Bruce D. Korant

Naturally occurring and artificially produced components of three rhinoviruses

Abstract Preparations of purified virions of human rhinoviruses type 2 and type 14 and of an equine rhinovirus are each homogeneous by the criteria of electron microscopy and rate-zonal and isopycnic gradient centrifugation. The rhinoviruses differ from each other in intrinsic infectivity and buoyant density in CsCl, while their sedimentation rates in sucrose gradients are similar, about 4% less than poliovirus. Purification in CsCl gradients produces an artifactual increase of 7% in the rhinovirus sedimentation constant, which is reversed with time after removal of CsCl from the preparation. Rhinovirus types differ in part in their four size classes of major polypeptides: VP1, VP2, VP3, and VP4. Crude preparations also contain empty capsids which sediment at half the rate of the virions, contain VP1 and VP3 and polypeptides larger than any in the virions (VPO). There is another naturally occurring component, the slow component, which sediments 90% as fast as intact virions. The slow component gives rise to a dense component in CsCl gradients which is 0.04–0.05 g/ml more dense than intact virions. The dense component contains RNA, VP1, and VP3. On the basis of indirect evidence, only the smallest polypeptide, VP4, is missing from the slow component, and VP2 is lost during CsCl centrifugation. Components which resemble the slow and dense components can be produced from purified virions in vitro by careful acidification.

A cell surface alteration in mouse L cells induced by interferon.

Abstract Mouse L cells grown in suspension culture when treated with L cell interferon have a greater electrophoretic mobility toward the anode than control cells. This change in electrophoretic mobility depends on the concentration of interferon in the medium and the duration of interferon interaction with the cells. It is concluded that the interferon-treated cells have a greater net negative charge on the cell surface than control cells and it is suggested that the cell surface is altered because of the interaction with interferon.

Fractionation of biologically active and inactive populations of human rhinovirus type 2

Abstract Infectious virions of human rhinovirus type 2 (HRV-2) migrate during electrophoresis to pH 6.4 in a sucrose-stabilized pH gradient. As already reported, acidification of HRV-2 produces two kinds of noninfectious components both of which have lost the smallest virion polypeptide, VP4: One has lost RNA in addition to VP4 and sediments at about 80 S, while the other retains RNA and sediments at 135 S. The 80 S particles migrate to pH 4.5 upon electrophoresis while the 135 S particles migrate to pH 4.2. Natural top component of HRV-2 is also separable into two subpopulations that are isoelectric at pH 6.3 and pH 4.5. Only the particles isoelectric at pH 6.3 react with virion (D) specific serum and attach to HeLa cells. The polypeptide compositions of both the attaching and nonattaching fractions of natural top component are the same, and therefore the differences in activity and isoelectric point may reflect the arrangement of the polypeptides in the capsids. After a preparation of purified infectious virus is focused to its isoelectric point some particles are found at pH 4.5. These particles are relatively noninfectious, yet contain RNA and a full complement of polypeptides, and they sediment at 140 S. They are unable to attach to host cells. These results argue against a direct role of VP4 or RNA in the attachment process and support the view that protein conformation changes occur during the inactivation of HRV-2 virions.

Poliovirus coat protein as the site of guanidine action.

Abstract The site of guanidine action on poliovirus type 2 was investigated. Physical-chemical and biological characterizations of viral mutants with altered guanidine sensitivities indicate changes in capsid polypeptides. Differences between mutants include electrophoretic mobilities of virions and of capsid subunits, stability of virions to high temperature, and chromatographic behavior of individual capsid polypeptides. No alterations were detected in nonstructural proteins of guanidine mutants. Aside from guanidine sensitivity of RNA synthesis, the differences between mutants in replication were limited to early events, particularly uncoating rates. No evidence for an effect of guanidine on processed capsid polypeptides was obtained; however, the capsid precursor polypeptide of sensitive virus is altered by guanidine treatment. A model is proposed in which a precursor to poliovirus structural polypeptides participates in viral RNA synthesis.

A Cellular Anti-Apoptosis Protein is Cleaved by the HIV-1 Protease

Cleavage of non-viral proteins is rarely observed with the HIV-1 protease (HIV pr). One such cleavage event occurs with bcl-2, an important cytoprotective protein. The loss of bcl-2 has biological consequences, leading to enhanced HIV replication and programmed death of the host cell. A strategy is proposed to suppress HIV with non-cleavable mutants of bcl-2.

Protease activity associated with HeLa cell ribosomes

Abstract HeLa cells contain endoprotease activity, detected by a sensitive, solid phase assay. The endoprotease has the ability to cleave a variety of protein substrates and is trypsin-like in its sensitivity to inhibitors. The activity is in part associated with cellular ribosomes and polysomes. A variety of biological and physical-chemical treatments which alter ribosomes or protein synthesis also directly affect the ribosomal protease activity.

Role of Cellular and Viral Proteases in the Processing of Picornavirus Proteins

The replication of picornaviruses requires the action of proteolytic enzymes on viral precursor proteins. The protein cleavages are often crucial to the virus replication process, so that a complete understanding of their role could lead to development of new types of antiviral agents. On a more fundamental level, the characterization of the proteolytic reactions in virus infections is leading to a fuller appreciation of regulation of function by limited proteolysis. The picornavirus is a model in which proteolytic modifications yield macromolecular complexes with altered conformational and antigenic properties, and altered affinities for nucleic acids and receptors on cell membranes. Obviously, there is a general significance of these phenomena in understanding similar events in cellular interactions and in the numerous proteolytic events occurring in the tissues and fluids of higher organisms. For recent reviews of protein cleavage in picornavirus replication, see ref. 1–3.

The HIV Protease and Therapies for AIDS

New, potent therapies for HIV disease are available, based on synthetic inhibitors of the viral protease, an essential viral enzyme. The results in clinical trials have been impressive with most treated individuals benefiting in terms of reduced quantity of detectable virus, enhanced numbers of CD4 lymphocytes and improvements in quality and duration of life. However, there are some remaining negatives associated with the new drugs, including high cost, side effects and appearance of drug-resistant strains of HIV. Problems and future prospects for use of protease inhibitors and alternate approaches in AIDS are discussed.

PROTEOLYTIC EVENTS IN REPLICATION OF ANIMAL VIRUSES

It has been clear for more than 10 years that proteolytic modifications of viral proteins occur during replication (reviewed in reference 1). Most commonly observed are cleavages of structural protein precursors, which cause profound modifications in the surface properties of the products, and lead to their aggregation into specific supermolecular structures with molecular weights of millions, and abilities to combine with viral nucleic acids and to recognize cellular surface receptors. With many of the viruses proteolytic cleavages occur during protein biosynthesis or just after translation is completed. The roles of these cleavages are virtually unknown. Until recently, the origin of the proteolytic enzymes that process the viral precursors was vague; however recent advances in studies of bacteriophages,* RNA tumor viru~es,~ togaviruses,‘ and picornaviru~e~~” have provided evidence for virus-coded proteases involved in the ultimate stages of processing. In this study we used precursors of picornavirus proteins to analyze the contribution of cellular and virusspecific proteases in the processing reactions.

Structural polypeptides of three rhinoviruses.

Abstract Growth, purification and electrophoretic analysis of three rhinoviruses and an enterovirus are described. It is shown that, with regard to structural polypeptides, the three rhinoviruses differ from other picornaviruses, and from each other. However, all three rhinoviruses resemble one another in possessing structural polypeptides of molecular weights 33,000 and 30,000 in similar proportions.