Kurt E. Gustin

Effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition

Infection of eukaryotic cells with lytic RNA viruses results in extensive interactions of viral gene products with macromolecular pathways of the host, ultimately leading to death of the infected cells. We show here that infection of cells with poliovirus results in the cytoplasmic accumulation of a variety of shuttling and non‐shuttling nuclear proteins that use multiple nuclear import pathways. In vitro nuclear import assays using semi‐permeabilized infected cells confirmed that nuclear import was blocked and demonstrated that docking of nuclear import receptor–cargo complexes at the cytoplasmic face of the nuclear pore complex (NPC) was prevented. Analysis of components of the NPC revealed that two proteins, Nup153 and p62, were proteolyzed during poliovirus infection. These results suggest that the cytoplasmic relocalization of numerous cellular proteins is caused by the inhibition of multiple nuclear import pathways via alterations in NPC composition in poliovirus‐infected cells. Blocking of nuclear import points to a novel strategy by which cytoplasmic RNA viruses can evade host immune defenses, by preventing signal transduction to the nucleus.

Inhibition of Nuclear Import and Alteration of Nuclear Pore Complex Composition by Rhinovirus

ABSTRACT Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.

Differential Targeting of Nuclear Pore Complex Proteins in Poliovirus-Infected Cells

ABSTRACT Poliovirus disrupts nucleocytoplasmic trafficking and results in the cleavage of two nuclear pore complex (NPC) proteins, Nup153 and Nup62. The NPC is a 125-MDa complex composed of multiple copies of 30 different proteins. Here we have extended the analysis of the NPC in infected cells by examining the status of Nup98, an interferon-induced NPC protein with a major role in mRNA export. Our results indicate that Nup98 is targeted for cleavage after infection but that this occurs much more rapidly than it does for Nup153 and Nup62. In addition, we find that cleavage of these NPC proteins displays differential sensitivity to the viral RNA synthesis inhibitor guanidine hydrochloride. Inhibition of nuclear import and relocalization of host nuclear proteins to the cytoplasm were only apparent at later times after infection when all three nucleoporins (Nups) were cleaved. Surprisingly, analysis of the distribution of mRNA in infected cells revealed that proteolysis of Nup98 did not result in an inhibition of mRNA export. Cleavage of Nup98 could be reconstituted by the addition of purified rhinovirus type 2 2Apro to whole-cell lysates prepared from uninfected cells, suggesting that the 2A protease has a role in this process in vivo. These results indicate that poliovirus differentially targets subsets of NPC proteins at early and late times postinfection. In addition, targeting of interferon-inducible NPC proteins, such as Nup98, may be an additional weapon in the arsenal of poliovirus and perhaps other picornaviruses to overcome host defense mechanisms.

Inhibition of nucleo-cytoplasmic trafficking by RNA viruses: targeting the nuclear pore complex

Abstract Analysis of virus–host interactions has revealed a variety of ways in which viruses utilize and/or alter host functions in an effort to facilitate efficient replication. Recent work has suggested that certain RNA viruses that replicate in the cytoplasm disrupt the normal trafficking of cellular RNAs and proteins within the host cell. This review will examine the recent evidence showing that poliovirus and vesicular stomatitis virus (VSV) can inhibit nucleo-cytoplasmic transport within cells. Interestingly, the data indicate that inhibition by both viruses involves targeting components of the nuclear pore complex (NPC). Following this, several possible explanations for why viruses might disrupt nucleo-cytoplasmic transport are discussed. Finally, the possibility that disruption of nucleo-cytoplasmic trafficking may be a more common feature of RNA virus–host interactions than previously thought is examined.

Inhibition of mRNA export and dimerization of interferon regulatory factor 3 by Theiler's virus leader protein.

Theilers murine encephalomyelitis virus (TMEV or Theilers virus) is a neurotropic picornavirus that can persist lifelong in the central nervous system of infected mice, causing a chronic inflammatory demyelinating disease. The leader (L) protein of the virus is an important determinant of viral persistence and has been shown to inhibit transcription of type I interferon (IFN) genes and to cause nucleocytoplasmic redistribution of host proteins. In this study, it was shown that expression of the L protein shuts off synthesis of the reporter proteins green fluorescent protein and firefly luciferase, suggesting that it induces a global shut-off of host protein expression. The L protein did not inhibit transcription or translation of the reporter genes, but blocked cellular mRNA export from the nucleus. This activity correlated with the phosphorylation of nucleoporin 98 (Nup98), an essential component of the nuclear pore complex. In contrast, the data confirmed that the L protein inhibited IFN expression at the transcriptional level, and showed that transcription of other chemokine or cytokine genes was affected by the L protein. This transcriptional inhibition correlated with inhibition of interferon regulatory factor 3 (IRF-3) dimerization. Whether inhibition of IRF-3 dimerization and dysfunction of the nuclear pore complex are related phenomena remains an open question. In vivo, IFN antagonism appears to be an important role of the L protein early in infection, as a virus bearing a mutation in the zinc finger of the L protein replicated as efficiently as the wild-type virus in type I IFN receptor-deficient mice, but had impaired fitness in IFN-competent mice.

