Samuel Dales

An electron microscopic study of moderate and virulent virus-cell interactions of the parainfluenza virus SV5☆☆☆

Abstract The multiplication of the parainfluenza virus SV5 was studied in primary rhesus monkey kidney (MK) cells and in a line of baby hamster kidney (BHK21-F) cells. Virus adsorbs to the cell surface and appears to penetrate by phagocytosis. Virus morphogenesis is similar in the two cells; the helical nucleocapsid forms in the cytoplasmic matrix and aligns under regions of cell membrane which acquire viral surface projections. Assembly and release of virus particles at the cell surface occurs by a budding process involving the incorporation into the viral envelope of a unit membrane which is continuous with and morphologically identical to that of the host cell. Both spherical and filamentous virus particles are formed; filaments frequently contain regularly coiled nucleocapsid throughout their length. In MK cells, which yield high titers of infective virus for many days but show little cytopathic effect, a balance appears to exist between synthesis of nucleocapsid and its continuous release within mature virus particles. In contrast, in BHK21-F cells, which produce little virus and disintegrate after extensive cell fusion, large aggregates of nucleocapsid accumulate in the cytoplasm, suggesting a block in maturation at the cell membrane. The present electron microscopic observations support previous studies which suggested that the virulence and yield of SV5 may depend, at least in part, on the response of the cell membrane to the virus.

Measles Virus Infection in a Transgenic Model: Virus-Induced Immunosuppression and Central Nervous System Disease

Measles virus (MV) infects 40 million persons and kills one million per year primarily by suppressing the immune system and afflicting the central nervous system (CNS). The lack of a suitable small animal model has impeded progress of understanding how MV causes disease and the development of novel therapies and improved vaccines. We tested a transgenic mouse line in which expression of the MV receptor CD46 closely mimicked the location and amount of CD46 found in humans. Virus replicated in and was recovered from these animals immune systems and was associated with suppression of humoral and cellular immune responses. Infectious virus was recovered from the CNS, replicated primarily in neurons, and spread to distal sites presumably by fast axonal transport. Thus, a small animal model is available for analysis of MV pathogenesis.

Depletion of a single nucleoporin, Nup107, prevents the assembly of a subset of nucleoporins into the nuclear pore complex

The nuclear pore complex (NPC) is a protein assembly that contains several distinct subcomplexes. The mammalian nucleoporin (Nup)-107 is part of a hetero-oligomeric complex, that also contains Nup160, Nup133, Nup96, and the mammalian homolog of yeast Sec13p. We used transfection of HeLa cells with small interfering RNAs to specifically deplete mRNA for Nup107. In a domino effect, Nup107 depletion caused codepletion of a subset of other Nups on their protein but not on their mRNA level. Among the affected Nups was a member of the Nup107 subcomplex, Nup133, whereas two other tested members of this complex, Nup96 and Sec13, were unaffected and assembled into Nup107/Nup133-deficient NPCs. We also tested several phenylalanine-glycine repeat-containing Nups that serve as docking sites for karyopherins. Some of these, such as Nup358, Nup214 on the cytoplasmic, and Nup153 on the nucleoplasmic side of the NPC, failed to assemble into Nup107/Nup133-depleted NPCs, whereas p62, a Nup at the center of the NPC, was unaffected. Interestingly, the filamentous, NPC-associated protein Tpr also failed to assemble into the NPCs of Nup107-depleted cells. These data indicate that Nup107 functions as a keystone Nup that is required for the assembly of a subset of Nups into the NPC. Despite the depletion of Nup107 and the accompanying effects on other Nups, there was no significant effect on the growth rate of these cells and only a partial inhibition of mRNA export. These data indicate redundancy of Nups in the function of the mammalian NPC.

