Human Papillomavirus Type 31b Infection of Human Keratinocytes Does Not Require Heparan Sulfate

Abstract

Oncogenic human papillomaviruses (HPVs) are difficult to study experimentally as they replicate at low levels in vivo. This has precluded the purification of high-risk HPV virions from in vivo lesions. Virus-like particles (VLPs) and pseudovirions from low-and high-risk HPV types can emulate various aspects of HPV virion attachment and infections. These studies suggest that HPV infection is mediated by ␣6-integrin and/or heparan-sulfonated receptors. However, whether VLPs and pseudovirions accurately reflect the infection process of HPV virions has not been verified. We generated infectious HPV31b virions from organotypic (raft) tissues and performed experimental infections in a variety of cells. Successful infection following viral attachment, internalization, and nuclear transport was assayed by detecting newly synthesized, spliced HPV transcripts using reverse transcription (RT)-PCR or RT-quantitative PCR. Most human epithelial cells were infected with HPV31b at a multiplicity of infection as low as 1 to 10 viral genome equivalents per cell. HPV31b infection was detected in other cell lines, including COS-7 monkey kidney cells, but higher viral multiplicities of infection were required. Heparin preparations of various molecular weights or heparinase I treatment of cells prevented HPV31b infection of COS-7 cells and C-33A human cervical cancer cells in reproducible and dose-dependent manners. However, these reagents were unable to block infection of human keratinocytes, including HaCaT and N/TERT-1 cells and low-passage human foreskin keratinocytes. These data suggest that HPV31b infection of human keratinocytes, the natural host cell for HPV infections in vivo, does not require a heparan-sulfonated receptor, whereas heparan sulfate is important for infection of some other cells.

Differential binding patterns to host cells associated with particles of several human alphapapillomavirus types

“…The importance of HS moieties as the primary binding receptor for HPVs has received general acceptance in the literature (reviewed by Sapp & Day, 2009), although several studies describing HS-independent uptake and infection in vitro and in vivo have also been reported (Day et al, 2008;Patterson et al, 2005). The HS receptors have been identified as present on both the cell surface (Giroglou et al, 2001;Joyce et al, 1999) and the ECM .…”

“…Earlier studies on HPV binding and infection focused predominantly on one HPV type and one cell type to identify papillomavirus binding receptors for HS, a-6 integrin, syndecan-1 and LN5 (Culp et al, 2006a;Evander et al, 1997;Joyce et al, 1999;Patterson et al, 2005;ShaftiKeramat et al, 2003). These earlier studies led investigators to propose general mechanisms for HPV infection that should be applicable to all HPV types.…”

“…Two main candidates have been identified: heparan sulfate (HS), found on heparan sulfate proteoglycans (HSPGs), and a-6 integrin (Evander et al, 1997;Giroglou et al, 2001;Patterson et al, 2005). HSPGs are expressed ubiquitously through all layers of the epithelium, whereas a-6 integrin is mainly restricted to basal keratinocytes (Andriessen et al, 1997; reviewed by Jones et al, 1998;Oksala et al, 1995).…”

Replication and Assembly of Human Papillomaviruses

“…These cells continuously divide and replenish cells that are lost due to desquamation (Kaur and Li, 2000). Controversies exist as to the primary and/or secondary receptors for HPV entry, with alpha integrin and heparin sulfate as key possibilities, in addition to a role for laminin 5 (Giroglou et al, 2001;Yoon et al, 2001;Culp and Christensen, 2004;Doorbar, 2005;Patterson et al, 2005). Heparin sulfate appears important for adsorption and infection of a variety of HPV-typed VLPs, PsV, and native virions in nonhost and transformed cell lines, whereas HPV31b organotypic culture-derived native virions (OTNV) do not require heparin sulfate for infection of HaCat or N/Tert-1 human keratinocytes, but do require it for infection of non-host and transformed cell lines (Giroglou et al, 2001;Yoon et al, 2001;Culp and Christensen, 2004;Doorbar, 2005;Patterson et al, 2005).…”

“…Controversies exist as to the primary and/or secondary receptors for HPV entry, with alpha integrin and heparin sulfate as key possibilities, in addition to a role for laminin 5 (Giroglou et al, 2001;Yoon et al, 2001;Culp and Christensen, 2004;Doorbar, 2005;Patterson et al, 2005). Heparin sulfate appears important for adsorption and infection of a variety of HPV-typed VLPs, PsV, and native virions in nonhost and transformed cell lines, whereas HPV31b organotypic culture-derived native virions (OTNV) do not require heparin sulfate for infection of HaCat or N/Tert-1 human keratinocytes, but do require it for infection of non-host and transformed cell lines (Giroglou et al, 2001;Yoon et al, 2001;Culp and Christensen, 2004;Doorbar, 2005;Patterson et al, 2005). Postadsorption to the cell surface, internalization of virions has been shown to take many hours, and, depending on the papillomavirus type, virions can enter via clathrin-coated pits or caveolae (Fligge et al, 2001;Bousarghin et al, 2003;Day et al, 2003;Hindmarsh and Laimins, 2007;Smith et al, 2007).…”