidney cancer. However, the molecular mechanisms underlying HIV-1-associated increase of epithelial malignancies are not fully understood. Here, we showed that HPV-16-immortalized anal AKC-2 and cervical CaSki epithelial cells that undergo prolonged exposure to cell-free HIV-1 virions or HIV-1 viral proteins gp120 and tat respond with the epithelial-mesenchymal transition (EMT) and increased invasiveness. Similar responses were observed in HPV-16-infected SCC-47 and HPV-16-negative HSC-3 oral epithelial cancer cells that were cultured with these viral proteins. EMT induced by gp120 and tat led to detachment of poorly adherent cells from the culture substratum; these cells remained capable of reattachment, upon which they coexpressed both E-cadherin and vimentin, indicative of an intermediate stage of EMT. The reattached cells also expressed stem cell markers CD133 and CD44, which may play a critical role in cancer cell invasion and metastasis. Inhibition of transforming growth factor (TGF)-ß1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasive activity of HPV-16-immortalized anal and cervical epithelial cells. Collectively, our results suggest that these approaches along with HIV viral suppression with antiretroviral therapy (ART) might be useful to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia. IMPORTANCE HPV-16-immortalized genital and oral epithelial cells and HPV-negative oral cancer cells that undergo prolonged contact with cell-free HIV-1 virions or with viral proteins gp120 and tat respond by becoming more invasive. EMT cells induced by HIV-1 in cultures of HPV-16-immortalized anal and cervical epithelial cells express the stem cell markers CD133 and CD44. These results suggest that the interaction of HIV-1 with neoplastic epithelial cells may lead to their de-differentiation into cancer stem cells that are resistant to apoptosis and anti-cancer drugs. Thus, this pathway may play a critical role in the development of invasive cancer. Inhibition of TGF-ß1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasiveness of HPV-16-immortalized anal and cervical epithelial cells. Taken together, these results suggest that these approaches might be exploited to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia.

Human Immunodeficiency Virus (HIV) and Human Cytomegalovirus (HCMV) Coinfection of Infant Tonsil Epithelium May Synergistically Promote both HIV-1 and HCMV Spread and Infection.

Mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) and human cytomegalovirus (HCMV) may occur during pregnancy, labor, or breastfeeding. These viruses from amniotic fluid, cervicovaginal secretions, and breast milk may simultaneously interact with oropharyngeal and tonsil epithelia; however, the molecular mechanism of HIV-1 and HCMV cotransmission through the oral mucosa and its role in MTCT are poorly understood. To study the molecular mechanism of HIV-1 and HCMV MTCT via oral epithelium, we established polarized infant tonsil epithelial cells and polarized-oriented ex vivo tonsil tissue explants. Using these models, we showed that cell-free HIV-1 and its proteins gp120 and tat induce the disruption of tonsil epithelial tight junctions and increase paracellular permeability, which facilitates HCMV spread within the tonsil mucosa. Inhibition of HIV-1 gp120-induced upregulation of mitogen-activated protein kinase (MAPK) and NF-κB signaling in tonsil epithelial cells, reduces HCMV infection, indicating that HIV-1-activated MAPK and NF-κB signaling may play a critical role in HCMV infection of tonsil epithelium. HCMV infection of tonsil epithelial cells also leads to the disruption of tight junctions and increases paracellular permeability, facilitating HIV-1 paracellular spread into tonsil mucosa. HCMV-promoted paracellular spread of HIV-1 increases its accessibility to tonsil CD4 T lymphocytes, macrophages, and dendritic cells. HIV-1-enhanced HCMV paracellular spread and infection of epithelial cells subsequently leads to the spread of HCMV to tonsil macrophages and dendritic cells. Our findings revealed that HIV-1- and HCMV-induced disruption of infant tonsil epithelial tight junctions promotes MTCT of these viruses through tonsil mucosal epithelium, and therapeutic intervention for both HIV-1 and HCMV infection may substantially reduce their MTCT. IMPORTANCE Most HIV-1 and HCMV MTCT occurs in infancy, and the cotransmission of these viruses may occur via infant oropharyngeal and tonsil epithelia, which are the first biological barriers for viral pathogens. We have shown that HIV-1 and HCMV disrupt epithelial junctions, reducing the barrier functions of epithelia and thus allowing paracellular penetration of both viruses via mucosal epithelia. Subsequently, HCMV infects epithelial cells, macrophages, and dendritic cells, and HIV-1 infects CD4+ lymphocytes, macrophages, and dendritic cells. Infection of these cells in HCMV- and HIV-1-coinfected tonsil tissues is much higher than that by HCMV or HIV-1 infection alone, promoting their MTCT at its initial stages via infant oropharyngeal and tonsil epithelia.

