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

Abstract

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.

Lamin B1 curtails early human papillomavirus infection by safeguarding nuclear compartmentalization and autophagic capacity

“…Despite their ample use in relevant HPV infection studies (7,25,42), HeLa cells contain multiple copies of integrated HPV18 DNA, which might in uence their response to HPV infection (43). However, we have here exposed cell-intrinsic mechanisms that foster the early phase of infection, and which may be less dependent of this status.…”

“…HPV16 PsV were produced by co-transfecting 293TT cells with p16SheLL and pClneo-eGFP or p8RwB plasmids (Addgene plasmid #37320, plasmid #46949 and plasmid #48733, all a kind gift of Dr. John Schiller) (22)(23)(24). The protocol used has been previously described (25). For PsV labeling experiments, the growth medium of the cells was supplemented with 20 µM 5-ethylnyl-2'-deoxyuridine (EdU) ve hours after the transfection.…”

“…Various particle detection algorithms are integrated in this version, including Stardist (31). However, to tweak the nuclear detection we pre-processed the images by exploiting StarDist-mediated nucleus detection included in our analysis pipeline (31) and conversion of the detected nuclear ROI into indexed spots of 2 µm radius (https://github.com/VerschuurenM/StarTrack) (25). Within TrackMate, we limited the linking distance during tracking to 20 µm, allowed gap closing of max.…”

“…To determine the role of nuclear lamins during HPV PsV infection, we selectively depleted nuclear lamins in HeLa human cervix carcinoma cells. While HPV has a natural tropism for keratinocytes, we have previously shown that HeLa is a much more susceptible and robust model for infection studies (25). A sustained knockdown (kd) of LMNA, LMNB1 and LMNB2 was obtained using repetitive siRNA-mediated gene silencing, while stable LMNA, LMNB1 and LMNB2 knockout (ko) cells were produced using targeted CRISPR/Cas9-mediated genome editing (Fig.…”

“…Since mitosis is crucial for HPV nuclear entry (7,8,25), a change in cell cycle kinetics may alter the infection probability. However, none of the lamin depleted cells -whether established by sustained knockdown or stable knockout -displayed an altered population doubling time (Fig.…”