Hans J. Eggers

STUDIES ON THE MECHANISM OF POLIOVIRUS-INDUCED CELL DAMAGE. I. THE RELATION BETWEEN POLIOVIRUS,-INDUCED METABOLIC AND MORPHOLOGICAL ALTERATIONS IN CULTURED CELLS.

Abstract Infection with the attenuated strain of poliovirus type 2 depressed cellular RNA and protein synthesis in parallel both in human embryonic lung cells (diploid) and in ERK cells. In HeLa cells protein synthesis was depressed more rapidly than RNA synthesis. In all experiments an input multiplicity of 100 PFU/cell was used. Guanidine, at a concentration of 1110 μ M , completely prevented production of new virus in human embryonic lung cells, but had only a slight delaying effect on the virus-induced inhibitions of RNA and protein synthesis. Guanidine did markedly delay the development of morphological changes in infected cells. Twenty-one hours after infection most of the drug-treated cells appeared morphologically normal, although the rates of cellular RNA and protein synthesis in these cells were as much depressed as in the untreated cells at 5–6 hours, at which time the untreated cells were already undergoing advanced morphological changes. The simplest hypothesis to explain these findings is that the virus-induced metabolic depressions are not the sole, immediate, and direct cause of the morphological changes, which in untreated infected cells accompany, or follow shortly after, the active replication of virus. However, the eventual degeneration of guanidine-treated infected cells, which occurs in the absence of virus multiplication, may indeed be due to the virus-induced inhibitions in cellular metabolism.

Inhibition of uncoating of poliovirus by arildone, a new antiviral drug

Abstract The antiviral effects of a new drug, arildone (4-[6-(2-chloro-4-methoxyphenoxy)hexyl]-3,5-heptanedione, on poliovirus type 2 replication and host cell functions are described. Arildone inhibits poliovirus replication at a minimal inhibitory concentration (MIC) of 0.2 μM, while transport of radioactively labeled precursors and synthesis of DNA, RNA, and protein in uninfected HeLa cells are not inhibited. This drug is not virucidal and does not interfere with adsorption or penetration. Arildone inhibits uncoating of poliovirus and thereby prevents virus-induced shutoff of host cell protein synthesis. The possible mechanisms by which arildone interacts with the poliovirus icosahedral capsid to prevent uncoating are discussed.

Detection of adenovirus nucleic acid sequences in human tonsils in the absence of infectious virus

Human tonsillar and adenoid tissues from surgical specimens and cell cultures established therefrom were screened for adenovirus type 2 (Ad2) sequences by in situ hybridization. For labeling we have utilized biotinylated DNA probes. We report detection of adenoviral sequences after hybridization with adenovirus type 2 DNA probes in tissues as well as in cell cultures from specimens without any signs of infectious virus even after long-term cultivation. In the infected tonsils only some of the cells appear to carry viral sequences. In conclusion, truly latent adenovirus infections in man seem to occur.

On the Mechanism of Selective Inhibition of Enterovirus Multiplication by 2-(α-Hydroxybenzyl)-Benzimidazole.

Abstract The mechanism of action of 2-(α-hydroxybenzyl)-benzimidazole (HBB), a selective inhibitor of enterovirus multiplication, was investigated. At a concentration of 219 μM or 49 μg/ml, HBB had no effect on adsorption or penetration of ECHO 12 virus, but inhibited a process which commenced during the second half of the latent period and continued into the second half of the exponential increase phase of virus reproduction. Given during the exponential increase in virus, HBB caused complete cessation of further virus production within 30–45 minutes of its addition. A possible biochemical basis for these findings was provided by the demonstration that HBB inhibits the synthesis of infective viral RNA of ECHO 12 virus and that the bulk of infective RNA of this virus is synthesized during the exponential increase in virus. HBB also inhibited the synthesis of infective viral RNA of Coxsackie A9 and Coxsackie B4 viruses. With these viruses the dose-effect curves describing inhibition of viral RNA synthesis followed closely those depicting inhibition of complete virus. In ECHO 12 virus-infected cells, however, a substantial amount of infective viral RNA was synthesized in the presence of 65 μM HBB, although no increase in infective virus was observed. HBB had no direct irreversible inactivating effect on the infectivity of viral RNA, nor did it interfere with its uptake by cells. In HBB-treated cells, in which replication of infective viral RNA was inhibited, production of complement-fixing antigen was also inhibited. Concentrations of HBB sufficient to inhibit production of infective viral RNA had no effect on host cell RNA synthesis as studied by uptake of radioactive precursors into RNA. Furthermore, the rate of host cell multiplication was unaffected by HBB.

