Inhibition of gamma secretase blocks HPV infection

Huang, Hao-Shun; Buck, Christopher B.; Lambert, Paul F.

doi:10.1016/j.virol.2010.09.002

Cited by 38 publications

(47 citation statements)

References 17 publications

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“…However, some of the hits-furin, the γ-secretase complex, and the vacuolar ATPase complex (which causes endosome acidification)-were reported by others to be required for HPV infection or are consistent with our prior understanding of this process (17)(18)(19)(20). The identification of these genes validated our screen design and suggested that our findings were relevant to HPV16 infection.…”

Section: Resultssupporting

confidence: 86%

“…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). After HPV is internalized, capsid disassembly is initiated in the endosome by acidification (11,15,20).…”

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confidence: 99%

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

Lipovsky

Popa

Pimienta

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

179

273

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Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/ Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed.

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Section: Resultssupporting

confidence: 86%

mentioning

confidence: 99%

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

Lipovsky

Popa

Pimienta

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

179

273

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“…Before we investigated the defect of these mutant PsVs in more detail, we tested whether the mutant viruses follow the same internalization pathway as wt HPV16. To test this, HeLa cells were infected with wt and mutant PsVs in the presence of well-established inhibitors of HPV16 infection targeting CyPs, ␥-secretase, tyrosine kinases, and endosomal acidification and trafficking (38,46,(48)(49)(50)(51)(52)(53)). All mutant PsVs tested displayed the same inhibition profile as wt HPV16 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Multiple Heparan Sulfate Binding Site Engagements Are Required for the Infectious Entry of Human Papillomavirus Type 16

Richards

Bienkowska-Haba

Dasgupta

et al. 2013

J Virol

102

120

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dHuman papillomavirus (HPV) entry is accompanied by multiple receptor-induced conformational changes (CCs) affecting both the major and minor capsid proteins, L1 and L2. Interaction of heparan sulfate (HS) with L1 is essential for successful HPV16 entry. Recently, cocrystallization of HPV16 with heparin revealed four distinct binding sites. Here we characterize mutant HPV16 to delineate the role of engagement with HS binding sites during infectious internalization. Site 1 (Lys278, Lys361), which mediates primary binding, is sufficient to trigger an L2 CC, exposing the amino terminus. Site 2 (Lys54, Lys356) and site 3 (Asn57, Lys59, Lys442, Lys443) are engaged following primary attachment and are required for infectious entry. Site 2 mutant particles are efficiently internalized but fail to undergo an L1 CC on the cell surface and subsequent uncoating in the endocytic compartment. After initial attachment to the cell, site 3 mutants undergo L1 and L2 CCs and then accumulate on the extracellular matrix (ECM). We conclude that the induction of CCs following site 1 and site 2 interactions results in reduced affinity for the primary HS binding site(s) on the cell surface, which allows engagement with site 3. Taken together, our findings suggest that HS binding site engagement induces CCs that prepare the virus for downstream events, such as the exposure of secondary binding sites, CCs, transfer to the uptake receptor, and uncoating.H uman papillomaviruses (HPVs) are small, nonenveloped epitheliotropic DNA viruses. HPV infection usually induces benign papillomas of the skin and mucosa. However, certain species, especially HPV16, are known as "high risk" due to their involvement in the progression to invasive carcinomas. Infection by HPV is considered necessary, though not sufficient, for the development of cervical cancer (1, 2). HPV infection is also associated with various anogenital and head and neck cancers (3). Despite the clear medical importance of preventing HPV-induced lesions, limited molecular detail regarding the attachment and entry of the virus is available. HPVs productively infect only epithelial cells in the skin and mucosa and depend on the differentiation of these cells for the completion of the viral life cycle (4). To bypass this obstacle, an in vitro surrogate system for viral propagation using a marker gene encapsidated into the viral capsid proteins was developed (5-7). This pseudovirus system overcomes the tropism and species specificity for viral propagation displayed by HPVs, allowing for study of the early events in the infection process.The papillomavirus virion is composed of the major and minor capsid proteins, L1 and L2, respectively. L1 is present in 360 copies organized into 72 pentamers, referred to as capsomeres (8-10). The L2 protein is present in an undetermined number of copies and is initially hidden inside the L1 structure prior to attachment to the cell surface (11). The outer virion shell, formed via pentavalent and hexavalent capsomere interactions between L1 molecules, medi...

