Inhibition of nuclear entry of HPV16 pseudovirus-packaged DNA by an anti-HPV16 L2 neutralizing antibody

Ishii, Yoshiyuki; Tanaka, Keiko; Kondo, Kazunari; Takeuchi, Takamasa; Mori, Shuji; Kanda, Tadahito

doi:10.1016/j.virol.2010.07.019

Cited by 38 publications

(41 citation statements)

References 25 publications

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“…We also generated quasivirions in 293TT cells that encapsidate the HPV16 genome (39). For most experiments, we used particles containing a pseudogenome labeled with 5-ethynyl-2′-deoxyuridine (EdU), a nucleotide analog that can be detected by immunofluorescent staining using Click-iT chemistry (40). Using differential permeabilization and epitope-mapped monoclonal antibodies (Fig.…”

Section: Resultsmentioning

confidence: 99%

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: Resultsmentioning

confidence: 99%

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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“…The RG-1 epitope is not exposed until after furin cleavage of L2 at Arg12 prior to cell entry (13). It is therefore possible that the predicted TM domain is also not exposed until after furin cleavage although this region has been previously described as being surface exposed, and a monoclonal antibody against an overlapping epitope (residues 56 to 75) blocked infection, albeit neutralization required a high concentration of antibody (45,46). To gain structural insight into the nature of this region of L2, we synthesized peptides corresponding to residues 45 to 67 and 45 to 61, both of which were predicted to be TM domains by separate prediction algorithms (Table 1), and performed circular dichroism (CD) spectroscopy in both aqueous and hydrophobic environments.…”

Section: Resultsmentioning

confidence: 99%

“…2C). Next, the viral genome was labeled with the thymidine analog EdU, allowing direct visualization of incoming vDNA in immunofluorescence experiments (44,45,49). Infection with wt HPV16 resulted in strong colocalization of EdU-labeled vDNA and nuclear PML bodies at late times postinfection (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Several features of our TM-based model are conceptually attractive. First, there is already existing evidence for involvement of this region of L2 in vDNA penetration as a monoclonal antibody against residues 56 to 75, encompassing part of the TM domain, exhibits postentry neutralization activity by blocking this late stage of infection (45,46). Second, the TM domain is in close proximity to the other N-terminal elements of L2 that are involved in vDNA translocation (Fig.…”

Section: Discussionmentioning

confidence: 99%

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A Transmembrane Domain and GxxxG Motifs within L2 Are Essential for Papillomavirus Infection

Bronnimann

Chapman

Park

et al. 2013

J Virol

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During cellular invasion, human papillomavirus type 16 (HPV16) must transfer its viral genome (vDNA) across the endosomal membrane prior to its accumulation at nuclear PML bodies for the establishment of infection. After cellular uptake, the capsid likely undergoes pH-dependent disassembly within the endo-/lysosomal compartment, thereby exposing hidden domains in L2 that facilitate membrane penetration of L2/vDNA complexes. In an effort to identify regions of L2 that might physically interact with membranes, we have subjected the L2 sequence to multiple transmembrane (TM) domain prediction algorithms. Here, we describe a conserved TM domain within L2 (residues 45 to 67) and investigate its role in HPV16 infection. In vitro, the predicted TM domain adopts an alpha-helical structure in lipid environments and can function as a real TM domain, although not as efficiently as the bona fide TM domain of PDGFR. An L2 double point mutant renders the TM domain nonfunctional and blocks HPV16 infection by preventing endosomal translocation of vDNA. The TM domain contains three highly conserved GxxxG motifs. These motifs can facilitate homotypic and heterotypic interactions between TM helices, activities that may be important for vDNA translocation. Disruption of some of these GxxxG motifs resulted in noninfectious viruses, indicating a critical role in infection. Using a ToxR-based homo-oligomerization assay, we show a propensity for this TM domain to self-associate in a GxxxG-dependent manner. These data suggest an important role for the self-associating L2 TM domain and the conserved GxxxG motifs in the transfer of vDNA across the endo-/lysosomal membrane.A ll nonenveloped viruses are faced with a cellular membrane barrier through which they must transfer their genetic material to establish a successful infection. These viruses behave as metastable particles, rigid enough to survive transmission through the extracellular milieu but structurally poised to undergo conformational rearrangements or partial disassembly upon encountering specific factors during cellular entry, including the engagement of host cell receptors, proteolytic and chaperone activity, and low pH of the intracellular endosomal compartment. In response to these factors, capsids rearrange to expose hidden and often hydrophobic membrane translocation domains (MTDs), thereby ensuring that the coordinated process of membrane penetration occurs only when the viral capsid has reached the appropriate intracellular locale. Through convergent evolution, viruses have become equipped with a variety of different MTDs, designed to accomplish the feat of membrane penetration in several ways (recently reviewed in reference 1). Amphipathic helices, myristoylated and/or hydrophobic peptides, and phospholipase activity have all been documented to facilitate membrane penetration through pore formation, local membrane disruption, or gross fragmentation of membranes although many molecular details of these MTD-driven activities remain obscure and are ongoing subjects of inve...

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“…Ultra-thin sections (70 nm) of cells were prepared and examined under a Hitachi HT-7700 transmission electron microscope (Hitachi High Technologies Co., Japan) as described previously [40]. Briefly, EECs were mock-infected or infected with PPRV Nigeria 75/1 at an MOI of 1 for 48 h. The cells were then washed three times with PBS and collected by centrifugation at 800 × g for 5 min; one drop of 2% preheated agarose was added to the cell pellet and uniformly mixed.…”

Section: Methodsmentioning

confidence: 99%

Autophagy enhances the replication of Peste des petits ruminants virus and inhibits caspase-dependent apoptosis in vitro

Yang

Xue

et al. 2018

Virulence

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Peste des petits ruminants (PPR) is an acute and highly contagious disease in small ruminants that causes significant economic losses in developing countries. An increasing number of studies have demonstrated that both autophagy and apoptosis are important cellular mechanisms for maintaining homeostasis, and they participate in the host response to pathogens. However, the crosstalk between apoptosis and autophagy in host cells during PPRV infection has not been clarified. In this study, autophagy was induced upon virus infection in caprine endometrial epithelial cells (EECs), as determined by the appearance of double- and single-membrane autophagy-like vesicles, LC3-I/LC3-II conversion, and p62 degradation. We also found that PPRV infection triggered a complete autophagic response, most likely mediated by the non-structural protein C and nucleoprotein N. Moreover, our results suggest that autophagy not only promotes the replication of PPRV in EECs but also provides a potential mechanism for inhibiting PPRV-induced apoptosis. Inhibiting autophagosome formation by wortmannin and knocking down the essential autophagic proteins Beclin-1 and ATG7 induces caspase-dependent apoptosis in EECs in PPRV infection. However, inhibiting autophagosome and lysosome fusion by NH4Cl and chloroquine did not increase the number of apoptotic cells. Collectively, these data are the first to indicate that PPRV-induced autophagy inhibits caspase-dependent apoptosis and thus contributes to the enhancement of viral replication and maturity in host cells.

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Inhibition of nuclear entry of HPV16 pseudovirus-packaged DNA by an anti-HPV16 L2 neutralizing antibody

Cited by 38 publications

References 25 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

A Transmembrane Domain and GxxxG Motifs within L2 Are Essential for Papillomavirus Infection

Autophagy enhances the replication of Peste des petits ruminants virus and inhibits caspase-dependent apoptosis in vitro

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