Human papillomavirus infection requires cell surface heparan sulfate

Using pseudoinfection of cell lines, we demonstrate that cell surface heparan sulfate is required for infection by human papillomavirus type 16 (HPV-16) and HPV-33 pseudovirions. Pseudoinfection was inhibited by heparin but not dermatan or chondroitin sulfate, reduced by reducing the level of surface sulfation, and abolished by heparinase treatment. Carboxy-terminally deleted HPV-33 virus-like particles still bound efficiently to heparin. The kinetics of postattachment neutralization by antiserum or heparin indicated that pseudovirions were shifted on the cell surface from a heparin-sensitive into a heparin-resistant mode of binding, possibly involving a secondary receptor. Alpha-6 integrin is not a receptor for HPV-33 pseudoinfection.     

Human papillomavirus type 31 uses a caveolin 1- and dynamin 2-mediated entry pathway for infection of human keratinocytes

Papillomaviruses are species-specific and epitheliotropic DNA viruses that cause tumors in their natural hosts. Certain infections with genital human papillomavirus (HPV) types are causally related to cervical cancer development. Most papillomaviruses are thought to infect cells via a clathrin-dependent pathway, yet no studies have determined the entry route in permissive host epithelial cells. Employing fluorescently labeled and native virions, we tested the effects of dominant-negative and biochemical inhibitors of cellular endocytosis pathways. Infections of human keratinocytes, a natural host cell type for HPVs, were assessed visually and by infectious entry assays. We found that HPV type 31 (HPV31) entry and initiation of early infection events require both caveolin 1 and dynamin 2 and occur independently of clathrin-mediated endocytosis. Treatment with chlorpromazine and filipin had opposing effects on HPV31 and HPV16 infection. HPV31 entry was remarkably slow, with a half-time of approximately 14 h, whereas the entry half-time of HPV16 was 4 h. Consistent with a caveola-mediated entry pathway for HPV31, the virions associated with detergent-resistant lipid rafts. During a 16-h microscopic tracking of HPV31 and HPV16 virions, no colocalization of the two viral types was observed. These data suggest that HPV31 and HPV16 virions use distinct routes for host epithelial cell entry.     

Long-term effect of interferon on keratinocytes that maintain human papillomavirus type 31

The long-term effects of interferon treatment on cell lines that maintain human papillomavirus type 31 (HPV-31) episomes have been examined. High doses and prolonged interferon treatment resulted in growth arrest of HPV-positive cells, with a high percentage of cells undergoing apoptosis. These effects were not seen with interferon treatment of either normal human keratinocytes or cells derived from HPV-negative squamous carcinomas, which exhibited only slight decreases in their rates of growth. Within 2 weeks of the initiation of treatment, a population of HPV-31-positive cells that were resistant to interferon appeared consistently and reproducibly. The resistant cells had growth and morphological characteristics similar to those of untreated cells. Long-term interferon treatment of HPV-positive cells also resulted in a reduction in HPV episome levels but did not significantly decrease the number of integrated copies of HPV. Cells that maintained HPV genomes lacking E5 were sensitive to interferon, while cells expressing only the E6/E7 genes were resistant. In contrast, cells that expressed E2 from a tetracycline-inducible promoter were found to be significantly more sensitive to interferon treatment than parental cells. This suggests that at least a portion of the sensitivity to interferon could be mediated through the E2 protein. These studies indicate that cells maintaining HPV episomes are highly sensitive to interferon treatment but that resistant populations arise quickly.     

Drosophila pipe protein activity in the ovary and the embryonic salivary gland does not require heparan sulfate glycosaminoglycans