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“…HeLa cells were infected with the reassembled particles in the presence of an inhibitor of furin (dec-RVKR-cmk), an inhibitor of ␥ secretase (compound XXI), or with NH 4 Cl. The first two inhibitors inhibit cellular enzymes known to be required for HPV16 PsV infection (38)(39)(40), while NH 4 Cl is a lysosomotropic inhibitor that neutralizes the pH of endosomes and is known to inhibit HPV16 PsV infection (41,42). All inhibitors prevented infection of HeLa cells with particles reassembled in nuclear extracts prepared from 293TT, 293H, or HaCaT cells (Fig.…”

Section: Characterization Of Assembly Reaction Componentsmentioning

confidence: 97%

A Cell-Free Assembly System for Generating Infectious Human Papillomavirus 16 Capsids Implicates a Size Discrimination Mechanism for Preferential Viral Genome Packaging

Cerqueira

Pang

Day

et al. 2016

J Virol

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We have established a cell-free in vitro system to study human papillomavirus type 16 (HPV16) assembly, a poorly understood process. L1/L2 capsomers, obtained from the disassembly of virus-like particles (VLPs), were incubated with nuclear extracts to provide access to the range of cellular proteins that would be available during assembly within the host cell. Incorporation of a reporter plasmid "pseudogenome" was dependent on the presence of both nuclear extract and ATP. Unexpectedly, L1/L2 VLPs that were not disassembled prior to incubation with a reassembly mixture containing nuclear extract also encapsidated a reporter plasmid. As with HPV pseudoviruses (PsV) generated intracellularly, infection by cell-free particles assembled in vitro required the presence of L2 and was susceptible to the same biochemical inhibitors, implying the cell-free assembled particles use the infectious pathway previously described for HPV16 produced in cell culture. Using biochemical and electron microscopy analyses, we observed that, in the presence of nuclear extract, intact VLPs partially disassemble, providing a mechanistic explanation to how the exogenous plasmid was packaged by these particles. Further, we provide evidence that capsids containing an <8-kb pseudogenome are resistant to the disassembly/reassembly reaction. Our results suggest a novel size discrimination mechanism for papillomavirus genome packaging in which particles undergo iterative rounds of disassembly/reassembly, seemingly sampling DNA until a suitably sized DNA is encountered, resulting in the formation of a stable virion structure. IMPORTANCELittle is known about papillomavirus assembly biology due to the difficulties in propagating virus in vitro. The cell-free assembly method established in this paper reveals a new mechanism for viral genome packaging and will provide a tractable system for further dissecting papillomavirus assembly. The knowledge gained will increase our understanding of virus-host interactions, help to identify new targets for antiviral therapy, and allow for the development of new gene delivery systems based on in vitrogenerated papillomavirus vectors. P apillomaviruses (PVs) are a group of nonenveloped viruses with small circular double-stranded DNA genomes that infect the skin and mucosa. PV replication is tightly coupled to the differentiation of the epithelium (1). Human papillomavirus 16 (HPV16) is medically the most important HPV type, as it is responsible for about 50% of cervical cancers and for the vast majority of noncervical HPV-positive anogenital and oropharyngeal cancers (2). The expression of viral early genes occurs in the lower layers of the epithelium, while vegetative replication of the viral genome, late gene expression, and virus assembly occur in more differentiated suprabasal cells (1). The strict dependency of viral replication on epithelial differentiation has made it difficult to produce virus in reasonable amounts and quality in cultured cells, although organotypic epithelial cell "raft" culture s...

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Inhibition of gamma secretase blocks HPV infection

Cited by 38 publications

References 17 publications

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

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

Multiple Heparan Sulfate Binding Site Engagements Are Required for the Infectious Entry of Human Papillomavirus Type 16

A Cell-Free Assembly System for Generating Infectious Human Papillomavirus 16 Capsids Implicates a Size Discrimination Mechanism for Preferential Viral Genome Packaging

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