Differences in the Selective Virus Inhibitory Action of 2-(α-Hydroxybenzyl)-Benzimidazole and Guanidine HCl.

Abstract Both 2-(α-hydroxybenzyl)-benzimidazole (HBB) and guanidine hydrochloride inhibit the multiplication of enteroviruses selectively. The present studies have revealed differences in the virus inhibitory action of HBB and guanidine. First, differences in the virus inhibitory spectra of these compounds were found; second, acquired resistance to one compound was associated with only a slight decrease in sensitivity to the other. Combined treatment with both HBB and guanidine had a greater viral suppressive effect than treatment with either compound alone. This was expected because there was little cross resistance between HBB and guanidine, and therefore emergence of resistant variants would have a lower probability in the presence of both compounds. On the basis of susceptibility to HBB and guanidine, the small, RNA-containing, ether-resistant viruses (“naniviruses,” Andrewes et al. , 1961) fall into three groups: (I) The guanidine- and HBB-susceptible group includes polio, Coxsackie B, and the great majority of ECHO viruses; (II) The guanidine-susceptible, HBB-insusceptible group includes Coxsackie A viruses and the HGP strain of Salisbury common cold virus; (III) The guanidine- and HBB-insusceptible group includes ECHO 22, 23, and 28, the B632 strain of Salisbury common cold virus. Coxsackie A9 virus, which is an atypical Coxsackie A virus, is guanidine- and HBB-susceptible and therefore falls into the first group.

Molecular cloning and sequence determination of the complete genome of the virulent echovirus 9 strain Barty

As part of a study of the molecular basis of pathogenicity of echovirus 9, the complete nucleotide sequence of the mouse-virulent echovirus 9 strain Barty was determined. Excluding the poly(A) tail, the complete RNA genome is composed of 7451 bases. The postulated open reading frame extends from nucleotide (nt) 741 to 7349 and predicts a polyprotein of 2203 amino acids (aa). As compared with the sequence of the echovirus 9 prototype strain Hill, which is apathogenic for newborn mice, 1492 nt are exchanged, leading to 9% divergence of the deduced amino acid sequence. The foremost difference between both strains is located at the C-terminus of the capsid protein VP1. In the case of strain Barty, an additional 10 aa fragment, including an RGD motif, is inserted.

STUDIES ON THE MECHANISM OF POLIOVIRUS-INDUCED CELL DAMAGE. II. THE RELATION BETWEEN POLIOVIRUS GROWTH AND VIRUS-INDUCED MORPHOLOGICAL CHANGES IN CELLS.

Abstract When human embryonic lung cells (diploid) were infected at an input multiplicity of 100:1 with the attenuated strain of poliovirus type 2, virus-induced morphological changes first became apparent between 4 and 4.5 hours after infection, and at 7 hours after infection 97% of the cells were affected. Thus, in this system, virus-induced cytopathological changes followed closely upon virus multiplication. When guanidine, a specific inhibitor of poliovirus multiplication, was added as late as 2 hours after virus inoculation in a single cycle of infection, virus multiplication and virus-induced cytopathological changes were still completely inhibited at 8.5 hours. However, the addition of guanidine 3.5 hours after infection no longer protected the cells from virus-induced morphological changes, although virus reproduction was largely prevented. Puromycin, at 200 μ M , caused no toxic changes in cells in 8.5 hours, and it also prevented viral multiplication and virus-induced morphological changes when added at 2 hours in a single cycle of poliovirus multiplication. However, puromycin, unlike guanidine, was capable of preventing the major part of virus-induced cytopathological changes even when added 3.5 hours after infection. All the available data are compatible with the hypothesis that virus-induced morphological changes in cells are caused by virus coat proteins whose synthesis begins toward the end of the latent period. However the possibility cannot be excluded that during viral replication some other virus-directed protein is made which is responsible for the morphological changes in cells.