HIV-1 proteins gp120 and tat induce the epithelial-mesenchymal transition in oral and genital mucosal epithelial cells.

The oral, cervical, and genital mucosa, covered by stratified squamous epithelia with polarized organization and strong tight and adherens junctions, play a critical role in preventing transmission of viral pathogens, including human immunodeficiency virus (HIV). HIV-1 interaction with mucosal epithelial cells may depolarize epithelia and disrupt their tight and adherens junctions; however, the molecular mechanism of HIV-induced epithelial disruption has not been completely understood. We showed that prolonged interaction of cell-free HIV-1 virions, and viral envelope and transactivator proteins gp120 and tat, respectively, with tonsil, cervical, and foreskin epithelial cells induces an epithelial-mesenchymal transition (EMT). EMT is an epigenetic process leading to the disruption of mucosal epithelia and allowing the paracellular spread of viral and other pathogens. Interaction of cell-free virions and gp120 and tat proteins with epithelial cells substantially reduced E-cadherin expression and activated vimentin and N-cadherin expression, which are well-known mesenchymal markers. HIV gp120- and tat-induced EMT was mediated by SMAD2 phosphorylation and activation of transcription factors Slug, Snail, Twist1 and ZEB1. Activation of TGF-ß and MAPK signaling by gp120, tat, and cell-free HIV virions revealed the critical roles of these signaling pathways in EMT induction. gp120- and tat-induced EMT cells were highly migratory via collagen-coated membranes, which is one of the main features of mesenchymal cells. Inhibitors of TGF-ß1 and MAPK signaling reduced HIV-induced EMT, suggesting that inactivation of these signaling pathways may restore the normal barrier function of mucosal epithelia.

Hedgehog signaling drives medulloblastoma growth via CDK6.

Medulloblastoma, an aggressive cancer of the cerebellum, is among the most common pediatric brain tumors. Approximately one-third of medulloblastomas are associated with misactivation of the Hedgehog (Hh) pathway. GLI family zinc finger 2 (GLI2) coordinates the Hh transcriptional program; however, the GLI2 targets that promote cancer cell proliferation are unknown. Here, we incorporated a Gli2-EGFP allele into 2 different genetic mouse models of Hh-associated medulloblastoma. Hh signaling induced GLI2 binding to the Cdk6 promoter and activated Cdk6 expression, thereby promoting uncontrolled cell proliferation. Genetic or pharmacological inhibition of CDK6 in mice repressed the growth of Hh-associated medulloblastoma and prolonged survival through inhibition of cell proliferation. In human medulloblastoma, misactivation of Hh signaling was associated with high levels of CDK6, pointing to CDK6 as a direct transcriptional target of the Hh pathway. These results suggest that CDK6 antagonists may be a promising therapeutic approach for Hh-associated medulloblastoma in humans.

Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I.

Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNß induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity.

Novel mutations in PXDN cause microphthalmia and anterior segment dysgenesis.

We used exome sequencing to study a non-consanguineous family with two children who had anterior segment dysgenesis, sclerocornea, microphthalmia, hypotonia and developmental delays. Sanger sequencing verified two Peroxidasin (PXDN) mutations in both sibs--a maternally inherited, nonsense mutation, c.1021C>T predicting p.(Arg341*), and a paternally inherited, 23-basepair deletion causing a frameshift and premature protein truncation, c.2375_2397del23, predicting p.(Leu792Hisfs*67). We re-examined exome data from 20 other patients with structural eye defects and identified two additional PXDN mutations in a sporadic male with bilateral microphthalmia, cataracts and anterior segment dysgenesis--a maternally inherited, frameshift mutation, c.1192delT, predicting p.(Tyr398Thrfs*40) and a paternally inherited, missense substitution that was predicted to be deleterious, c.947 A>C, predicting p.(Gln316Pro). Mutations in PXDN were previously reported in three families with congenital cataracts, microcornea, sclerocornea and developmental glaucoma. The gene is expressed in corneal epithelium and is secreted into the extracellular matrix. Defective peroxidasin has been shown to impair sulfilimine bond formation in collagen IV, a constituent of the basement membrane, implying that the eye defects result because of loss of basement membrane integrity in the developing eye. Our finding of a broader phenotype than previously appreciated for PXDN mutations is typical for exome-sequencing studies, which have proven to be highly effective for mutation detection in patients with atypical presentations. We conclude that PXDN sequencing should be considered in microphthalmia with anterior segment dysgenesis.

