Nada Ledinko

Electron Microscope Study of Human NB and SMH Cells Infected with the Parvovirus, H-1: Involvement of the Nucleolus*

SummarynThe sequence of changes in human NB and SMH cells that occurred after H-1 virus infection, was studied with the electron microscope. The earliest alterations in NB cells after infection were detected in the nucleolus, after 31 hr. The pars fibrosa appeared devoid of its formed elements and was occupied instead with mainly ‘incomplete’ H-1 virus. The pars granulosa was more diffuse than normal and its nucleolar granules more sharply defined. A few ‘incomplete’ and ‘complete’ virus particles were scattered about the nucleus at approximately the same time. The cytoplasm was intact. Shortly thereafter, margination of the nuclear chromatin occurred. The nucleoli became increasingly condensed and shrunken, and apparently formed a doughnut-shaped body with condensed walls containing empty virus. Eventually, most of the nucleolar elements disappeared as the nuclei filled with complete and incomplete virus particles as well as occasional crystals possibly of protein nature. As the nucleolus disappeared, the cytoplasm disintegrated except for a few scattered mitochondria and some recognizable areas of endoplasmic reticulum which contained complete virus particles in linear array. Seventy-seven hr after infection when the nucleus had also broken down, virus was found associated with the disintegrated nucleus, within fragments of endoplasmic reticulum, and attached to thickened and adjoining plasma membranes of the few remaining, apparently uninfected cells.nIn SMH cells basically similar changes occurred except that the nucleoli appeared to fragment 31 hr after infection. Later, though most of the nucleolar sections disappeared, a few persisted and formed ‘nucleolar inclusions’ identical to those observed in NB cells. In contrast to the mixture of ‘complete’ and ‘incomplete’ virus observed in NB cells, the virus found in SMH cells was almost exclusively ‘empty’. Virus seen outside the nucleus, either in NB or SMH cells, appeared complete.

Relationship between Potentiation of H-1 Growth by Human Adenovirus 12 and Inhibition of the 'Helper' Adenovirus by H-1

Summary H-1 virus underwent an abortive cycle of replication in secondary human embryonic lung-cell cultures, characterized by formation of immunofluorescent H-1 antigen, without the production of infective virus. After mixed infection with adenovirus 12, approximately 30 to 40% of cells yielded infective H-1. A single infective adenovirus particle was capable of acting as helper. Comparison of the growth cycles of H-1 and ‘helper’ adenovirus showed that the two viruses had a similar latent period of about 24 hr. Inoculation of cells 24 hr before with adenovirus shortened the latent period for H-1 by at least 12 hr. Particles with serological characteristics of both viruses (mixed coats) were not found in virus yields from mixed infections. H-1 inhibited yields of adenovirus p.f.u. as well as virus antigens, provided that a low adenovirus input was used. The decrease in adenovirus yield was due to a reduction in the number of cells producing virus. With a high adenovirus input (10 or more p.f.u. per cell), no interference by H-1 was found. Maximal or nearly maximal potentiation of H-1 growth apparently occurred even when the H-1 caused virtually complete interference with formation of its ‘helper’ adenovirus.

Relationship between induction of thymidine kinase and potentiation of growth of H-1 virus by human adenovirus 12.

The parvovirus, H-1, undergoes an abortive cycle of growth in secondary human embryonic lung (HEL) cell cultures, but replicates in several stable cell lines of human origin (Toolan & Ledinko, 1965; Toolan, 1968). In HEL cells, H-1 infection causes the formation of capsid proteins, without the production of infective virus (Ledinko, Hopkins & Toolan, 1969). Mixed infection with human adenovirus type 12 allows H-1 virus to complete its development (Ledinko & Toolan, 1968; Ledinko, Hopkins & Toolan, 1969). At present, the events of the adenovirus growth cycle required for H-1 virus are not known. The restriction in HEL cells of H-1 virus, which contains single-stranded DNA (Usategui-Gomez et al. 1969), may involve an inhibition of viral DNA synthesis. It is possible that some enzyme concerned with DNA synthesis, and induced by adenovirus, can function in the synthesis of active H-1 virus.

Requirement of adenovirus type 12 gene 401 function for initiation of virus DNA synthesis.

The highly oncogenic human adenovirus type 12 temperature sensitive mutant. H12ts401, is unable to maintain the growth characteristics of transformed cells at the non-permissive temperature. In lytic infection, the 401 gene function is required to produce virus DNA. In the present study, virus DNA synthesized in ts401-infected human cells after temperature shift-up was characterized. No apparent suppression of DNA chain elongation or ligation occurs at the non-permissive temperature, but, as shown by density labelling, new initiation of virus DNA replication is inhibited under this condition. The results indicate that the 401 gene function is involved in the initiation of virus DNA synthesis in the lytic cycle.

Thymidine Kinase Activity and DNA Synthesis in Cells Infected with the Parvovirus, H-1

Summary Infection of SV40-transformed newborn human kidney (NB) cell cultures with H-1 virus resulted in a marked drop in the rate of incorporation of 3H-thymidine into DNA relatively late in infection. Autoradiographic measurements revealed that the inhibition of total DNA synthesis was due to a decrease in the capacity of an infected cell to synthesize DNA. Thymidine kinase activity increased 1.5- to 2.6-fold during a period of virus maturation (20–48 hr), but, later in infection, it decreased to 11–43% of the control cell activity. A similar decline in enzyme activity was also observed in “Salk monkey heart” (SMH) cells late after H-1 infection. Extracts prepared from NB cells infected for different times had no effect on the enzyme activity from uninfected cells. The decrease in thymidine kinase activity found after H-1 infection relative to control cell activity was apparently dependent on de novo protein and DNA-dependent RNA synthesis.

Polynucleotide Ligase Activity in Adenovirus-Infected Human Embryonic Kidney Cell Cultures

Summary Cell-free extracts prepared from human embryonic kidney (HEK) cultures after infection with adenovirus 2 or 12 had a decreased ability to repair single-strand breaks in the HEK cell DNA substrate. The apparent loss of polynucleotide ligase activity found in infected cells may be related to the adenovirus-induced inhibition of HEK cell DNA synthesis.

Temperature-sensitive mutants of type 12 adenovirus defective in a late function: protein synthesis and evidence for recombination between mutants in complementation group D.

Four temperature sensitive mutants of adenovirus type 12, classified into complementation group D, synthesize most or all of the late virus-specific polypeptides at the restrictive temperature, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Infections by these mutants under restrictive conditions apparently result in some over production of the virion hexon polypeptide compared to wild type infection. Genetic recombination between the four mutants has been demonstrated. A partial linear genetic map of the D cistron is presented.