Inhibition of type 5 adenovirus infectivity by periodate oxidation.

Periodate oxidation of purified type 5 Adenovirus (Ad5) led to a mean loss of infectivity of 6.84 logs. There were no significant differences in adsorption and penetration between oxidized and mock-oxidized virus. However, after infection with oxidized virus, no synthesis of viral structural proteins could be detected and a 78.5% inhibition of viral DNA synthesis was observed. Labelling experiments performed by treating oxidized and mock-oxidized virus with tritiated sodium borohydride revealed that the fiber glycoprotein was one of the proteins labelled in oxidized virus whereas no labelled proteins were detected in non oxidized virus. In addition, it was found that one mol of formaldehyde generated during oxidation of sugar residues was bound per 500 base pairs in oxidized virus. One consequence of this in situ generation of formaldehyde is the formation of DNA-protein crosslinks. The DNA so crosslinked showed different patterns of restriction fragments with endonucleases such as Hpa I, Hind III and Kpn I but not with Xho I.

Inactivation of human cytomegalovirus by sodium periodate oxidation.

Human cytomegalovirus (HCMV), oxidized by sodium periodate (NaIO4), is incapable of giving rise to viral progeny in cell culture. At a NaIO4 concentration as low as 5 mM, there is a loss of at least 6 logs of viral infectivity which occurs very rapidly (less than 5 min). Further, the inactivation is a first-order reaction depending on the periodate concentration. Adsorption to the cell surface, penetration into cells, and penetration of the viral DNA into cell nuclei were found to occur identically in mock oxidized and oxidized HCMV. Since the carbohydrate moiety of viral glycoproteins was the target of periodate attack, these observations strongly suggest that the structural integrity of the sugar residues is not a prerequisite for adsorption and penetration. Nevertheless, no evidence for viral DNA or protein synthesis was detected in cells infected with oxidized virus, and even after 3 weeks in culture, no cytopathic effect was observed.

Contribution of 4-methylthio-2-oxobutanoate and its transaminase to the growth of methionine-dependent cells in culture. Effect of transaminase inhibitors.

The growth in culture of methionine-dependent transformed cells of human, rat and mouse origin was arrested in the absence of L-methionine (Met) but took place in the presence of 4-methylthio-2-oxobutanoic acid (MTOB), the keto acid of Met. From 24 hr after seeding, cells grew in 0.1 mM MTOB medium at a rate comparable to that in 0.1 mM Met medium. Using [35S]MTOB, it was found that the Met synthesized was used in normal MRC-5 cells and in transformed HeLa cells to the same extent for protein, adenosylmethionine and adenosylhomocysteine syntheses. However, when the free Met content was examined, it was found to be 3-fold greater in HeLa than in MRC-5 cells. To examine the importance of this free Met for the growth of transformed cells, the transaminase responsible for converting MTOB to Met was chosen as a target enzyme for the synthesis of compounds with potential inhibitory activity. Since this is a multisubstrate enzyme, reduced Schiff bases were prepared containing both pyridoxal or other aromatic groups, as one constituent, and L-Met or other amino-acids in the free acid or ester or amide form, as the other constituent. Only esters containing the pyridoxal moiety and Met or certain of its structural analogues exhibited good selective growth inhibitory activity in that there was little (20%) or no effect on the growth of normal MRC-5 and derm cells, respectively, while that of transformed HeLa, HEp-2 and L1210 cells was strongly inhibited (80%). This inhibition was accompanied by a concomitant decrease in the activity of the MTOB transaminase in both HeLa and MRC-5 cells treated with 3c the most potent inhibitor. However, using [35S]MTOB it was found that MTOB itself accumulated 48% in HeLa but only 12% in MRC-5 cells treated with 3c. On the contrary [35S]Met formed from [35S]MTOB increased 3.7-fold in MRC-5 inhibitor-treated cells showing 20% growth inhibition whereas it decreased 38% in HeLa-treated cells showing 80% growth inhibition. This decrease in cellular Met in HeLa is not responsible for growth arrest. Indeed the growth of HeLa cells could not be restored by adding a 10-fold excess of Met. Since MTOB can alleviate Met-dependence, the intracellular homeostasis of this metabolite may play a hitherto unsuspected role in controlling cell growth.

The effect of a novel inhibitor of aldehyde dehydrogenase on viral replication.

