Martin Fraiberk

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.

VP1, the major structural protein of the mouse polyomavirus (MPyV), is the major architectural component of the viral capsid. Its pentamers are able to self-assemble into capsid-like particles and to non-specifically bind DNA. Surface loops of the protein interact with sialic acid of ganglioside receptors. Although the replication cycle of the virus, including virion morphogenesis, proceeds in the cell nucleus, a substantial fraction of the protein is detected in the cytoplasm of late-phase MPyV-infected cells. In this work, we detected VP1 mainly in the cytoplasm of mammalian cells transfected with plasmid expressing VP1. In the cytoplasm, VP1-bound microtubules, including the mitotic spindle, and the interaction of VP1 with microtubules resulted in cell cycle block at the G2/M phase. Furthermore, in the late phase of MPyV infection and in cells expressing VP1, microtubules were found to be hyperacetylated. We then sought to understand how VP1 interacts with microtubules. Dynein is not responsible for the VP1–microtubule association, as neither overexpression of p53/dynamitin nor treatment with ciliobrevin-D (an inhibitor of dynein activity) prevented binding of VP1 to microtubules. A pull-down assay for VP1-interacting proteins identified the heat shock protein 90 (Hsp90) chaperone, and Hsp90 was also detected in the VP1–microtubule complexes. Although Hsp90 is known to be associated with acetylated microtubules, it does not mediate the interaction between VP1 and microtubules. Our study provides insight into the role of the major structural protein in MPyV replication, indicating that VP1 is a multifunctional protein that participates in the regulation of cell cycle progression in MPyV-infected cells.

A simple nanoprecipitation method was used for preparation of stable photoactive polystyrene nanoparticles (NPs, diameter 30 ± 10 nm) from sulfonated electrospun polystyrene nanofiber membranes with encapsulated 5,10,15,20-tetraphenylporphyrin (TPP) or platinum octaethylporphyrin (Pt-OEP). The NPs prepared with TPP have strong antibacterial and antiviral properties and can be applied to the photooxidation of external substrates based on photogenerated singlet oxygen. In contrast to nanofiber membranes, which have limited photooxidation ability near the surface, NPs are able to travel toward target species/structures. NPs with Pt-OEP were used for oxygen sensing in aqueous media, and they presented strong linear responses to a broad range of oxygen concentrations. The nanofiber membranes can be applied not only as a source of NPs but also as an effective filter for their removal from solution.

JC and BK polyomaviruses (JCV and BKV) infect humans and can cause severe illnesses in immunocompromised patients. Merkel cell polyomavirus (MCPyV) can be found in skin carcinomas. In this study, we assessed the occurrence of serum antibodies against MCPyV, BKV, and JCV polyomaviruses in a healthy population of the Czech Republic. Serum samples from 991 healthy individuals (age range: 6–64 years) were examined by enzyme-linked immunoassay (ELISA) using virus-like particles (VLPs) based on the major VP1 capsid proteins of these viruses. Overall, serum antibodies against MCPyV, JCV, and BKV were found in 63%, 57%, and 69%, respectively, of this population. For all three viruses, these rates were associated with age; the occurrence of antibodies against MCPyV and JCV was highest for those older than 59 years, while the occurrence of antibodies against BKV was highest in those aged 10–19 years and 20–29 years. This is the first large study to determine the seroprevalence rates for BKV, JCV, and MCPyV polyomaviruses in the general Czech Republic population. J. Med. Virol. 86:1560–1568, 2014.

Lamins are the best characterized cytoskeletal components of the cell nucleus that help to maintain the nuclear shape and participate in diverse nuclear processes including replication or transcription. Nuclear actin is now widely accepted to be another cytoskeletal protein present in the nucleus that fulfills important functions in the gene expression. Some viruses replicating in the nucleus evolved the ability to interact with and probably utilize nuclear actin for their replication, e.g., for the assembly and transport of capsids or mRNA export. On the other hand, lamins play a role in the propagation of other viruses since nuclear lamina may represent a barrier for virions entering or escaping the nucleus. This review will summarize the current knowledge about the roles of nuclear actin and lamins in viral infections.