Large Scale RNAi Reveals the Requirement of Nuclear Envelope Breakdown for Nuclear Import of Human Papillomaviruses

Aydin, Inci; Weber, Susanne N.; Snijder, Berend; Ventayol, Pilar Samperio; Kühbacher, Andreas; Becker, Miriam; Day, Patricia M.; Schiller, John T.; Kann, Michael; Pelkmans, Lucas; Helenius, Ari; Schelhaas, Mario

doi:10.1371/journal.ppat.1004162

Cited by 135 publications

(222 citation statements)

References 84 publications

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“…Our observations are somewhat contradictory to recently published findings purporting that arresting cells in a monoastral phenotype does not block HPV16 infection (34). However, a careful analysis reveals that Eg5 inhibition is leaky and that a minority of cells complete an additional cell cycle rather than being arrested in mitosis.…”

Section: Discussioncontrasting

confidence: 99%

“…In contrast to our observations, a previous study reported that inhibition of Eg5 has no effect on pseudoviral infectivity in both HeLa and HaCaT cells (34). In line with that study, we also observed no significant inhibition of infection in the presence of Eg5i (Fig.…”

Section: Incoming Viral Genome Is Protected From Nuclease Digestion Dsupporting

confidence: 62%

“…Previous observations have suggested that L2 protein can associate with condensed chromosomes during mitosis (34); therefore, it is reasonable to speculate that L2 may interact directly with nuclear resident proteins or with the condensed chromosomes to ensure that the transport vesicle remains associated with the mitotic bodies until the completion of mitosis. We have provided evidence that the 16L2-R302/5A mutant protein is defective for deposition of HPV-harboring vesicles on condensed chromosomes.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, siRNA screens have identified several phospholipases required for HPV infection (27,34). Another nonenveloped virus, parvovirus, uses phospholipases to penetrate intracellular membranes.…”

Section: Discussionmentioning

confidence: 99%

“…A large portion of the L2 protein translocates across the endocytic membrane to engage factors, including the retromer complex, dynein, sorting nexins, and rab GTPases, that mediate transport to the trans-Golgi network (TGN) (25)(26)(27)(28)(29)(30)(31)(32)(33)). An siRNA screen has suggested that nuclear pore complexes are not required for nuclear entry, but that nuclear envelope breakdown during mitosis is necessary (34,35).…”

mentioning

confidence: 99%

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Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis

DiGiuseppe

Luszczek

Keiffer

et al. 2016

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

140

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During the entry process, the human papillomavirus (HPV) capsid is trafficked to the trans-Golgi network (TGN), whereupon it enters the nucleus during mitosis. We previously demonstrated that the minor capsid protein L2 assumes a transmembranous conformation in the TGN. Here we provide evidence that the incoming viral genome dissociates from the TGN and associates with microtubules after the onset of mitosis. Deposition onto mitotic chromosomes is L2-mediated. Using differential staining of an incoming viral genome by small molecular dyes in selectively permeabilized cells, nuclease protection, and flotation assays, we found that HPV resides in a membrane-bound vesicle until mitosis is completed and the nuclear envelope has reformed. As a result, expression of the incoming viral genome is delayed. Taken together, these data provide evidence that HPV has evolved a unique strategy for delivering the viral genome to the nucleus of dividing cells. Furthermore, it is unlikely that nuclear vesicles are unique to HPV, and thus we may have uncovered a hitherto unrecognized cellular pathway that may be of interest for future cell biological studies.HPV entry | vesicular transport | mitosis | digitonin | nuclear vesicle A major hurdle of DNA viruses during a primary infection is successful navigation of the cytoplasm to deliver the viral genome to the nucleus. Foreign DNA that enters the cytoplasm is susceptible to being sensed by innate immune sensors (1). Not surprisingly, viruses have evolved mechanisms to evade detection by these sensors. For example, herpesviruses and adenoviruses both egress into the cytoplasm, yet protect their viral DNA by keeping it encased in viral capsids until directly transferring it into the nucleus through the nuclear pore complex. In contrast, the capsid is unlikely to protect papillomavirus (PV) genomes while traversing the cytoplasm, because the major capsid protein is lost in the endocytic compartment (2).The PV capsid is composed of two viral proteins, the major capsid protein L1 and the minor capsid protein L2, which enclose a chromatinized, circular, double-stranded DNA genome ∼8 kb in size (3-6). Following primary attachment and internalization, acidification of early endosomes triggers capsid disassembly (7-22). Host cell cyclophilins release the majority of L1 from the L2 protein, which remains in complex with the viral genome (2,23,24). A large portion of the L2 protein translocates across the endocytic membrane to engage factors, including the retromer complex, dynein, sorting nexins, and rab GTPases, that mediate transport to the trans-Golgi network (TGN) (25)(26)(27)(28)(29)(30)(31)(32)(33)). An siRNA screen has suggested that nuclear pore complexes are not required for nuclear entry, but that nuclear envelope breakdown during mitosis is necessary (34, 35).Currently, when and how the human PV (HPV) genome egresses from the membranous compartment is unclear. Here we present evidence indicating that after the onset of mitosis, the viral genome of HPV type 16 (HPV16), an HPV type...

