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Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV) infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants) to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus) with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na⁺/H⁺ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH.

High-risk human papillomaviruses (HPVs) cause various cancers. While type-specific prophylactic vaccines are available, additional anti-viral strategies are highly desirable. Initial HPV cell entry involves receptor-switching induced by structural capsid modifications. These modifications are initiated by interactions with cellular heparan sulphates (HS), however, their molecular nature and functional consequences remain elusive. Combining virological assays with hydrogen/deuterium exchange mass spectrometry, and atomic force microscopy, we investigate the effect of capsid-HS binding and structural activation. We show how HS-induced structural activation requires a minimal HS-chain length and simultaneous engagement of several binding sites by a single HS molecule. This engagement introduces a pincer-like force that stabilizes the capsid in a conformation with extended capsomer linkers. It results in capsid enlargement and softening, thereby likely facilitating L1 proteolytic cleavage and subsequent L2-externalization, as needed for cell entry. Our data supports the further devising of prophylactic strategies against HPV infections. The study shows that HPV activation requires a minimum glycan length, which enlarges and softens the virus. Using virological assays, AFM, and HDX-MS, the authors propose a model explaining HPV structural activation and its role in infection.

In persistent human papillomavirus (HPV) infection, the viral genome is maintained at a constant copy number, replicates in synchrony with host DNA during S-phase, and is partitioned into daughter cells. The exact method by which HPVs partition to daughter cells is not well understood, and the elucidation of such mechanisms may reveal relevant pharmacological targets to combat persistent HPV infection.

A two-step, high-throughput RNAi silencing screen was used to identify host cell factors required during human papillomavirus type 16 (HPV16) infection. Analysis of validated hits implicated a cluster of mitotic genes and revealed a previously undetermined mechanism for import of the viral DNA (vDNA) into the nucleus. In interphase cells, viruses were endocytosed, routed to the perinuclear area, and uncoated, but the vDNA failed to be imported into the nucleus. Upon nuclear envelope perforation in interphase cells HPV16 infection occured. During mitosis, the vDNA and L2 associated with host cell chromatin on the metaphase plate. Hence, we propose that HPV16 requires nuclear envelope breakdown during mitosis for access of the vDNA to the nucleoplasm. The results accentuate the value of genes found by RNAi screens for investigation of viral infections. The list of cell functions required during HPV16 infection will, moreover, provide a resource for future virus-host cell interaction studies.

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.

Human papillomaviruses (HPVs) utilize an atypical mode of nuclear import during cell entry. Residing in the Golgi apparatus until mitosis onset, a subviral complex composed of the minor capsid protein L2 and viral DNA (L2/vDNA) is imported into the nucleus after nuclear envelope breakdown by associating with mitotic chromatin. In this complex, L2 plays a crucial role in the interactions with cellular factors that enable delivery and ultimately tethering of the viral genome to mitotic chromatin. To date, the cellular proteins facilitating these steps remain unknown. Here, we addressed which cellular proteins may be required for this process. Using label-free mass spectrometry, biochemical assays, microscopy, and functional virological assays, we discovered that L2 engages a hitherto unknown protein complex of Ran-binding protein 10 (RanBP10), karyopherin alpha2 (KPNA2), and dynein light chain DYNLT3 to facilitate transport towards mitotic chromatin. Thus, our study not only identifies novel cellular interactors and mechanism that facilitate a poorly understood step in HPV entry, but also a novel cellular transport complex.

