Pyridine hydroxamic acids are specific anti-HCV agents affecting HDAC6.

Recently we reported benzohydroxamic acids (BHAs) as potent and selective inhibitors of hepatitis C virus (HCV) replicon propagation. In this work 12 pyridine hydroxamic acids (PHAs) were synthesized and tested in full-genome replicon assay. It was found that PHAs possessed very similar anti-HCV properties compared to BHAs. Both classes of hydroxamic acids caused hyperacetylation of α-tubulin pointing to inhibition of histone deacetylase 6 (HDAC6) as part of their antiviral activity. The tested compounds did not inhibit the growth of poliovirus, displaying high selectivity against HCV.

Benzohydroxamic acids as potent and selective anti-HCV agents.

A diverse collection of 40 derivatives of benzohydroxamic acid (BHAs) of various structural groups were synthesized and tested against hepatitis C virus (HCV) in full-genome replicon assay. Some of these compounds demonstrated an exceptional activity, suppressing viral replication at sub-micromolar concentrations. The compounds were inactive against key viral enzymes NS3, and NS5B in vitro assays, suggesting host cell inhibition target(s). The testing results were consistent with metal coordination by the BHAs hydroxamic group in complex with a target(s). Remarkably, this class of compounds did not suppress poliomyelitis virus (PV) propagation in RD cells indicating a specific antiviral activity of BHAs against HCV.

Interactions between viral and prokaryotic pathogens in a mixed infection with cardiovirus and mycoplasma.

In the natural environment, animal and plant viruses often share ecological niches with microorganisms, but the interactions between these pathogens, although potentially having important implications, are poorly investigated. The present report demonstrates, in a model system, profound mutual effects of mycoplasma and cardioviruses in animal cell cultures. In contrast to mycoplasma-free cells, cultures contaminated with Mycoplasma hyorhinis responded to infection with encephalomyocarditis virus (EMCV), a picornavirus, but not with poliovirus (also a picornavirus), with a strong activation of a DNase(s), as evidenced by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) immunofluorescence assay and electrophoretic analysis of host DNA. This degradation was reminiscent of that observed upon apoptosis but was caspase independent, judging by the failure of the specific pan-caspase inhibitor Q-VD-OPh to prevent it. The electrophoretic mobility of the enzyme responsible for DNA degradation and dependence of its activity on ionic conditions strongly suggested that it was represented by a DNase(s) of mycoplasma origin. In cells not infected with EMCV, the relevant DNase was dormant. The possibility is discussed that activation of the mycoplasma DNase might be linked to a relatively early increase in permeability of plasma membrane of the infected cells caused by EMCV. This type of unanticipated virus-mycoplasma "cooperation" may exemplify the complexity of pathogen-host interactions under conditions when viruses and microorganisms are infecting the same host. In the course of the present study, it was also demonstrated that pan-caspase inhibitor zVAD(OMe).fmk strongly suppressed cardiovirus polyprotein processing, illustrating an additional pitfall in investigations of viral effects on the apoptotic system of host cells.

Antiapoptotic activity of the cardiovirus leader protein, a viral "security" protein.

Apoptosis is a common antiviral defensive mechanism that potentially limits viral reproduction and spread. Many viruses possess apoptosis-suppressing tools. Here, we show that the productive infection of HeLa cells with encephalomyocarditis virus (a cardiovirus) was not accompanied by full-fledged apoptosis (although the activation of caspases was detected late in infection) but rather elicited a strong antiapoptotic state, as evidenced by the resistance of infected cells to viral and nonviral apoptosis inducers. The development of the antiapoptotic state appeared to depend on a function(s) of the viral leader (L) protein, since its mutational inactivation resulted in the efflux of cytochrome c from mitochondria, the early activation of caspases, and the appearance of morphological and biochemical signs of apoptosis in a significant proportion of infected cells. Infection with both wild-type and L-deficient viruses induced the fragmentation of mitochondria, which in the former case was not accompanied with cytochrome c efflux. Although the exact nature of the antiapoptotic function(s) of cardioviruses remains obscure, our results suggested that it includes previously undescribed mechanisms operating upstream and possibly downstream of the mitochondrial level, and that L is involved in the control of these mechanisms. We propose that cardiovirus L belongs to a class of viral proteins, dubbed here security proteins, whose roles consist solely, or largely, in counteracting host antidefenses. Unrelated L proteins of other picornaviruses as well as their highly variable 2A proteins also may be security proteins. These proteins appear to be independent acquisitions in the evolution of picornaviruses, implying multiple cases of functional (though not structural) convergence.

