Subcellular Trafficking of the Papillomavirus Genome during Initial Infection: The Remarkable Abilities of Minor Capsid Protein L2

Campos, Samuel K.

doi:10.3390/v9120370

Cited by 54 publications

(72 citation statements)

References 122 publications

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“…During HPV16 infection, the N-terminal 12 amino acids of L2 are removed by furin cleavage (54), an obligatory step for proper retrograde trafficking and infection (reviewed in (18)). To determine if BSO affects furin processing of L2 we utilized a previously described system to easily monitor L2 cleavage called PSTCD-L2 (54).…”

Section: Resultsmentioning

confidence: 99%

“…Internalized virions initially traffic through the endosomal compartment where low pH and the intramembrane protease complex γ-secretase trigger membrane insertion and protrusion of L2 through the vesicular membrane into the cytosol for recruitment of sorting nexins and retromer (14-18). A C-terminal charged region of L2, with inherent “cell penetration peptide” properties is also important for protrusion of L2 across the endosomal membranes (19).…”

Section: Introductionmentioning

confidence: 99%

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Glutathione is Required for Efficient Post-Golgi Trafficking of Incoming HPV16 Genome

Bronnimann

Williams

et al. 2019

Preprint

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24Human papillomavirus (HPV) is the most common sexually transmitted pathogen in the 25 United States, causing 99% of cervical cancers and 5% of all human cancers worldwide. 26 HPV infection requires transport of the viral genome (vDNA) into the nucleus of basal 27 keratinocytes. During this process, minor capsid protein L2 facilitates subcellular 28 retrograde trafficking of the vDNA from endosomes to the Golgi, and accumulation at 29 host chromosomes during mitosis for nuclear retention and localization during 30 interphase. Here we investigated the relationship between cytosolic GSH and HPV16 31 infection. siRNA knockdown of GSH biosynthetic enzymes results in a partial decrease 32 of HPV16 infection. Likewise, infection of HPV16 in GSH depleted keratinocytes is 33 inefficient, an effect that was not seen with adenoviral vectors. Analysis of trafficking 34 revealed no defects in cellular binding, entry, furin cleavage of L2, or retrograde 35 trafficking of HPV16, but GSH depletion hindered post-Golgi trafficking and 36 translocation, decreasing nuclear accumulation of vDNA. Although precise mechanisms 37have yet to be defined, this work suggests that GSH is required for a specific post-Golgi 38 trafficking step in HPV16 infection. 39 40 42 in the United States (1). Currently there are >300 HPV types that have been deposited 43 and annotated in the Papillomavirus Episteme (PAVE) database (2). Based on disease 44 association, HPV can be divided into low-risk HPV and cancer-associated high-risk HPV 45 (3). Most HPVs are considered as low-risk HPV, causing cutaneous and mucosal warts 46 3(4). However, 99% of cervical cancers and 5% of all human cancers worldwide are 47 caused by high-risk HPV infection (5,6). HPV16 belongs to the high-risk HPV, and 48 HPV16 alone is responsible for >50% cervical cancers (7). There are highly effective 49 vaccines being used to prevent high-risk HPV infection, but the vaccine cannot protect 50 against all types of high-risk HPV infection and the high cost prevents people from 51 developing world to get access to the vaccine (7). 52HPVs are small non-enveloped DNA viruses. The icosahedral capsid is built from 53 72 pentamers (360 molecules) of the major capsid protein L1. Within this particle, 54 variable copies (<72 molecules but typically 20-40) of minor capsid protein L2 are 55 complexed to the ~8kb dsDNA genome (vDNA). At the cellular level, HPV infection 56 begins with virion attachment to keratinocytes via heparin sulfate proteoglycans 57 (HSPGs) followed by furin-and kalleikrein-dependent cleavage of the capsid (8,9), 58 likely causing conformational changes and transfer to secondary entry receptor 59 complex(es). The nature of the entry complex(es) is still debatable but likely include 60growth factor receptor tyrosine kinases, integrins, tetraspanins, and annexin A2 (10,11). 61Asynchronous cellular uptake of virion occurs by macropinocytosis-like mechanisms 62 after this potentially prolonged residence on the cell surface (12,13). 63Internalized virions initially traf...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Glutathione is Required for Efficient Post-Golgi Trafficking of Incoming HPV16 Genome

Bronnimann

Williams

et al. 2019

Preprint

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“…HPV infectious entry involves a complex series of steps, including the attachment of virus to the extracellular matrix, the structural remodelling of the virion, receptor attachment, endocytic uptake, intracellular trafficking through the endosomal compartment, uncoating, and the recruitment of different components of the cellular transport machinery to ensure delivery of the incoming viral genomes to the nucleus through the trans-Golgi network (25)(26)(27). However, no studies have been performed to investigate whether potential phosphorylation of the HPV capsid proteins can play any roles in any of these processes.…”

Section: Discussionmentioning

confidence: 99%

“…Interactions of the viral capsid proteins with cellular components play an important role in papillomavirus infectious entry (25)(26)(27). Intracellular processing of the viral capsid is dependent on a number of different host factors, such as furin (13), cyclophilin (16), and gamma-secretase (21,(28)(29)(30), among others.…”

mentioning

confidence: 99%

Phosphorylation of Human Papillomavirus Type 16 L2 Contributes to Efficient Virus Infectious Entry

