The Interplay between Viruses and Host DNA Sensors.

DNA virus infections are often lifelong and can cause serious diseases in their hosts. Their recognition by the sensors of the innate immune system represents the front line of host defence. Understanding the molecular mechanisms of innate immunity responses is an important prerequisite for the design of effective antivirotics. This review focuses on the present state of knowledge surrounding the mechanisms of viral DNA genome sensing and the main induced pathways of innate immunity responses. The studies that have been performed to date indicate that herpesviruses, adenoviruses, and polyomaviruses are sensed by various DNA sensors. In non-immune cells, STING pathways have been shown to be activated by cGAS, IFI16, DDX41, or DNA-PK. The activation of TLR9 has mainly been described in pDCs and in other immune cells. Importantly, studies on herpesviruses have unveiled novel participants (BRCA1, H2B, or DNA-PK) in the IFI16 sensing pathway. Polyomavirus studies have revealed that, in addition to viral DNA, micronuclei are released into the cytosol due to genotoxic stress. Papillomaviruses, HBV, and HIV have been shown to evade DNA sensing by sophisticated intracellular trafficking, unique cell tropism, and viral or cellular protein actions that prevent or block DNA sensing. Further research is required to fully understand the interplay between viruses and DNA sensors.

Nuclear Cytoskeleton in Virus Infection.

The nuclear lamina is the main component of the nuclear cytoskeleton that maintains the integrity of the nucleus. However, it represents a natural barrier for viruses replicating in the cell nucleus. The lamina blocks viruses from being trafficked to the nucleus for replication, but it also impedes the nuclear egress of the progeny of viral particles. Thus, viruses have evolved mechanisms to overcome this obstacle. Large viruses induce the assembly of multiprotein complexes that are anchored to the inner nuclear membrane. Important components of these complexes are the viral and cellular kinases phosphorylating the lamina and promoting its disaggregation, therefore allowing virus egress. Small viruses also use cellular kinases to induce lamina phosphorylation and the subsequent disruption in order to facilitate the import of viral particles during the early stages of infection or during their nuclear egress. Another component of the nuclear cytoskeleton, nuclear actin, is exploited by viruses for the intranuclear movement of their particles from the replication sites to the nuclear periphery. This study focuses on exploitation of the nuclear cytoskeleton by viruses, although this is just the beginning for many viruses, and promises to reveal the mechanisms and dynamic of physiological and pathological processes in the nucleus.

The Major Capsid Protein, VP1, of the Mouse Polyomavirus Stimulates the Activity of Tubulin Acetyltransferase 1 by Microtubule Stabilization.

Viruses have evolved mechanisms to manipulate microtubules (MTs) for the efficient realization of their replication programs. Studying the mechanisms of replication of mouse polyomavirus (MPyV), we observed previously that in the late phase of infection, a considerable amount of the main structural protein, VP1, remains in the cytoplasm associated with hyperacetylated microtubules. VP1-microtubule interactions resulted in blocking the cell cycle in the G2/M phase. We are interested in the mechanism leading to microtubule hyperacetylation and stabilization and the roles of tubulin acetyltransferase 1 (αTAT1) and deacetylase histone deacetylase 6 (HDAC6) and VP1 in this mechanism. Therefore, HDAC6 inhibition assays, αTAT1 knock out cell infections, in situ cell fractionation, and confocal and TIRF microscopy were used. The experiments revealed that the direct interaction of isolated microtubules and VP1 results in MT stabilization and a restriction of their dynamics. VP1 leads to an increase in polymerized tubulin in cells, thus favoring αTAT1 activity. The acetylation status of MTs did not affect MPyV infection. However, the stabilization of MTs by VP1 in the late phase of infection may compensate for the previously described cytoskeleton destabilization by MPyV early gene products and is important for the observed inhibition of the G2→M transition of infected cells to prolong the S phase.

Microtubules in Polyomavirus Infection.

Microtubules, part of the cytoskeleton, are indispensable for intracellular movement, cell division, and maintaining cell shape and polarity. In addition, microtubules play an important role in viral infection. In this review, we summarize the role of the microtubules' network during polyomavirus infection. Polyomaviruses usurp microtubules and their motors to travel via early and late acidic endosomes to the endoplasmic reticulum. As shown for SV40, kinesin-1 and microtubules are engaged in the release of partially disassembled virus from the endoplasmic reticulum to the cytosol, and dynein apparently assists in the further disassembly of virions prior to their translocation to the cell nucleus-the place of their replication. Polyomavirus gene products affect the regulation of microtubule dynamics. Early T antigens destabilize microtubules and cause aberrant mitosis. The role of these activities in tumorigenesis has been documented. However, its importance for productive infection remains elusive. On the other hand, in the late phase of infection, the major capsid protein, VP1, of the mouse polyomavirus, counteracts T-antigen-induced destabilization. It physically binds microtubules and stabilizes them. The interaction results in the G2/M block of the cell cycle and prolonged S phase, which is apparently required for successful completion of the viral replication cycle.

Cell-Based Reporter System for High-Throughput Screening of MicroRNA Pathway Inhibitors and Its Limitations.

MicroRNAs (miRNAs) are small RNAs repressing gene expression. They contribute to many physiological processes and pathologies. Consequently, strategies for manipulation of the miRNA pathway are of interest as they could provide tools for experimental or therapeutic interventions. One of such tools could be small chemical compounds identified through high-throughput screening (HTS) with reporter assays. While a number of chemical compounds have been identified in such high-throughput screens, their application potential remains elusive. Here, we report our experience with cell-based HTS of a library of 12,816 chemical compounds to identify miRNA pathway modulators. We used human HeLa and mouse NIH 3T3 cell lines with stably integrated or transiently expressed luciferase reporters repressed by endogenous miR-30 and let-7 miRNAs and identified 163 putative miRNA inhibitors. We report that compounds relieving miRNA-mediated repression via stress induction are infrequent; we have found only two compounds that reproducibly induced stress granules and relieved miRNA-targeted reporter repression. However, we have found that this assay type readily yields non-specific (miRNA-independent) stimulators of luciferase reporter activity. Furthermore, our data provide partial support for previously published miRNA pathway modulators; the most notable intersections were found among anthracyclines, dopamine derivatives, flavones, and stilbenes. Altogether, our results underscore the importance of appropriate negative controls in development of small compound inhibitors of the miRNA pathway. This particularly concerns validation strategies, which would greatly profit from assays that fundamentally differ from the routinely employed miRNA-targeted reporter assays.