Variability in apoptotic response to poliovirus infection.

In several cell types, poliovirus activates the apoptotic program, implementation of which is suppressed by viral antiapoptotic functions. In such cells, productive infection leads to a necrotic cytopathic effect (CPE), while abortive reproduction, associated with inadequate viral antiapoptotic functions, results in apoptosis. Here, we describe two other types of cell response to poliovirus infection. Murine L20B cells expressing human poliovirus receptor responded to the infection by both CPE and apoptosis concurrently. Interruption of productive infection decreased rather than increased the proportion of apoptotic cells. Productive infection was accompanied by the early efflux of cytochrome c from the mitochondria in a proportion of cells and by activation of DEVD-specific caspases. Inactivation of caspase-9 resulted in a marked, but incomplete, prevention of the apoptotic response of these cells to viral infection. Thus, the poliovirus-triggered apoptotic program in L20B cells was not completely suppressed by the viral antiapoptotic functions. In contrast, human rhabdomyosarcoma RD cells did not develop appreciable apoptosis during productive or abortive infection, exhibiting inefficient efflux of cytochrome c from mitochondria and no marked activation of DEVD-specific caspases. The cells were also refractory to several nonviral apoptosis inducers. Nevertheless, typical caspase-dependent signs of apoptosis in a proportion of RD cells were observed after cessation of viral reproduction. Such "late" apoptosis was also observed in productively infected HeLa cells. In addition, a tiny proportion of all studied cells were TUNEL positive even in the presence of a caspase inhibitor. Degradation of DNA in such cells appeared to be a postmortem phenomenon. Biological relevance of variable host responses to viral infection is discussed.

The major apoptotic pathway activated and suppressed by poliovirus.

Cells respond to poliovirus infection by switching on the apoptotic program, implementation of which is usually suppressed by viral antiapoptotic functions. We show here that poliovirus infection of HeLa cells or derivatives of MCF-7 cells was accompanied by the efflux of cytochrome c from mitochondria. This efflux occurred during both abortive infection (e.g., interrupted by guanidine-HCl and ending with apoptosis) and productive infection (leading to cytopathic effect). The former type of infection, but not the latter, was accompanied by truncation of the proapoptotic protein Bid. The virus-triggered cytochrome c efflux was suppressed by overexpression of Bcl-2. Both abortive and productive infections also resulted in a decreased level of procaspase-9, as revealed by Western blotting. In the former case, this decrease was accompanied by the accumulation of a protein with the electrophoretic mobility of active caspase-9. In contrast, in the productively infected cells, the latter protein was absent but caspase-9-related polypeptides with altered mobility could be detected. Both caspase-9 and caspase-3 were shown to be essential for the development of such hallmarks of virus-induced apoptosis as chromatin condensation, DNA degradation, and nuclear fragmentation. These and some other results suggest the following scenario. Poliovirus infection activates the apoptotic pathway, involving mitochondrial damage, cytochrome c efflux, and consecutive activation of caspase-9 and caspase-3. The apoptotic signal appears to be amplified by a loop which includes secondary processing of Bid. The implementation of the apoptotic program in productively infected cells may be suppressed, however, by the viral antiapoptotic functions, which act at a step(s) downstream of the cytochrome c efflux. The suppression appears to be caused, at least in part, by aberrant processing and degradation of procaspase-9.