Integrin alpha(v)beta3 (vitronectin receptor) is a candidate receptor for the virulent echovirus 9 strain Barty.

The enterovirus echovirus 9 strain Barty (E9/Barty) is pathogenic for newborn mice as well as for humans. In contrast to the apathogenic prototype strain Hill, strain Barty encodes an RGD motif in the C-terminal part of the structural protein VP1. Data are presented that show that E9/Barty binds its target cells via contact of the RGD motif to the αvβ3 integrin (vitronectin receptor), whereas prototype Hill uses a different, still unidentified receptor site. Furthermore, virus titres of murine muscle tissue were compared after infection of newborn and 1-, 2-, 3- and 12-week-old mice. The replication capacity of the virus decreased dramatically with age of the infected mice. Since E9/Barty does not replicate or replicates only poorly in mice older than about 5 days, and expression of the vitronectin receptor is reported to be down-regulated in striated muscle tissue during development, it is suggested that susceptibility of mice to this echovirus infection is controlled by the availability of αvβ3 integrin.

Heterogeneity of Coxsackie B4 Virus: Two Kinds of Particles which differ in Antibody Sensitivity, Growth Rate, and Plaque Size.

Abstract Two kinds of virus particles which are antigenically similar but which differ in several biological properties have been isolated from a strain of Coxsackie B4 virus. These virus particles have been designated as and ai. The as particles are sensitive to specific antibody and to an inhibitor present in agar, and form small plaques; the ai particles are relatively insensitive to antibody, insensitive to the agar inhibitor, and form large plaques. The addition of DEAE dextran to agar abolishes the inhibition of as particles by the agar inhibitor and results in the formation of large plaques by these particles. The as virus adsorbs, multiplies, is released more rapidly, and causes more complete cell destruction, than the ai virus. The properties which distinguish the two kinds of virus were stable on passage. Early passages of Coxsackie B4 strains consisted predominantly of virus particles which formed plaques resembling those formed by ai virus; whereas the Powers strain, after many passages, consisted predominantly of as virus, presumably because of the rapid growth rate of as particles. The presence of two kinds of particles in varying proportions in Coxsackie B4 virus strains provides an explanation for some of the problems encountered in previous immunological studies with these strains.

Selective inhibition of uncoating of echovirus 12 by rhodanine A study on early virus-cell interactions

Abstract 2-Thio-4-oxothiazolidine, rhodanine, a selective inhibitor of echovirus 12 multiplication, appears to act by blocking uncoating of the virus. Uncoating is measured as the acquisition of light resistance of neutral red-sensitized virus (NR-virus) in infected cells. Rhodanine does not affect virus adsorption and penetration nor viral RNA-initiated multiplication of echovirus 12. On the basis of the data presented it does not appear necessary to invoke any mechanism other than inhibition of virus uncoating to explain the inhibition of echovirus 12 multiplication. From rhodanine-treated cells echovirus 12 can be quantitatively recovered in infective form even 4 hr postinfection. Rhodanine-inhibited virus cannot be rescued by superinfection with the rhodanine-insusceptible poliovirus 1 nor with a rhodanine-resistant echovirus 12 mutant isolated in the course of the present work. De nova protein synthesis is not required for uncoating of echovirus 12 to occur. Rhodanine, at virus-inhibitory concentrations, protects the echo 12 virion from alkaline degradation, whereas poliovirus is not stabilized by the compound. The use of NR-virus permits direct measurement without extrapolations of the time of eclipse of echovirus 12 during its multiplication cycle.