The Drosophila pipe gene encodes ten related proteins that exhibit amino acid sequence similarity to vertebrate heparan sulfate 2-O-sulfotransferase. One of the Pipe isoforms, which is expressed in the ventral follicular epithelium, is a key determinant of embryonic dorsoventral polarity, suggesting that Pipe-mediated sulfation of a heparan sulfate proteoglycan provides a spatial cue for dorsoventral axis formation. We used several approaches to investigate this possibility in the work described here. We determined the nucleotide alterations in 11 different pipe alleles. Ten of the mutations specifically affect the pipe isoform that is expressed in the ovary. Among these ten mutations, two alter an amino acid in the putative binding site for 3'-phosphoadenosine 5'-phosphosulfate, the universal sulfate donor. Using Alcian Blue, a histochemical stain that detects sulfated glycans, we observed a novel, pipe-dependent macromolecule in the embryonic salivary glands. Genes known to participate in the formation of heparan sulfate in Drosophila are not required for the production of this material. To investigate whether a heparan sulfate proteoglycan is involved in pipe function in dorsoventral patterning, we generated females carrying follicle cell clones mutant for heparan sulfate synthesis-related genes. Embryos from follicles with mutant clones did not exhibit a dorsalized phenotype. Taken together, our data provide evidence that Pipe acts as a sulfotransferase, but argue against the hypothesis that the target of Pipe is a heparan sulfate glycosaminoglycan.     

Human papillomavirus genotype 31 does not express detectable microRNA levels during latent or productive virus replication

It has recently become clear that several pathogenic DNA viruses express virally encoded microRNAs in infected cells. In particular, numerous microRNAs have been identified in a range of human and animal herpesviruses, and individual microRNAs have also been identified in members of the polyoma- and adenovirus families. Although their functions remain largely unknown, it seems likely that these viral microRNAs play an important role in viral replication in vivo. Here we present an analysis of the microRNAs expressed in human cells during the latent and productive phases of the human papillomavirus genotype 31 (HPV31) replication cycle. Although over 500 cellular microRNAs were cloned and identified, not a single HPV31-specific microRNA was obtained. We therefore concluded that HPV31, and possibly human papillomaviruses in general, does not express viral microRNAs.     

Human helper-T-cell function does not require T4 antigen expression

The relationship between immunoregulatory T-cell function and the expression of T-cell subset-specific differentiation antigens was examined using a phenotypically anomalous human T-cell line (TCL), termed H-1. H-1 cells were found to express T11, extremely high levels of T3, but no T4 nor T8 antigen. Despite their lack of T4 antigen expression, H-1 cells could be activated by coculture with pokeweed mitogen (PWM), anti-T3 antibody, or autologous B cells to provide potent help for B-cell differentiation into plaque-forming cells (PFC). In contrast, H-1 cells did not suppress the PFC response triggered by PWM-activated T4+ cells. These results demonstrate that the expression of the T-cell subclass-specific differentiation antigen, T4, is not required for a T cell to become activated and to implement the program for helper function. In addition, enhanced expression of T3 on the T4-, T8-, H-1 cell surface may reflect a compensatory upregulation of the T3/Ti receptor complex on T cells which are deficient in these nonpolymorphic associative recognition structures.     

Epidermal growth factor receptor down-regulation induced by UVA in human keratinocytes does not require the receptor kinase activity

Activation of the epidermal growth factor (EGF) receptor by EGF, its ligand, results in receptor internalization and down-regulation, which requires receptor kinase activity, phosphorylation, and ubiquitination. In contrast, we have found here in human HaCaT keratinocytes that exposure to UVA induces EGF receptor internalization and down-regulation without receptor phosphorylation and ubiquitination. The presence of the receptor kinase activity inhibitor AG1478 increased UVA-induced receptor down-regulation, whereas it inhibited EGF-induced receptor down-regulation. These observations demonstrate that, in contrast to EGF, receptor kinase activity is not required for receptor down-regulation by UVA. Concurrent with receptor down-regulation, caspases were activated by UVA exposure. The presence of caspase inhibitors blocked receptor down-regulation in a pattern similar to poly(ADP)-ribose polymerase cleavage. Much more receptor down-regulation was observed after UVA exposure in apoptotic detached cells in which caspase is activated completely. These results indicate that UVA-induced receptor down-regulation is dependent on caspase activation. Similar to UVA, both UVB and UVC induced receptor down-regulation, in which receptor kinase activity is not required, whereas caspase activation is involved. Inhibition of EGF receptor down-regulation increased receptor activation and activation of its downstream survival signaling ERK and AKT after UVA exposure. Preventing the activation of each of these pathways enhanced apoptosis induced by UVA. These findings suggest that EGF receptor down-regulation by UVA may play an important role in the execution of the cell suicide program by attenuating its anti-apoptotic function and thereby preventing cell transformation and tumorigenesis in vivo.     

