Bernard N. Fields

Isolation and preliminary genetic and biochemical characterization of temperature-sensitive mutants of reovirus

Abstract A set of temperature-sensitive mutants of reovirus type 3 has been isolated. The mutagens used were nitrous acid, nitrosoguanidine, and proflavine. Thirty-five mutants have been tested for their ability to recombine following pairwise infection. Five recombination groups have been detected. Twenty-nine mutants fall into one group, four into another, and three mutants recombine efficiently with every other mutant tested. Recombination frequencies are either zero or in excess of 2%. This result supports chemical evidence that the genome of reovirus consists of a number of segments. The mutants have been tested for their ability to induce the formation of virus-specific RNA at the nonpermissive temperature. The classification of mutants according to this criterion agrees exactly with that deduced from recombination tests.

Distinct pathways of viral spread in the host determined by reovirus S1 gene segment

The genetic and molecular mechanisms that determine the capacity of a virus to utilize distinct pathways of spread in an infected host were examined by using reoviruses. Both reovirus type 1 and reovirus type 3 spread to the spinal cord following inoculation into the hindlimb or forelimb footpad of newborn mice. For type 3 this spread is through nerves and occurs via the microtubule-associated system of fast axonal transport. By contrast, type 1 spreads to the spinal cord through the bloodstream. With the use of reassortant viruses containing various combinations of double-stranded RNA segments (genes) derived from type 1 and type 3, the viral S1 double-stranded RNA segment was shown to be responsible for determining the capacity of reoviruses to spread to the central nervous system through these distinct pathways.

Evidence for functional domains on the reovirus type 3 hemagglutinin

Abstract The reovirus σ1 protein (HA) is the polypeptide responsible for specific cell surface binding and hemagglutination. In order to define the biochemical basis of these properties, we have isolated a number of hybridomas secreting monoclonal antibodies directed against the serotype 1 and 3 HA proteins. Using neutralization and hemagglutination inhibition (HI) testing, we have been able to define at least four distinct antigenic regions (epitopes) on the type 3 HA. One class of antibodies had exclusively neutralizing activity; a second class had only HI activity. One antibody had neutralizing and HI activity and the fourth class of monoclonal antibodies had no detectable neutralization or HI activity. Thus there appears to be marked functional specialization within regions of the reovirus type 3 HA.

Identification of the gene coding for the hemagglutinin of reovirus

Abstract A genetic approach has been used to identify the gene which codes for the hemagglutinin of reovirus. Hemagglutination by reovirus serotypes is type specific; type 1 hemagglutinates human but not bovine erythrocytes and type 3 hemagglutinates bovine but not human erythrocytes. Using recombinants derived from mixed infections of reovirus types 1 and 3, we have determined that the hemagglutinating properties of reovirus are a function of the σ1 outer capsid polypeptide, the polypeptide encoded in the S1 dsRNA segment of the virus.

Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma 3.

By atomic absorption analysis, we determined that the reovirus outer capsid protein sigma 3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, we localized the zinc- and dsRNA-binding activities of sigma 3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxy-terminal fragment. By these techniques, new zinc- and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor IIIA-like zinc-binding site within sigma 3. We suggest that the zinc- and dsRNA-binding activities of sigma 3 may be important for its proposed regulatory effects on viral and host cell transcription and translation.

The interaction of mammalian reoviruses with the cytoskeleton of monkey kidney CV-1 cells

Abstract We have examined the effect of reovirus infection on the CV-1 cytoskeleton. Reovirus infection produces a major disruption of vimentin filaments without producing a discernable disorganization of microtubules or microfilament bundles in CV-1 cells. In addition to disrupting the organization of vimentin filaments, reovirus infection appears to cause a reorganization of vimentin filaments. Viral inclusions contain vimentin filamentous structures. Viral infection also alters the cytoplasmic distribution of mitochondria, consistent with the proposed role of vimentin filaments in determining the distribution of mitochondria.

Pathogenesis of viral infections. Basic concepts derived from the reovirus model.

INFECTIOUS agents such as bacteria, viruses, fungi, and protozoa are the most frequent causes of acute illnesses. However, only a small proportion of the microorganisms associated with human beings...

Reovirus inhibition of cellular RNA and protein synthesis: Role of the S4 gene

Abstract Type 2 reovirus inhibits L cell RNA and protein synthesis more rapidly and efficiently than type 3. By using recombinant reoviruses containing various combinations of double-stranded RNA segments derived from reovirus type 2 and 3 we have found that the S4 double-stranded RNA segment (the segment that encodes the σ3 polypeptide, the major outer capsid polypeptide) is responsible for the capacity of type 2 to inhibit L cell RNA and protein synthesis.

