Human Papillomavirus Type 16 Entry: Retrograde Cell Surface Transport along Actin-Rich Protrusions

The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30–60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing

Essential Roles for Soluble Virion-Associated Heparan Sulfonated Proteoglycans and Growth Factors in Human Papillomavirus Infections

A subset of human papillomavirus (HPV) infections is causally related to the development of human epithelial tumors and cancers. Like a number of pathogens, HPV entry into target cells is initiated by first binding to heparan sulfonated proteoglycan (HSPG) cell surface attachment factors. The virus must then move to distinct secondary receptors, which are responsible for particle internalization. Despite intensive investigation, the mechanism of HPV movement to and the nature of the secondary receptors have been unclear. We report that HPV16 particles are not liberated from bound HSPG attachment factors by dissociation, but rather are released by a process previously unreported for pathogen-host cell interactions. Virus particles reside in infectious soluble high molecular weight complexes with HSPG, including syndecan-1 and bioactive compounds, like growth factors. Matrix mellatoproteinase inhibitors that block HSPG and virus release from cells interfere with virus infection. Employing a co-culture assay, we demonstrate HPV associated with soluble HSPG-growth factor complexes can infect cells lacking HSPG. Interaction of HPV-HSPG-growth factor complexes with growth factor receptors leads to rapid activation of signaling pathways important for infection, whereas a variety of growth factor receptor inhibitors impede virus-induced signaling and infection. Depletion of syndecan-1 or epidermal growth factor and removal of serum factors reduce infection, while replenishment of growth factors restores infection. Our findings support an infection model whereby HPV usurps normal host mechanisms for presenting growth factors to cells via soluble HSPG complexes as a novel method for interacting with entry receptors independent of direct virus-cell receptor interactions

Entry of Human Papillomavirus Type 16 by Actin-Dependent, Clathrin- and Lipid Raft-Independent Endocytosis

Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV) infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants) to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus) with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na+/H+ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH

Emerging concepts in biomarker discovery; The US-Japan workshop on immunological molecular markers in oncology

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations

Modelling polar auxin transport in developmental patterning

Auxin interacts with its own polar transport to influence cell polarity and tissue patterning. Research over the past decade has started to deliver new insights into the molecular mechanisms that drive and regulate polar auxin transport. The most prominent auxin efflux protein, PIN1, has subsequently become a crucial component of auxin transport models because it is now known to direct auxin flow and maintain local auxin gradients. Recent molecular and genetic experiments have allowed the formulation of conceptual models that are able to interpret the role of (i) auxin, (ii) its transport, and (iii) the dynamics of PIN1 in generating temporal and spatial patterns. Here we review the current mathematical models of patterning in two specific developmental contexts: lateral shoot and vein formation, focusing on how these models can help to untangle the details of auxin transport-mediated patterning

Adenovirus vectors based on human adenovirus type 19a have high potential for human muscle-directed gene therapy.

Until recently, adenovirus-based gene therapy has been almost exclusively based on human adenovirus serotype 5 (Ad5). The aim of this study was to systematically compare the efficiency of transduction of primary muscle cells from various species by two adenoviral vectors from subgroups C and D. Transduction of a panel of myoblasts demonstrated a striking specificity of an Ad19a-based replication-defective E1-deleted vector (Ad19aEGFP) for human cells, whereas the Ad5-based vector had high affinity for nonhuman primate myoblasts. Transgene expression correlated well with cell-associated vector genomes. Up to 6.59% of the initially applied Ad19aEGFP vector particles were taken up by human myoblasts, as compared with 0.1% of the corresponding Ad5 vector. Remarkably, Ad19aEGFP but not Ad5EGFP efficiently transduced differentiated human myotubes, an in vitro model for skeletal muscle transduction. Uptake of Ad19aEGFP vector particles in human myotubes was 12-fold more efficient than that of Ad5EGFP. Moreover, both vectors demonstrated an early block at the level of vector uptake in mouse myoblasts and rat L6 cells. Investigation of the underlying mechanism for binding and uptake of the two vectors by human myoblasts showed high susceptibility for Ad19a to neuraminidase and wheat germ agglutinin (WGA) lectin, whereas Ad5-mediated transduction was dependent on binding to the coxsackie-adenovirus receptor (CAR) and sensitive to soluble RGD peptide and heparin. Our study offers insights into species-dependent factors that determine Ad tropism and, moreover, provides a basis for application of the novel Ad19a-based vector for gene transfer into human skeletal muscle