Evaluation of nanolipoprotein particles (NLPs) as an in vivo delivery platform.

Nanoparticles hold great promise for the delivery of therapeutics, yet limitations remain with regards to the use of these nanosystems for efficient long-lasting targeted delivery of therapeutics, including imparting functionality to the platform, in vivo stability, drug entrapment efficiency and toxicity. To begin to address these limitations, we evaluated the functionality, stability, cytotoxicity, toxicity, immunogenicity and in vivo biodistribution of nanolipoprotein particles (NLPs), which are mimetics of naturally occurring high-density lipoproteins (HDLs). We found that a wide range of molecules could be reliably conjugated to the NLP, including proteins, single-stranded DNA, and small molecules. The NLP was also found to be relatively stable in complex biological fluids and displayed no cytotoxicity in vitro at doses as high as 320 µg/ml. In addition, we observed that in vivo administration of the NLP daily for 14 consecutive days did not induce significant weight loss or result in lesions on excised organs. Furthermore, the NLPs did not display overt immunogenicity with respect to antibody generation. Finally, the biodistribution of the NLP in vivo was found to be highly dependent on the route of administration, where intranasal administration resulted in prolonged retention in the lung tissue. Although only a select number of NLP compositions were evaluated, the findings of this study suggest that the NLP platform holds promise for use as both a targeted and non-targeted in vivo delivery vehicle for a range of therapeutics.

Clonal growth and fine-scale genetic structure in tanoak (Notholithocarpus densiflorus: Fagaceae).

The combination of sprouting and reproduction by seed can have important consequences on fine-scale spatial distribution of genetic structure (SGS). SGS is an important consideration for species' restoration because it determines the minimum distance among seed trees to maximize genetic diversity while not prejudicing locally adapted genotypes. Local environmental conditions can be expected to influence levels of clonal spread and SGS, particularly in the case of disturbance regimes such as fire. Here, we characterize fine-scale genetic structure and clonal spread in tanoak from drier upland sites and more mesic lowland woodlands. Clonal spread was a significant mode of stand development, but spread was limited on average to about 5-6 m. Gene dispersal was decomposed into clonal and sexual components. The latter varied according to whether it was estimated from all ramets with the clonal component removed or for a single ramet per genet. We used the difference in these 2 estimates of gene dispersal as a measure of the effect of clonality on effective population size in this species. Although upland sites had a greater number of ramets per genet, most of the other indices computed were not significantly different. However, they tended to show greater heterozygote excess and shorter gene dispersal distances than the lowland sites. The average distance among inferred sibships on upland sites was approximately at the scale of maximum clonal range. This was not the case on lowland sites, where sibs were more dispersed. We recommend minimum distances among seed trees to avoid selecting clones and to maximize genetic diversity for restoration.

Evidence for the role of synchronicity between host phenology and pathogen activity in the distribution of sudden oak death canker disease.

Variations in synchronicity between colonization rate by the pathogen and host phenology may account for unexplained spatial distribution of canker disease. The hypothesis that synchronous pathogenicity and host development are necessary for incidence of sudden oak death disease was tested by correlating seasonal variations in host cambial phenology and response to inoculation with Phytophthora ramorum. Response to infection was estimated by inoculating branch cuttings from coast live oak (Quercus agrifolia) trees at nine dates through a full annual cycle in 2003-2004. Host phenology was estimated from measurements of bud burst and cambial activity in spring 2006. Lesions were largest in the spring soon after the cambium resumed activity. A moderate genetic component to lesion size was detected. Variation among trees in date of largest lesions correlated with variation in timing of bud burst and cambial phenology. The data support the hypothesis that active host cambial tissue is a necessary requisite for successful infection with the pathogen that causes sudden oak death canker disease. Genetic variation in host phenology will buffer coast live oak against epidemics of this disease.