Crossing the nuclear envelope border

Subtitle:systematic single cell analysis of early human papillomavirus infection

Human papillomaviruses (HPV) are small, non-enveloped DNA viruses that infect cutaneous or mucosal epithelial cells. HPV infections are the prime elicitor of cervical cancer and head-and-neck cancers in humans, representing 5% of all cancers worldwide. Because of its severity, the infectious life cycle of HPV has been studied thoroughly in the last decades and vaccines have been developed for the most prevalent cancer-causing HPV types. However, no antiviral agent therapies are available to treat infected people. Hence, there is a need for novel therapeutic entry points and means to identify them. We established a systematic image-based single cell analysis to quantitatively investigate the early phase of HPV infection by making use of high-throughput microscopy. This allowed us to accurately measure infection kinetics in human cervical carcinoma cells and immortalized keratinocytes, which were exposed to pseudoviruses of the widespread HPV type 16 encoding an EGFP reporter. Next to cell type and cell cycle dependent differences in infection kinetics, we also found alterations in nuclear organization upon HPV PsV infection. In line with other studies, we found a major dependence on cell division for successful infection. This is because the HPV genome can only enter the nucleus when the nuclear envelope dismantles. Since lamins safeguard nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. Using Crispr technology, we established knockout cells (ko) for all three major lamin-encoding genes (LMNA, LMNB1, LMNB2) and challenged them with HPV16 PsV. High-throughput and correlative live/fixed cell microscopy revealed a conspicuous increase in the infection rate of LMNB1 ko cells. The higher nuclear PsV load in these cells was linked to their prolonged mitotic window and extensive nuclear rupture propensity during interphase. Surprisingly, we found no change in EGFP transcript levels. We attribute this to the lower basal number of PML bodies – transcriptional facilitators for HPV – and more compact chromatin status of these cells. While TBK1 signaling was still intact, we found that the DNA sensor cGAS was enriched at the nuclear periphery of LMNB1ko cells. Since the autophagic capacity - target of activated cGAS/STING - was significantly decreased in LMNB1ko cells, we speculate that the continuous activation of cGAS by self-DNA desensitizes these cells to its downstream signaling. From this, we conclude that the loss of lamin B1 increases nuclear perviousness and blunts the autophagic capacity, which primes cells for unrestrained buildup of HPV particles. These findings illuminate novel cell-intrinsic mechanisms that restrain viral infection, but also expose vulnerabilities that may be of relevance to a broad class of DNA viruses that demand nuclear access for their successful reproduction. Furthermore, the tools that we have developed for systematic single cell analysis hold promise for antiviral drug screening as their multiparametric readout provide important additional information beyond sheer infection potential such as cellular heterogeneity or putative side effects.

Publication year:2022

Keywords:Doctoral thesis

Accessibility:Open