Incoming papillomaviruses (PVs) depend on mitotic nuclear envelope breakdown to gain initial access to the nucleus for viral transcription and replication. In our previous work, we hypothesized that the minor capsid protein L2 of PVs tethers the incoming vDNA to mitotic chromosomes to direct them into the nascent nuclei. To re-evaluate how dynamic L2 recruitment to cellular chromosomes occurs specifically during prometaphase, we developed a quantitative, microscopy-based assay for measuring the degree of chromosome recruitment of L2-EGFP. Analyzing various HPV16 L2 truncation-mutants revealed a central chromosome-binding region (CBR) of 147 amino acids that confers binding to mitotic chromosomes. Specific mutations of conserved motifs (IVAL286AAAA, RR302/5AA, and RTR313EEE) within the CBR interfered with chromosomal binding. Moreover, assembly-competent HPV16 containing the chromosome-binding deficient L2(RTR313EEE) or L2(IVAL286AAAA) were inhibited for infection despite their ability to be transported to intracellular compartments. Since vDNA and L2 were not associated with mitotic chromosomes either, the infectivity was likely impaired by a defect in tethering of the vDNA to mitotic chromosomes. However, L2 mutations that abrogated chromatin association also compromised translocation of L2 across membranes of intracellular organelles. Thus, chromatin recruitment of L2 may in itself be a requirement for successful penetration of the limiting membrane thereby linking both processes mechanistically. Furthermore, we demonstrate that the association of L2 with mitotic chromosomes is conserved among the alpha, beta, gamma, and iota genera of Papillomaviridae. However, different binding patterns point to a certain variance amongst the different genera. Overall, our data suggest a common strategy among various PVs, in which a central region of L2 mediates tethering of vDNA to mitotic chromosomes during cell division thereby coordinating membrane translocation and delivery to daughter nuclei.

Mitosis induces cellular rearrangements like spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Similar to certain retroviruses, nuclear delivery during entry of human papillomavirus (HPV) genomes is facilitated by mitosis, during which minor capsid protein L2 tethers viral DNA to mitotic chromosomes. However, the mechanism of viral genome delivery and tethering to condensed chromosomes is barely understood. It is unclear, which cellular proteins facilitate this process or how this process is regulated. This work identifies crucial phosphorylations on HPV minor capsid protein L2 occurring at mitosis onset. L2’s chromosome binding region (CBR) is sequentially phosphorylated by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, regulates timely delivery of HPV vDNA to mitotic chromatin during mitosis. In summary, our work demonstrates a crucial role of mitotic kinases for nuclear delivery of viral DNA and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis. Human papillomavirus (HPV) coopts mitosis for nuclear entry by tethering the viral DNA to mitotic chromosomes, a process facilitated by the viral minor capsid protein L2. Here, Rizzato et al. show that L2 contains conserved phosphorylation motifs within the chromosome-binding region and provide evidence that host master mitotic kinases CDK1 and PLK1 sequentially mediate phosphorylation of L2 at mitosis onset to allow timely tethering of viral DNA to mitotic chromosomes.

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image‐based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single‐cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome‐wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell‐based screens at this depth reveals widespread RNAi‐induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell‐to‐cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large‐scale RNAi screens are increasingly performed to reach a systems‐level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single‐cell microenvironment. A large set of high‐content RNAi screens investigating mammalian virus infection and multiple cellular activities is analysed to reveal the impact of population context on phenotypic variability and to identify indirect RNAi effects. Synopsis A large set of high‐content RNAi screens investigating mammalian virus infection and multiple cellular activities is analysed to reveal the impact of population context on phenotypic variability and to identify indirect RNAi effects. Cell population context determines phenotypes in RNAi screens of multiple cellular activities (including virus infection, cell size regulation, endocytosis, and lipid homeostasis), which can be accounted for by a combination of novel image analysis and multivariate statistical methods. Accounting for cell population context‐mediated effects strongly changes the reproducibility and consistency of RNAi screens across cell lines as well as of siRNAs targeting the same gene. Such analyses can identify the perturbed regulation of population context dependent cell‐to‐cell variability, a novel perturbation phenotype. Overall, these methods advance the use of large‐scale RNAi screening for a systems‐level understanding of cellular processes.

Glycans are, with nucleic acids, proteins and lipids, one of the four founding structures of cellular life. Due to their non-template synthesis, they are inherently heterogeneous and difficult to study with regards to their structure and function. Since 2016, the research group ViroCarb, funded by the German Research Foundation, has investigated the role of glycans in non-enveloped virus infections with a highly interdisciplinary approach. The core idea was to bring together scientists and students from various disciplines such as structural biology, cell biology, virology and chemistry to advance research by an interdisciplinary means. In 2018, ViroCarb hosted the 1st International Symposium on Glycovirology in Schöntal, Germany, with a similar aim. Scientists from various disciplines gathered to discuss their area of study, present recent findings, establish or strengthen collaborations, and mentor the next generation of glycovirologists through formal presentations and informal discussions. The secluded meeting at the monastery of Schöntal gave ample time for in-depth discussions. On behalf of ViroCarb, this report summarizes the reports and highlights advances in the field.