The function of a Drosophila glypican does not depend entirely on heparan sulfate modification

Division abnormally delayed (Dally) is one of two glycosylphosphatidylinositol (GPI)-linked heparan sulfate proteoglycans in Drosophila. Numerous studies have shown that it influences Decapentaplegic (Dpp) and Wingless signaling. It has been generally assumed that Dally affects signaling by directly interacting with these growth factors, primarily through its heparan sulfate (HS) chains. To understand the functional contributions of HS chains and protein core we have (1) assessed the growth factor binding properties of purified Dally using surface plasmon resonance, (2) generated a form of Dally that is not HS modified and evaluated its signaling capacity in vivo. Purified Dally binds directly to FGF2, FGF10, and the functional Dpp homolog BMP4. FGF binding is abolished by preincubation with HS, but BMP4 association is partially HS-resistant, suggesting the Dally protein core contributes to binding. Cell binding and co-immunoprecipitation studies suggest that non-HS-modified Dally retains some ability to bind Dpp or BMP4. Expression of HS-deficient Dally in vivo showed it does not promote signaling as well as wild-type Dally, yet it can rescue several dally mutant phenotypes. These data reveal that heparan sulfate modification of Dally is not required for all in vivo activities and that significant functional capacity resides in the protein core.     

Characterization of primary human keratinocytes transformed by human papillomavirus type 18.

Primary human epithelial cells were cotransfected with pHPV-18 and pSV2neo, and cell strains were generated by selecting in G418. One cell strain (FE-A), which exhibits an extended life span, is currently in its 30th passage. In comparison, control cultures can only be maintained up to the seventh passage. Southern blot analysis revealed the presence of at least one intact, integrated viral genome in these cells. FE-A cells showed altered growth properties, characterized by a change in morphology, and clonal density. Differentiation markers analyzed by Western blotting (immunoblotting), such as cytokeratins and involucrin, indicated that the cells resembled a partially differentiated epithelial population. Increased expression of the 40-kilodalton cytokeratin was observed in FE-A cells, similar to that observed in simian virus 40-immortalized human keratinocytes (M. Steinberg and V. Defendi, J. Cell Physiol. 123:117-125, 1985). FE-A cells were also found to be defective in their response to terminal differentiation stimuli. Calcium and 12-O-tetradecanoyl-phorbol-13-acetate treatment induced normal epithelial cells to differentiate, whereas the human papillomavirus 18 (HPV-18)-containing keratinocytes were resistant to these signals, indicating their partially transformed nature. These cells were not able to induce tumors in nude mice over a period of up to 8 months. A second cell strain, FE-H18L, also generated by transfecting HPV-18, also exhibited an extended life span and similar alterations in morphology. Viral RNA transcribed from the early region of HPV-18 was detected in both cell strains by Northern (RNA) blot analysis. These cell strains should provide a useful model for determining the role of HPV in carcinogenesis.     

Human rhinovirus type 89 variants use heparan sulfate proteoglycan for cell attachment

We have previously isolated mutants of the major-group human rhinovirus type 89 that grow in cells deficient in intercellular adhesion molecule 1 (ICAM-1), the receptor used by the wild-type virus for cell entry [A. Reischl, M. Reithmayer, G. Winsauer, R. Moser, I. Goesler, and D. Blaas., J. Virol. 75:9312-9319, 2001]. We now demonstrate that one of these variants utilizes heparan sulfate proteoglycan (HSPG) as a cellular receptor. Adaptation to ICAM-1-deficient cells not only resulted in the newly acquired receptor specificity but also rendered the virus less stable at low pH and at elevated temperatures. This instability might compensate for the absence of the uncoating activity of ICAM-1. Whereas wild-type virus infection via ICAM-1 proceeded in the presence of the vesicular H(+)-ATPase inhibitor bafilomycin A1, infection by the mutant via HSPG was prevented by the drug. This suggests that the low pH prevailing in endosomal compartments is required for uncoating in the absence of the catalytic activity of ICAM-1.     

