A Dual Role for the Nonreceptor Tyrosine Kinase Pyk2 during the Intracellular Trafficking of Human Papillomavirus 16

Abstract

The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. IMPORTANCE In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study advances our knowledge of intracellular trafficking processes in keratinocytes.P apillomaviridae is a family of small, nonenveloped DNA viruses with tropism for epithelial tissue. Low-risk human papillomavirus (HPV) types cause benign warts, and high-risk types cause anogenital or oropharyngeal area cancers (1-3). Studying the tactics used by viruses during infection has a 2-fold importance: (i) the potential to identify drug targets and (ii) understanding processes of endocytosis and intracellular trafficking.The HPV capsid is composed of the major capsid protein L1 and the minor capsid protein L2. Infection begins when the virus capsid binds to heparan sulfate proteoglycans (HSPGs) at the cell surface (in vitro) or on the basement membrane (in vivo) (4-8). The HSPG syndecan-1 has been shown to be unnecessary in a mouse model; however, it was suggested that other HSPGs may compensate (9). Conformational changes to the viral capsid mediated by cyclophilin B (a peptidyl-prolyl cis-trans isomerase) and furin or PC5/6 (proprotein convertase) occur during initial...

HPV entry into cells

“…Proximity ligation assays (PLA) in HaCaT cells showed colocalization between EEA1 and L1 (HPV16 PsVs) at 8 hours and showed a decrease in colocalization between the two proteins at 16 hours [74]. The EdU-labeled pseudogenome of HPV16 PsVs colocalized with EEA1 in HaCaT cells at 2 hours post-infection [75]. In HeLa cells, HA-tagged L2 in HPV16 PsVs colocalized with EEA1 at 12 hours post-infection [76].…”

“…Colocalization of the HPV L1 protein with LAMP1 seems to occur slightly later; starting at approximately 6–8h post-infection [54]. The level of colocalization between HPV L1 and LAMP1 increases as infection progresses, which suggests the majority of the L1 protein is retained in the lysosome for degradation [75, 79]. AF488-labeled HPV16 PsVs colocalized with LAMP1 at 8h post-infection in HeLa cells [67].…”

“…AF488-labeled HPV16 PsVs colocalized with LAMP1 at 8h post-infection in HeLa cells [67]. In HaCaT cells, HPV16 PsV L1 (using the conformational-dependent antibody H16.V5 against L1 [80, 81]) colocalized with LAMP1 starting at 8 h post-infection and increased at 16h [75]. AF488-labeled HPV31 virions colocalized with LAMP1 at 16h and 24h post-infection in HaCaT cells [79].…”

“…The use of the L1–7 antibody, which recognizes an epitope of L1 located in the interior of intact viral capsids, has been used to assess the disassembly/degradation of viral capsids during infection [45, 83, 84]. The L1–7 epitope begins to become accessible approximately 4 hours post-infection, increasing in accessibility over the next 12 hours of infection in HaCaT and HeLa cells, and colocalizes with LAMP1 in HaCaTs [45, 54, 75, 85]. …”

Cruising the cellular highways: How human papillomavirus travels from the surface to the nucleus

“…A recent study suggests that egress from the TGN may involve Pyk2 signaling in cells. While the exact contribution of Pyk2 signaling during intracellular trafficking remains to be determined (Gottschalk and Meneses, 2015), it would be interesting for future studies to investigate whether Pyk2 plays a role in trafficking following the onset of mitosis. There is also some evidence to support the idea that the viral genome traffics to the ER.…”