Radiobiological and biochemical investigations of polyoma virus-cell interactions

The cytolytic interaction of Polyoma virus with mouse embryo cells has been studied by radiobiological methods known to distinguish temperate from virulent bacteriophage. No evidence for "temperate" properties of Polyoma was found. During the course of these studies, it was observed that the curve of inactivation of Polyoma virus by ultraviolet light had two components - a more sensitive one at low doses, and a less sensitive one at higher doses. Virus which survives a low dose has an eclipse period similar to that of unirradiated virus, while virus surviving higher doses shows a significantly longer eclipse period. If Puromycin is present during the early part of the eclipse period, the survival curve becomes a single exponential with the sensitivity of the less sensitive component. These results suggest a repair mechanism in mouse cells which operates more effectively if virus development is delayed.

A comparison of the rates of inactivation of the cytolytic and transforming abilities of Polyoma by ultraviolet light, X-rays, nitrous acid treatment, or the decay of incorporated P³², showed that the transforming ability has a target size roughly 60% of that of the plaque-forming ability. It is thus concluded that only a fraction of the viral genes are necessary for causing transformation.

The appearance of virus-specific RNA in productively infected mouse kidney cells has been followed by means of hybridization between pulse-labelled RNA from the infected cells and the purified virus DNA. The results show a sharp increase in the amount of virus-specific RNA around the time of virus DNA synthesis. The presence of a small amount of virus-specific RNA in virus-free transformed cells has also been shown. This result offers strong evidence for the persistence of at least part of the viral genome in transformed cells.

Cell fate specification during Caenorhabditis elegans male tail development

The cells of the specialized mating structures of the nematode Caenorhabditis elegans adult male tail develop from sex-specific divisions of postembryonic blast cells. One male-specific blast cell, B, is the precursor to all the cells of the copulatory spicules. Both cell interactions and autonomous fate specification mechanisms are utilized in the B lineage to specify fate.

During development the anterior daughter of B, B.a, generates four distinct pairs of cells. Cell ablation experiments indicate that the cells of each pair respond to positional cues provided by other male-specific blast cells. F and U promote anterior fates, Y.p promotes some posterior fates, and the B.a progeny promote posterior fates. The cells within each pair may also interact.

The lin-3/let-23 signalling pathway, identified for its function in C. elegans hermaphrodite vulval induction, mediates the signal from F and U. Reduction-of-function mutations in lin-3 (EGF-like signal), let-23 (receptor), sem-5 (adaptor), let-60 (ras), or lin-45 (raf) disrupt the fates of the anterior cells, and mimic F and U ablation. In addition, ectopically expressed lin-3 disrupts the fates of the posterior cells, and can promote anterior fates even in the absence of F and U.

A genetic screen of over 9000 mutagenized gametes recovered 22 mutations in 20 loci that disrupt fate specification in male tail lineages. Seven of these mutations may represent new genes that play a role in male tail development.

The first division of the B cell is asymmetric. The gene vab-3 is required for specification of B.a fates, and it may represent a factor whose activity is localized to the B.a cell via the gene lin-17. lin-17 acts both at the first division of the B cell and at specific other cell divisions in the lineage.

Studies of temperature sensitive mutants of polyoma virus

Polyoma virus can undergo two different types of interactions with susceptible cells; one type of interaction leads to the production of new infectious virus and eventual cell death while the other leads to a neoplastically transformed cell which is able to continue to divide under conditions that inhibit the multiplication of uninfected normal cells. In order to study the viral genes involved in both of these virus-cell interactions the isolation of temperature sensitive mutants of polyoma virus was undertaken.

Two strains (TS-a, TS-b) which were temperature sensitive in their plaque forming ability at 38.5˚C, but not at 31.5˚C, were isolated from a mutagenized stock of the polyoma wild type virus (PY). TS-a was studied in further detail.

TS-a grown at 31.5˚C was found to be indistinguishable from PY in a number of physical characteristics including the heat sensitivity of the completed viral components. TS-a was inhibited in its ability to produce infectious virus in mouse cells when incubated at 38.5˚C; this inhibition could be overcome by infection with high multiplicities.

The nature of the intracellular temperature sensitive step of TS-a was analysed to some degree. It was found that this step occurs after uncoating of the infecting virus particles and about the time of new viral DNA synthesis. New infectious viral DNA does not appear to be made at the nonpermissive temperature; in contrast noninfectious capsids are made at 38.5˚C, but in amounts smaller than a full yield, such as made by TS-a at 31.5˚C or by PY at both the high and low temperature.

TS-a has also been found to be temperature sensitive in its transforming ability in vitro. Cells transformed at 31.5˚C by TS-a retain their transformed characteristics upon cultivation at 38.5˚C. Thus the temperature sensitive function seems to be important for the initiation of transformation, but not essential for the maintenance of the transformed state. TS-a also appears to be temperature sensitive in the production of tumors in newborn hamsters.

Gut microbiota promote hematopoiesis to control bacterial infection

The commensal microbiota impacts specific immune cell populations and their functions at peripheral sites, such as gut mucosal tissues. However, it remains unknown whether gut microbiota control immunity through regulation of hematopoiesis at primary immune sites. We reveal that germ-free mice display reduced proportions and differentiation potential of specific myeloid cell progenitors of both yolk sac and bone marrow origin. Homeostatic innate immune defects may lead to impaired early responses to pathogens. Indeed, following systemic infection with Listeria monocytogenes, germ-free and oral antibiotic-treated mice display increased pathogen burden and acute death. Recolonization of germ-free mice with a complex microbiota restores defects in myelopoiesis and resistance to Listeria. These findings reveal that gut bacteria direct innate immune cell development via promoting hematopoiesis, contributing to our appreciation of the deep evolutionary connection between mammals and their microbiota.