Human immunodeficiency virus type 1 infection and replication in normal human oral keratinocytes

Recent epidemiologic studies show increasing human immunodeficiency virus type 1 (HIV-1) transmission through oral-genital contact. This paper examines the possibility that normal human oral keratinocytes (NHOKs) might be directly infected by HIV or might convey infectious HIV virions to adjacent leukocytes. PCR analysis of proviral DNA constructs showed that NHOKs can be infected by CXCR4-tropic (NL4-3 and ELI) and dualtropic (89.6) strains of HIV-1 to generate a weak but productive infection. CCR5-tropic strain Ba-L sustained minimal viral replication. Antibody inhibition studies showed that infection by CXCR4-tropic viral strains is mediated by the galactosylceramide receptor and the CXCR4 chemokine coreceptor. Coculture studies showed that infectious HIV-1 virions can also be conveyed from NHOKs to activated peripheral blood lymphocytes, suggesting a potential role of oral epithelial cells in the transmission of HIV infection.     

Caveolin-1-dependent infectious entry of human papillomavirus type 31 in human keratinocytes proceeds to the endosomal pathway for pH-dependent uncoating

High-risk human papillomaviruses (HPVs) are small nonenveloped DNA viruses with a strict tropism for squamous epithelium. The viruses are causative agents of cervical cancer and some head and neck cancers, but their differentiation-dependent life cycles have made them difficult to study in simple cell culture. Thus, many aspects of early HPV infection remain mysterious. We recently showed the high-risk HPV type 31 (HPV31) enters its natural host cell type via caveola-dependent endocytosis, a distinct mechanism from that of the closely related HPV16 (Smith et al., J. Virol. 81:9922-9931, 2007). Here, we determined the downstream trafficking events after caveolar entry of HPV31 into human keratinocytes. After initial plasma membrane binding, HPV31 associates with caveolin-1 and transiently localizes to the caveosome before trafficking to the early endosome and proceeding through the endosomal pathway. Caveosome-to-endosome transport was found to be Rab5 GTPase dependent. Although HPV31 capsids were observed in the lysosome, Rab7 GTPase was dispensable for HPV31 infection, suggesting that viral genomes escape from the endosomal pathway prior to Rab7-mediated capsid transport. Consistent with this, the acidic pH encountered by HPV31 within the early endosomal pathway induces a conformational change in the capsid resulting in increased DNase susceptibility of the viral genome, which likely aids in uncoating and/or endosomal escape. The entry and trafficking route of HPV31 into human keratinocytes represents a unique viral pathway by which the virions use caveolar entry to eventually access a low-pH site that appears to facilitate endosomal escape of genomes.     

HIV infection does not require endocytosis of its receptor, CD4

The T cell surface molecule CD4 interacts with class II MHC molecules on the surface of target cells as well as with the envelope glycoprotein of human immunodeficiency virus (HIV). Internalization of CD4 molecules is observed after exposure of CD4+ T cells to either phorbol esters or appropriate antigen-bearing target cells. To determine whether HIV entry proceeds via receptor-mediated endocytosis or direct viral fusion with the cell membrane, we have constructed two mutants in the cytoplasmic domain of the CD4 protein that severely impair the ability of CD4 molecules to undergo endocytosis. Quantitative infectivity studies reveal that HeLa cell lines expressing wild-type or mutant CD4 molecules are equally susceptible to HIV infection. In addition, HIV binding does not lead to CD4 endocytosis. These studies indicate that although the CD4 molecule can be internalized, HIV entry proceeds via direct fusion of the viral envelope with the cell membrane.     

Human rhinovirus type 54 infection via heparan sulfate is less efficient and strictly dependent on low endosomal pH

K-type major-group human rhinoviruses (HRVs) (including HRV54) share a prominent lysine residue in the HI surface loop of VP1 with all minor-group HRVs. Despite the presence of this residue, they cannot use members of the low-density lipoprotein receptor family for productive infection. Reexamining all K-type viruses for receptor usage, we noticed that HRV54 is able to replicate in RD cells that lack the major-group receptor intercellular adhesion molecule 1 (ICAM-1). By using receptor blocking assays, inhibition of sulfation, enzymatic digestion, and proteoglycan-deficient cell lines, we show here that wild-type HRV54, without any adaptation, uses heparan sulfate (HS) proteoglycan as an alternate receptor. However, infection via HS is less efficient than infection via ICAM-1. Moreover, HRV54 has an acid lability profile similar to that of the minor-group virus HRV2. In ICAM-1-deficient cells its replication is completely blocked by the H(+)-ATPase inhibitor bafilomycin A1, whereas in ICAM-1-expressing cells it replicates in the presence of the drug. Thus, use of a "noncatalytic" receptor requires the virus to be highly unstable at low pH.     