The effect of AMPAL (4-amino 4-methyl 2-pentyne 1-al), an inhibitor of aldehyde dehydrogenase, on adenovirus type 5 (Ad5) replication was studied. AMPAL at 2 x 10(-4) M clearly reduced the cytopathic effect on HeLa cells but had no effect on cell growth at this concentration. Viral adsorption, penetration and protein synthesis were not affected by adding AMPAL at 2 hr post infection. When viral DNA synthesized in the presence of AMPAL was investigated, no significant inhibition was observed on either synthesis or the physicochemical properties of the neosynthesized DNA. However, there was a 4-fold increase in the amount of condensed DNA. In addition, AMPAL inhibited intracellular viral production (40%) and brought about concomitant inhibition of virus release (70%) into the medium. The absence of a quantitative relationship between the inhibition of viral DNA synthesis on one hand and that of viral production on the other may imply that the antiviral effect of AMPAL is indirectly mediated by the action of the malondialdehyde which has accumulated on some, as not yet identified, membrane constituent.

[Incidence of Mycoplasma pneumoniae infection in a population of hospitalized patients during an epidemic]. / Incidence de l'infection à Mycoplasma pneumoniae dans une population de malades hospitalisés au cours d'une épidémie.

During an outbreak with Mycoplasma pneumoniae, a serological survey was performed with 3,165 sera from 1,900 hospitalized patients over a 33 months period. Four hundred and eleven patients exhibit serological pattern suggestive for a recent infection. The main points are the following: the infection was more frequently (21.6%) detected in females than males, in the patients 5 to 19 years than in the other age groups; the incidence of the infection is the same in the group of patients hospitalized for extra-respiratory syndromes compared to respiratory infections; children and teen-agers exhibit the higher antibody titers; no modification of the type of infection was found in relation with age groups; the antibody titers are higher when patients are hospitalized with respiratory diseases, whatever their age group may be; there is no relation between sex and the type of infection, pulmonary or extra-pulmonary; a surprisingly high incidence of infections was detected in patients hospitalized with renal failure.

Changes in human adenovirus 5 propagated in Burkitt's lymphoma cells.

The effect of host cells on human adenovirus 5 properties was studied with the use of Burkitt's lymphoma cell lines (Raji and Jijoye) that poorly replicate the virus. Only a small fraction of the cell population was producing adenovirus 5. The major virus components were synthesized; however, the purified virus particles differed from those produced in HeLa cells by lower density in CsCl gradient, lower content in DNA, limited changes in DNA restriction enzyme pattern, and polypeptide composition. After one passage in HeLa cells, modifications of polypeptides reversed.

The fate of type 7 adenovirions in lysosomes of HeLa cells.

Properties of type 7 adenovirions in lysosomes of HeLa cells were studied 12 hr postinfection. Viral particles were transferred to the lysosomes very quickly after initiation of penetration, i.e., after 10 min of incubation at 37 degrees. No morphological modification of the virion was detected for 6 hr postinfection. However, by 12 hr postinfection, the virion was no longer recognizable. Most of the virus remained infectious for 2 hr, whereas after 12 hr the infectivity was abolished. Soon after the adsorption of the virus on the cell membrane at 4 degrees, the viral DNA in the virion became sensitive to pancreatic DNase, and this sensitivity increased during the first 2 hr of incubation at 37 degrees. This result suggests that some modification in the architecture of the virion occurred before transfer to the lysosomes. The adenovirus 7 (Ad 7) DNA extracted from the lysosomes appeared intact for 6 hr postinfection and was found to cosediment at 34 S with the Ad 2 DNA marker. Comparable activities of free acid phosphatase were found in lysosomes isolated from uninfected control cells and from infected cells. In in vitro experiments, lysosomal acid DNase and pancreatic DNase were shown to degrade Ad 7 DNA at similar rates; however, in vivo, intralysosomal Ad 7 DNA was only partially sensitive to lysosomal DNase.

[Transfer of adenovirus DNA into the nucleus at the beginning of infection of HeLa cells]. / Transfert de l'ADN d'adénovirus dans le noyau au début de l'infection des cellules HeLa

2 hrs after infection of HeLa cells with Ad 7, viral DNA was found in the nuclei. Inactivation of the virus with ultraviolet or antiserum, or pretreatment of the cells with chloroquine, did not affect the localisation of the DNA. The intranuclear DNA originated at least partly from viral particles passing through lysosomes. The appearance of Ad 7 DNA in the nucleus was delayed as compared to Ad 5 DNA.