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Section: Discussioncontrasting

confidence: 99%

Section: Incoming Viral Genome Is Protected From Nuclease Digestion Dsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis

DiGiuseppe

Luszczek

Keiffer

et al. 2016

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

140

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Fluorescently Labeled Human Papillomavirus Pseudovirions for Use in Virus Entry Experiments

Ventayol

Schelhaas

2015

CP Microbiology

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Human papillomaviruses (HPV) infect skin or mucosal epidermis. The simplistic capsid consists of a major capsid protein L1, a minor capsid protein L2, and a double‐stranded circular DNA of about 8kB in size. The development of HPV‐based vectors [i.e., pseudovirions (PsV)] as tools to study the initial infection has facilitated our understanding of HPV entry. The covalent coupling of fluorescent molecules to these PsV allows following the viruses en route to the nucleus, i.e., the site of replication. In the first section, we describe a facile method to covalently label HPV PsV that retain their infectivity. In this method, fluorophores coupled to a reactive succinimidyl ester are covalently attached to amine residues in the virion in a one‐step chemical reaction. In the second section of this unit, several assays are outlined that use the fluorescently labeled virions for entry studies in live and fixed cells. © 2015 by John Wiley & Sons, Inc.

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Cells infected with human papilloma pseudovirus display nuclear reorganization and heterogenous infection kinetics

et al. 2022

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Human papillomaviruses (HPV) are small, non‐enveloped DNA viruses, which upon chronic infection can provoke cervical and head‐and‐neck cancers. Although the infectious life cycle of HPV has been studied and a vaccine is available for the most prevalent cancer‐causing HPV types, there are no antiviral agents to treat infected patients. Hence, there is a need for novel therapeutic entry points and a means to identify them. In this work, we have used high‐content microscopy to quantitatively investigate the early phase of HPV infection. Human cervical cancer cells and immortalized keratinocytes were exposed to pseudoviruses (PsV) of the widespread HPV type 16, in which the viral genome was replaced by a pseudogenome encoding a fluorescent reporter protein. Using the fluorescent signal as readout, we measured differences in infection between cell lines, which directly correlated with host cell proliferation rate. Parallel multiparametric analysis of nuclear organization revealed that HPV PsV infection alters nuclear organization and inflates promyelocytic leukemia protein body content, positioning these events at the early stage of HPV infection, upstream of viral replication. Time‐resolved analysis revealed a marked heterogeneity in infection kinetics even between two daughter cells, which we attribute to differences in viral load. Consistent with the requirement for mitotic nuclear envelope breakdown, pharmacological inhibition of the cell cycle dramatically blunted infection efficiency. Thus, by systematic image‐based single cell analysis, we revealed phenotypic alterations that accompany HPV PsV infection in individual cells, and which may be relevant for therapeutic drug screens.

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Large Scale RNAi Reveals the Requirement of Nuclear Envelope Breakdown for Nuclear Import of Human Papillomaviruses

Cited by 135 publications

References 84 publications

Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis

Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis

Fluorescently Labeled Human Papillomavirus Pseudovirions for Use in Virus Entry Experiments

Cells infected with human papilloma pseudovirus display nuclear reorganization and heterogenous infection kinetics

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