Human Papillomavirus Type 16 Infection of Human Keratinocytes Requires Clathrin and Caveolin-1 and Is Brefeldin A Sensitive

Abstract

Human papillomavirus type 16 (HPV16) has been identified as being the most common etiological agent leading to cervical cancer. Despite having a clear understanding of the role of HPV16 in oncogenesis, details of how HPV16 traffics during infection are poorly understood. HPV16 has been determined to enter via clathrin-mediated endocytosis, but the subsequent steps of HPV16 infection remain unclear. There is emerging evidence that several viruses take advantage of cross talk between routes of endocytosis. Specifically, JCV and bovine papillomavirus type 1 have been shown to enter cells by clathrin-dependent endocytosis and then require caveolin-1-mediated trafficking for infection. In this paper, we show that HPV16 is dependent on caveolin-1 after clathrin-mediated endocytosis. We provide evidence for the first time that HPV16 infection is dependent on trafficking to the endoplasmic reticulum (ER). This novel trafficking may explain the requirement for the caveolar pathway in HPV16 infection because clathrin-mediated endocytosis typically does not lead to the ER. Our data indicate that the infectious route for HPV16 following clathrin-mediated entry is caveolin-1 and COPI dependent. An understanding of the steps involved in HPV16 sorting and trafficking opens up the possibility of developing novel approaches to interfere with HPV16 infection and reduce the burden of papillomavirus diseases including cervical cancer.

Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus

“…HPV is then thought to escape into the cytoplasm and travel by an unknown, microtubule-dependent route to the nucleus where virus replication occurs (12,19). One study concluded that HPV16 traffics through the endoplasmic reticulum (ER) (11). The L2 protein appears to be important for proper intracellular trafficking, as does binding of sorting nexin 17 to an internal segment of L2 (21)(22)(23).…”

“…Cyclophilin B and the proteases furin and γ-secretase play essential but not clearly understood roles during HPV entry (16)(17)(18)(19). After HPV is internalized, capsid disassembly is initiated in the endosome by acidification (11,15,20). HPV is then thought to escape into the cytoplasm and travel by an unknown, microtubule-dependent route to the nucleus where virus replication occurs (12,19).…”

“…After an initial association of L1 with heparan sulfate proteoglycans, capsids undergo conformational changes, and proteolytic cleavage occurs in L2 (5)(6)(7). HPV is then thought to be transferred to an as-yetunidentified cell-surface receptor, followed by endocytosis and intracellular trafficking (8)(9)(10)(11)(12)(13)(14)(15). Cyclophilin B and the proteases furin and γ-secretase play essential but not clearly understood roles during HPV entry (16)(17)(18)(19).…”

Phospholipid Scramblase 1 Is Secreted by a Lipid Raft-dependent Pathway and Interacts with the Extracellular Matrix Protein 1 in the Dermal Epidermal Junction Zone of Human Skin

“…Cultures of HaCaT cells, a spontaneously immortalized human keratinocyte cell line, were maintained in DMEM (Sigma-Aldrich), supplemented with 10% (v/v) sterile-filtered fetal bovine serum (Sigma-Aldrich), 2% L-glutamine (Invitrogen), 100 g/ml streptomycin, and 100 IU/ml penicillin (Invitrogen) at 37°C in a humidified atmosphere with 5% CO 2 . For inhibition of the endoplasmic reticulum/Golgi secretory transport, 25 ng/ml brefeldin A (Sigma-Aldrich) was added to a HaCaT cell culture for 48 h (25). Palmitoylation was metabolically inhibited by treating HaCaT cultures with 2-bromo-palmitate (SigmaAldrich) overnight at 50 M (26).…”

“…In order to further clarify the membrane localization of PLSCR1, we next investigated whether palmitoylation is necessary for the secretion of PLSCR1. Therefore, HaCaT cells were cultured under the condition where palmitoylation was metabolically prevented by 2-bromo-palmitate, a competitive inhibitor of the transfer of palmitoyl-CoA to the cysteinoyl thiol (25). However, no altered secretion of PLSCR1 was observed when comparing the supernatant and cell lysate of HaCaT cells stably overexpressing eGFP-PLSCR1 in the presence or absence of 2-bromo-palmitate (2-BP) (Fig.…”