Attenuation of the type I interferon response in cells infected with human rhinovirus.

The type I interferon (IFN) response requires the coordinated activation of the latent transcription factors NF-kappaB, IRF-3 and ATF-2 which in turn activate transcription from the IFN-beta promoter. Here we have examined the type I interferon response in rhinovirus type 14-infected A549 cells, with particular emphasis on the status of the transcription factor IRF-3. Our results indicate that although rhinovirus type 14 (RV14) infection induces the activation of NF-kappaB and ATF-2, only very low levels of IFN-beta mRNA are detected. Analysis of ISG54 mRNA levels revealed very little induction of this IRF-3 responsive transcript and suggested that IRF-3 activation might be impaired. Examination of IRF-3 in RV14-infected cells demonstrated only low levels of phosphorylation, a lack of homodimer formation and an absence of nuclear accumulation indicating that this transcription factor is not activated. Inhibition of viral protein synthesis following infection resulted in an increase in IFN-beta mRNA levels indicating that viral gene products prevent induction of this pathway. Collectively, these results indicate that RV14 infection inhibits the host type I interferon response by interfering with IRF-3 activation.

Differential cytotoxicity exhibited by silica nanowires and nanoparticles

Silica nanowires are one-dimensional nanomaterials that are being developed for use in biological systems. Unfortunately, little is known regarding the cytotoxic potential of this type of nanomaterial. Here, using two different human epithelial cell lines we have examined the cytotoxicity of silica nanowires over a broad concentration range. The results indicate that silica nanowires are nontoxic at concentrations below 190 µg/ml but exhibit considerable cytotoxicity at higher concentrations. Examination of the mechanisms responsible for nanowire-induced cytotoxicity indicates that apoptotic pathways are not activated. Instead, cytotoxicity appears to be primarily due to increased necrosis in cells exposed to high concentrations of nanowires. In contrast to what was seen with silica nanowires, analysis of silica nanoparticles revealed very little cytotoxicity even at the highest concentrations tested. These results indicate that structural differences between silica nanomaterials can have dramatic effects on interaction of these nanomaterials with cells.

Proteolytic Cleavage of the Catalytic Subunit of DNA-Dependent Protein Kinase during Poliovirus Infection

ABSTRACT DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PKCS. Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PKCS is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PKCS enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PKCS is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PKCS is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PKCS is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PKCS during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or “immunocryptic,” protein fragments.

Utilization of solid nanomaterials for drug delivery.

Background: Solid nanostructures are versatile platforms for constructing hybrid drug delivery systems that have tremendous potential for improving disease prevention and treatment. The rationale and application of solid nanostructures in the context of drug delivery are explored in this article. Objective: The purpose of this paper is to provide a concise review of the major attributes of solid nanostructures as they relate to drug delivery and to describe the outstanding issues that need to be addressed in order to develop these materials into clinically useful reagents. Methods: The scope of this opinion has been restricted to solid nanostructures, where solid nanostructures are defined as those that are not biodegradable. The opinion has been further limited to the three primary types of nanostructures: nanoparticles, nanowires and nanotubes. Results/conclusion: There is a need for cross-disciplinary training and standardized protocols for developing and evaluating the efficacy of solid nanomaterials.

Rapid detection and quantitation of poliovirus and rhinovirus sequences in viral stocks and infected cells.

Laboratories working with closely related viruses need simple and cost-effective ways to rapidly validate viral stocks, detect contamination and measure the abundance of viral RNA species. Using RT-PCR and specific primers an approach for the specific detection of rhinovirus type 14 (RV14) or poliovirus type 1 (PV1) is presented. It is demonstrated that viral sequences can be amplified directly from viral stocks or from infected cells. In addition, the utility of this protocol for the detection of low levels of contaminating PV1 in RV14 stocks is shown. Further, using quantitative real-time PCR It is shown that this approach can be used for the quantitative analysis of viral RNA and replication kinetics in infected cells. This method should be useful for laboratories working with PV and RV14 and could be adapted easily for use by laboratories working with other rhinovirus and enterovirus serotypes.