Broniarczyk

Massimi

Pim

et al. 2019

J Virol

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The human papillomavirus (HPV) capsid comprises two viral proteins, L1 and L2, with the L2 component being essential to ensure efficient endocytic transport of incoming viral genomes. Several studies have previously reported that L1 and L2 are posttranslationally modified, but it is uncertain whether these modifications affect HPV infectious entry. Using a proteomic screen, we identified a highly conserved phospho-acceptor site on the HPV-16 and bovine papillomavirus 1 (BPV-1) L2 proteins. The phospho-modification of L2 and its presence in HPV pseudovirions (PsVs) were confirmed using anti-phospho-L2-specific antibodies. Mutation of the phosphoacceptor sites of both HPV-16 and BPV-1 L2 resulted in the production of infectious virus particles, with no differences in efficiencies of packaging the reporter DNA. However, these mutated PsVs showed marked defects in infectious entry. Further analysis revealed a defect in uncoating, characterized by a delay in the exposure of a conformational epitope on L1 that indicates capsid uncoating. This uncoating defect was accompanied by a delay in the proteolysis of both L1 and L2 in mutated HPV-16 PsVs. Taken together, these studies indicate that phosphorylation of L2 during virus assembly plays an important role in optimal uncoating of virions during infection, suggesting that phosphorylation of the viral capsid proteins contributes to infectious entry. IMPORTANCE The papillomavirus L2 capsid protein plays an essential role in infectious entry, where it directs the successful trafficking of incoming viral genomes to the nucleus. However, nothing is known about how potential posttranslational modifications may affect different aspects of capsid assembly or infectious entry. In this study, we report the first phospho-specific modification of the BPV-1 and HPV-16 L2 capsid proteins. The phospho-acceptor site is very highly conserved across multiple papillomavirus types, indicating a highly conserved function within the L2 protein and the viral capsid. We show that this modification plays an essential role in infectious entry, where it modulates susceptibility of the incoming virus to capsid disassembly. These studies therefore define a completely new means of regulating the papillomavirus L2 proteins, a regulation that optimizes endocytic processing and subsequent completion of the infectious entry pathway.

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“…This review summaries the unique entry mechanism used by papillomaviruses, with a focus on recent results that have clarified several key aspects of intracellular HPV trafficking during virus entry, and suggests potential therapeutic approaches. For comprehensive reviews on HPV entry, readers are referred to the excellent reviews by Campos, Sapp, and Banks and their colleagues [1][2][3].…”

Section: Introductionmentioning

confidence: 99%

Papillomaviruses Go Retro

et al. 2020

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Human papillomaviruses are important pathogens responsible for approximately 5% of cancer as well as other important human diseases, but many aspects of the papillomavirus life cycle are poorly understood. To undergo genome replication, HPV DNA must traffic from the cell surface to the nucleus. Recent findings have revolutionized our understanding of HPV entry, showing that it requires numerous cellular proteins and proceeds via a series of intracellular membrane-bound vesicles that comprise the retrograde transport pathway. This paper reviews the evidence supporting this unique entry mechanism with a focus on the crucial step by which the incoming virus particle is transferred from the endosome into the retrograde pathway. This new understanding provides novel insights into basic cellular biology and suggests novel rational approaches to inhibit HPV infection.Pathogens 2020, 9, 267 2 of 11 with wild-type or mutant capsid proteins, and the convenience of assaying successful infection by measuring reporter gene expression. Nevertheless, authentic papillomaviruses should be used to validate crucial findings obtained with pseudoviruses because there may be important differences between authentic viruses produced in stratified epithelial cells and pseudoviruses produced in a monolayer cell culture [13]. Role of Retrograde Trafficking and Retromer in HPV EntryKnockdown and inhibitor studies have identified numerous cell proteins required for HPV entry and trafficking, including but not limited to furin, kallikrein-8, ADAM17, cyclophilin B, SNX17, SNX27, γ-secretase, VPS4, obscurin-like1, TSG101,TRAPPC8, growth factor receptors, DCT, annexin A2/S100A10, and tetraspanins CD131 and CD63 [8,10,[14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29]. The proteases furin and kallikrein-8 cleave L2 and L1, respectively. Furin-catalyzed cleavage near the N terminus of L2 occurs at the cell surface, but in the absence of cleavage, intracellular trafficking events are disrupted [30]. ADAM17 appears to assemble an entry platform containing CDC151 and an epidermal growth factor receptor that mediates internalization of HPV [27]. In most cases, however, little is known about the mechanism by which these proteins support HPV infection.To comprehensively identify cellular proteins involved in HPV entry, we conducted a genome-wide siRNA screen. In this screen,~20,000 different siRNA pools were transfected into HeLa cells in a microtiter plate format to repress individual cellular genes, and the ability of an HPV16 pseudovirus to express GFP from the encapsidated reporter plasmid was measured in each well of cells [31]. The results showed that efficient HPV infection requires numerous cellular proteins localized to vesicular compartments such as the trans-Golgi network (TGN) and the endoplasmic reticulum (ER). Subsequent validation studies and more targeted analysis of candidate genes confirmed that these proteins were required for HPV entry [30,31]. Among the required factors identified in these experiments were Rab proteins...

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Subcellular Trafficking of the Papillomavirus Genome during Initial Infection: The Remarkable Abilities of Minor Capsid Protein L2

Cited by 54 publications

References 122 publications

Glutathione is Required for Efficient Post-Golgi Trafficking of Incoming HPV16 Genome

Glutathione is Required for Efficient Post-Golgi Trafficking of Incoming HPV16 Genome

Phosphorylation of Human Papillomavirus Type 16 L2 Contributes to Efficient Virus Infectious Entry

Papillomaviruses Go Retro

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