Molecular basis of viral neurotropism: experimental reovirus infection.

Clinical sequelae of human infection by neurotropic viruses have been recognized for nearly 4,000 years. A funeral stela of a XIXth dynasty Egyptian priest showed the effects of prior poliomyelitis.’ The epitome of classic clinical and pathologic studies of neurotropic viruses can be found in Pasteur’s work on rabies2 Until recently, it was not possible to explain the fundamental clinical properties of viruses at the level of the viral gene. Nowhere is this difficulty better exemplified than in the problems of neurotropism and the spread of virus within the nervous system. It has been known for over a century that the striking clinical variability in infection by polio or rabies implies selective infection of discrete neuronal populations. Using experimental reovirus infection, it has been possible to establish the molecular and genetic basis of viral neurotropi~m.”.~ Reoviruses are nonencapsulated viruses 600 to 900 . & in diameter, with icosahedral symmetry.5 “Reo” is an acronym for respiratory and enteric orphan virus and reflects the original sites of isolation of these viruses as well as the initial lack of associated disease (hence “orphan”). The viral core contains 10 unique segments of double-stranded RNA (dsRNA) that can be subdivided into three classes based on molecular weight. Each dsRNA segment encodes a single strand of messenger RNA that is translated on host ribosomes into a unique single polypeptide. Two of the small dsRNA segments (S1 and S4) and one medium segment (M2) code for structural polypeptides (o1, a{, and pic), which together form the outer capsid The three distinct serotypes (types 1 ,2 , and 3) of reovirus identified by hemagglutination inhibition and antibody neutralization tests also differ in electrophoretic mobility of the RNA segments and proteins. Stable recombinants containing specific desired gene combinations can be isolated and perpetuated unchanged in tissue culture.R,9 If two serotypes differ in a biologic property, such as the ability to infect a discrete population of nerve cells, it is possible to identify the molecular and genetic basis of the property by studying recombinant viruses of known genetic and structural composition. Neurovirulence has been attributed to the S1 gene:3.*.10-ie, the type 1 S1 gene segment is responsible for reovirus tropism for ependymal cells, and the type 3 S1 gene segment is responsible for tropism for neurons leading to the necrotizing encephalitis induced by reovirus type 3.:1.4J(’ We wished to extend our prior studies of neurotropism to discrete populations of neurons in the CNS. The spread of reovirus from brain to retina provides an ideal situation to study neurotropism more directly and to examine a related property-ie, spread of virus within the nervous system. The discrete and orderly arrangement of structurally distinct groups of cells in the retina provides an excellent background for investigating both hematogenous and neural spread of viruses, and allows us to quickly and reproducibly evaluate specific patterns of viral tropism. In this report, we have found that reovirus type 3 infects a discrete population of neural cells, the retinal ganglion cells, and that this property is a function of a single gene (Sl) that encodes the a1 polypeptide on the viral surface. The differing patterns of viral growth and tropism shown by reovirus type 1 and type 3 suggest that these reovirus serotypes spreadby different routes. Type 3 may reach the eye by neural pathways. By contrast, the rapidity of spread and the lack of viral infection of neural cells by type 1 suggest that this type spreads through vascular routes.

Cytochalasin B inhibits the maturation of measles virus

The release of measles virus was studied in the presence of cytochalasin B (CB), a drug that disrupts actin microfilaments. In the presence of CB, infected cells accumulated infectious virus while virus released from these cultures decreased drastically (up to 99% inhibition). Electron micrographs showed that viral buds were reduced and had an unusual distribution along the cell membrane in CB-treated cultures. CB inhibition of released virus occurred rapidly (within 30 min) and to a full extent even when the drug was added during the final 2 hr of a 48-hr replicative cycle. CB inhibition of cellular functions is reversible and, similarly, inhibition of virus release could be almost completely reversed within 30 min after the drug was removed. Since CB can also inhibit sugar transport and protein glycosylation, 2-deoxy-D-glucose (DG) was used to study the manifestations of glycosylation inhibition. DG inhibited virus production only when added during the first one-third of the replicative cycle and inhibited cell-associated and released virus to an equal extent. Cytochalasin D, which disrupts microfilaments without affecting protein glycosylation, caused an inhibition of virus release analogous to the inhibition caused by CB. Thus, alteration of microfilament structure alters the normal budding process of measles virions. This suggests that microfilaments may play a role in the release of budding virions.