The nucleoporin Nup98 associates with the intranuclear filamentous protein network of TPR

The Nup98 gene codes for several alternatively spliced protein precursors. Two in vitro translated and autoproteolytically cleaved precursors yielded heterodimers of Nup98-6kDa peptide and Nup98-Nup96. TPR (translocated promoter region) is a protein that forms filamentous structures extending from nuclear pore complexes (NPCs) to intranuclear sites. We found that in vitro translated TPR bound to in vitro translated Nup98 and, via Nup98, to Nup96. Double-immunofluorescence microscopy with antibodies to TPR and Nup98 showed colocalization. In confocal sections the nucleolus itself was only weakly stained but there was intensive perinucleolar staining. Striking spike-like structures emanated from this perinucleolar ring and attenuated into thinner structures as they extended to the nuclear periphery. This characteristic staining pattern of the TPR network was considerably enhanced when a myc-tagged pyruvate kinase-6kDa fusion protein was overexpressed in HeLa cells. Double-immunoelectron microscopy of these cells using anti-myc and anti-TPR antibodies and secondary gold-coupled antibodies yielded row-like arrangements of gold particles. Taken together, the immunolocalization data support previous electron microscopical data, suggesting that TPR forms filaments that extend from the NPC to the nucleolus. We discuss the possible implications of the association of Nup98 with this intranuclear TPR network for an intranuclear phase of transport.

Cytopathology of mengovirus infection II. Proliferation of membranous cisternae

Abstract During cytopathic degeneration of host cells infected by picornaviruses, synthesis of lipids is elevated, and rapidly developed vacuoles become prominent in the centrosphere region. To discover whether such enhanced formation of lipid provides structural material for proliferation of new cisternae, we examined biochemically and autoradiographically the synthesis of phospholipid. In a series of chemical tests with an isotopically labeled compound, it was established that choline is a highly specific precursor of phospholipid, especially of lecithin. Following infection with Mengovirus, incorporation of choline is generally stimulated, the microsomes possessing the highest concentrations of nascent phospholipid. This result is supported by counts of silver grains made in radioautograms of thinly sectioned cells, which showed that nascent phospholipid was concentrated in the region occupied by newly developed cisternae. However, if L cells are labeled in their phospholipid by choline prior to inoculation and then are examined several hours after infection, it is found that labeled phospholipid is also concentrated over the recently formed vacuoles. From these results we hypothesize that phospholipids of new membranes assembled in infected host cells are partly derived from nascent macromolecules and partly from preexisting phospholipids.

Cytopathology of mengovirus infection I. Relationship between cellular disintegration and virulence

Abstract Characterization of the viral factor(s) that regulate the degree of virulence in animals and toxicity for cells in vitro , was attempted. Comparative studies, employing a large- and small-plaque variant isolated from stock lysate, were conducted on: the development of cytopathic alterations; rapidity of rupturing outer membranes of cultured cells; and efficiency for killing animals. The variant which makes large plaques is the more virulent, as judged by its capacity to kill animals and cultured cells more rapidly. Experiments with a fast-acting inhibitor, streptovitacin A, indicate that a cytotoxic principle responsible for initiating cell degeneration is a protein, possibly a viral coat protein, synthesized under the regulation of a late viral function. We have considered the mechanism whereby such a protein, once it has been synthesized, could trigger cytopathic changes in host cells by initiating a leakage of hydrolases from lysosomes.

A role for dual viral hits in causation of subacute sclerosing panencephalitis

Subacute sclerosing panencephalitis (SSPE) is a progressive fatal neurodegenerative disease associated with persistent infection of the central nervous system (CNS) by measles virus (MV), biased hypermutations of the viral genome affecting primarily the matrix (M) gene with the conversion of U to C and A to G bases, high titers of antibodies to MV, and infiltration of B cells and T cells into the CNS. Neither the precipitating event nor biology underlying the MV infection is understood, nor is their any satisfactory treatment. We report the creation of a transgenic mouse model that mimics the cardinal features of SSPE. This was achieved by initially infecting mice expressing the MV receptor with lymphocytic choriomeningitis virus Cl 13, a virus that transiently suppressed their immune system. Infection by MV 10 days later resulted in persistent MV infection of neurons. Analysis of brains from infected mice showed the biased U to C hypermutations in the MV M gene and T and B lymphocyte infiltration. These sera contained high titers of antibodies to MV. Thus, a small animal model is now available to both molecularly probe the pathogenesis of SSPE and to test a variety of therapies to treat the disease.

Absence of surface projections on some noninfectious forms of RSV

Abstract Electron microscopic examination was made of the purified virus particles of two strains of Rous sarcoma virus that are defective in glycoprotein synthesis. Under conditions where the projections are detectable on the surface of regular, nondefective avian leukosis or sarcoma virus, no corresponding structures were observed on these defective virions. Projections were present on the surface of virions of these RSV strains when they were produced from helper factor (chf)-positive chicken cells.