Lyudmila I. Romanova

Competing Death Programs in Poliovirus-Infected Cells: Commitment Switch in the Middle of the Infectious Cycle

ABSTRACT Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first ∼2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors.

The Major Apoptotic Pathway Activated and Suppressed by Poliovirus

ABSTRACT 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.

Biochemical evidence for intertypic genetic recombination of polioviruses

Two basically different kinds of genetic recombination are known to occur in RNA-containing viruses. Firstly, there is a high-frequency recombination among viruses with segmented RNA genomes, such as reoviruses [ 1 J or orthomyxoviruses [2]. This type of genetic recombination is due to physical exchange of separate RNA molecules (‘segments’) between parental genomes and, therefore, may be designated ‘genetic reassortment’ [3]. Secondly, relatively low-frequency recombination is reported to exist in picornaviruses, whose genome is represented by a single RNA molecule [4,5]. The nature of the latter type of recombination is poorly understood, but it is believed to be true inheritance in a single RNA molecule of genetic properties of both parents [4,5]. Evidence for this interpretation has only been provided by purely genetic techniques and direct biochemical proof that recombinants do acquire genetic information from more than one parent is lacking. Experimental solution of this problem became more feasible when recombinants between different poliovirus serotypes were obtained (E. A. T., M. S. K., in preparation). Well-established differences between the genomes of different types of polioviruses ([6-lo], L. I. R., E. A. T., V. I. A., submitted) make possible a direct comparison of recombinants with each of their parents. Here we report results showing that some polypeptides coded for by the genome of a type l/type 3 poliovirus recombinant are indistinguishable from the corresponding polypeptides of the type 1 parent, whereas some other recombinant-specific polypeptides appear to be of the type 3 origin. These results are the first direct biochemical evidence for intermolecular recombination between RNA genomes.

Antiapoptotic Activity of the Cardiovirus Leader Protein, a Viral “Security” Protein

ABSTRACT 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.

Final checkpoint in the drug-promoted and poliovirus-promoted apoptosis is under post-translational control by growth factors.

The treatment of HeLa subline (HeLa‐B) cells with cycloheximide or Actinomycin D resulted in a rapid (∼︁ 1.5 h and ∼︁ 2.5 h, respectively) development of morphological and biochemical signs of apoptosis. The addition of fetal bovine serum to the cycloheximide‐treated or Actinomycin D‐treated cells suppressed the apoptotic reaction, as evidenced by the postponement of the DNA fragmentation for at least 9 and 5 h, respectively. A similar suppressive effect was observed upon the serum addition to cells undergoing abortive infection with poliovirus, which died of apoptosis in the absence of the serum. The serum appeared to exert its anti‐apoptotic effect without any appreciable lag and even immediately blocked further progress of ongoing DNA fragmentation. The epidermal growth factor also suppressed, although less efficiently and more transiently, the apoptotic reaction promoted by the metabolic inhibitors. It is concluded that growth factors may affect, without modulating either transcription or translation, the balance of pro‐apoptotic and anti‐apoptotic activities at a final checkpoint, just preceding the irreversible effector step of apoptosis.

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.

Circularity and cross-linking in preparations of replicative form of encephalomyocarditis virus RNA

Abstract Electron microscopy of the purified replicative form RNA of encephalomyocarditis virus revealed three categories of molecules: linear (2.33 ± 0.21 μm long), circular (2.42 ± 0.13 μm) and compact entities of unknown structure. The circular molecules were either in an extended form or had several superhelical twists. At least two components could be discerned in preparations of replicative form RNA by analytical velocity band centrifugation in 3 m -CsCl; the uncorrected sedimentation coefficients of these components were 24.7 s and 17.4 s, respectively. The effect of an intercalating dye, ethidium bromide, on the sedimentation rate of these components was studied. The plot of the sedimentation coefficient of the fastest component versus ethidium bromide concentration was shown to be qualitatively similar to that reported for supercoiled covalently closed circular DNA, i.e. it had a minimum at a certain concentration of ethidium bromide. This fact suggests that a portion of the replicative form RNA is probably represented by closed circular double-stranded molecules. Thermal denaturation-renaturation experiments suggested that both chains, at least in a portion of the isolated replicative form RNA molecules, were connected to each other in such a way that complete separation could not be achieved under the denaturing conditions. The binding between chains appeared to be due, at least to a large extent, to some kind of cross-linking, rather than to the existence of covalently closed circular molecules, since the fragmented linear molecules (fragmented by hydrodynamic shearing) possessed essentially the same ability to renature upon rapid cooling as did non-fragmented molecules of the replicative form RNA.

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.

Interconversion of linear and circular forms of double-stranded RNA of encephalomyocarditis virus.

Abstract Preparations of encephalomyocarditis (EMC) virus replicative form (RF) RNA, when spread for electron microscopy under nondenaturing conditions, contained roughly 10% of circular molecules. When spread under partially denaturing conditions, these preparations contained very few, if any, circles. The circular molecules reappeared immediately after transfer of partially melted RF RNA preparations to nondenaturing conditions. The reappearance of circles was prevented in the presence of poly(A), poly(U), poly(I), or, to a lesser extent, poly(G) · poly(C) but it was not inhibited in the presence of poly(G) or poly(C). It is concluded that the circles of EMC virus RF RNA are formed, at least predominantly, by a noncovalent linkage between the ends of the molecule.

Defect in poliovirus maturation under hypotonic conditions

Abstract Reduction of osmotic pressure in the medium in which poliovirus-infected cells are incubated results in inhibition of the formation of infectious virions and virus-specific particles sedimenting with the rate of mature virions. Under hypotonic conditions, however, synthesis of “soluble” virus-specific proteins and 74 S particles (procapsids) is observed. The 74 S particles formed under hypotonic and isotonic conditions do not differ from each other by sedimentation in sucrose density gradients, by buoyant density in CsCl, or by the spectrum of polypeptides detected by electrophoresis in polyacrylamide gel. No significant differences have been found in the set of polypeptides in the fraction of “soluble” proteins in extracts of productively or abortively infected cells. Taking into consideration earlier evidence that infectious poliovirus RNA is formed under hypotonic conditions (Tolskaya et al. , 1966), a conclusion is drawn that under these conditions maturation of virions from the available precursors is disturbed.