Apoptotic cell-mediated immunoregulation of dendritic cells does not require iC3b opsonization

A number of recent studies show that activation of CR3 on dendritic cells (DCs) suppresses TLR-induced TNF-alpha and IL-12 production and inhibits effective Ag presentation. Although the proposed physiologic role for these phenomena is immune suppression due to recognition of iC3b opsonized apoptotic cells by CR3, all of the aforementioned investigations used artificial means of activating CR3. We investigated whether iC3b opsonized apoptotic cells could induce the same changes reported with artificial ligands such as mAbs or iC3b-opsonized RBC. We explored the kinetics of iC3b opsonization in two models of murine cell apoptosis, gamma-irradiated thymocytes and cytokine deprivation of the IL-3 dependent cell line BaF3. Using a relatively homogenous population of early apoptotic cells (IL-3 deprived BaF3 cells), we show that iC3b opsonized apoptotic cells engage CR3, but this interaction is dispensable in mediating the anti-inflammatory effects of apoptotic cells. TLR-induced TNF-alpha and IL-12 production by bone marrow-derived DCs occurs heterogeneously, with apoptotic cells inhibiting only certain populations depending on the TLR agonist. In contrast, although apoptotic cells induced homogeneous IL-10 production by DCs, IL-10 was not necessary for the inhibition of TNF-alpha and IL-12. Furthermore, because the ability of iC3b opsonization to enhance phagocytosis of apoptotic cells has been controversial, we report that iC3b opsonization does not significantly affect apoptotic cell ingestion by DCs. We conclude that the apoptotic cell receptor system on DCs is sufficiently redundant such that the absence of CR3 engagement does not significantly affect the normal anti-inflammatory processing of apoptotic cells.     

Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes.

The E6 proteins of the high-risk human papillomaviruses (HPVs) have been shown to form a complex with and induce the degradation of human p53 in vitro. To determine whether p53 is degraded more rapidly in cells expressing E6 in vivo, the half-life of p53 was determined by pulse-chase analysis in early-passage normal human keratinocytes and fibroblasts, human keratinocytes immortalized with HPV type 16 (HPV16) E6 plus E7, and nonimmortal keratinocytes transfected with E6. The results of these experiments indicate that (i) the half-life of newly synthesized p53 is relatively long (4 h) in early-passage human keratinocytes and fibroblasts but short in keratinocytes expressing E6 (15 to 30 min), (ii) a similar increased rate of p53 degradation was measured in lines immortalized with HPV16 E6 plus E7 and senescent cells expressing E6, indicating that this increase is not simply the result of selection in the immortalized lines, and (iii) very low levels of expression of E6 result in a greatly decreased half-life of p53, suggesting that E6 acts in a catalytic manner.     

Human complement factor I does not require cofactors for cleavage of synthetic substrates

Complement factor I (fI) plays a major role in the regulation of the complement system. It circulates in an active form and has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH), complement receptor type 1, membrane cofactor protein, or C4-binding protein. Using peptide-7-amino-4-methylcoumarin derivatives, we investigated the substrate specificity of fI. There is no previous report of synthetic substrate cleavage by fI, but five substrates were found in this study. A survey of 15 substrates and a range of inhibitors showed that fI has specificity similar to that of thrombin, but with much lower catalytic activity than that of thrombin. fI amidolytic activity has a pH optimum of 8.25, typical of serine proteases and is insensitive to ionic strength. This is in contrast to its proteolytic activity within the fI-C3b-fH reaction, in which the pH optimum for C3b cleavage is <5.5 and the reaction rate is highly dependent on ionic strength. The rate of cleavage of tripeptide 7-amino-4-methylcoumarins by fI is unaffected by the presence of fH or C3(NH(3)). The amidolytic activity is inhibited by the synthetic thrombin inhibitor Z-D-Phe-Pro-methoxypropylboroglycinepinanediol ester, consistent with previous reports, and by benzenesulfonyl fluorides such as Pefabloc SC. Suramin inhibits fI directly at concentration of 1 mM. Within a range of metal ions tested, only Cr(2+) and Fe(3+) were found to inhibit both the proteolytic and amidolytic activity of fI.