Biochemical Evidence for the Oligomeric (Possibly Trimeric) Structure of the Major Inner Capsid Polypeptide (45K) of Rotaviruses

Abstract

SUMMARYThe major structural polypeptide of rotaviruses is p45K (VP6), which forms the morphological subunit of the inner capsid. Such subunits show a trimeric structure when examined with the electron microscope. Treatment of single-capsid rotavirus particles with 1.5 M-CaCI~ removes p45K, resulting in the generation of smooth cores. Sucrose density gradient centrifugation analysis of the removed p45K revealed that it has a sedimentation coefficient close to 7.3S, compatible with an oligomeric (possibly trimeric) structure. Polyacrylamide gel electrophoresis under reducing or non-reducing conditions indicated that p45K has intramolecular but not intermolecular disulphide bonds, suggesting that interactions between p45K monomers may be due to some other type of association, such as hydrophobic or charge interactions. Velocity sedimentation of infected cell extracts revealed that native p45K also behaves as an oligomeric protein. Such results were confirmed using p45K partially purified by DEAE-cellulose chromatography. The evidence obtained indicated that all p45K present in the virion is in the oligomerie form, not associated by disulphide bonding, and that most native p45K present in the infected cells is also in the oligomeric form, probably as a consequence of early protein-protein interaction in rotavirus morphogenesis.

Biological Characterization of the Omp1-like Protein from Actinobacillus actinomycetemcomitans

“…[9][10][11][12] Additional thermal denaturation experiments for the folded and unfolded Omp1-like proteins were carried out using CD spectroscopy ( Fig. 4A and B).…”

The Structure of the Rotavirus Inner Capsid Studied by Electron Microscopy of Chemically Disrupted Particles

“…Neither formamide nor urea appear to affect the association between the core proteins and the major inner capsid polypeptide (p45K or VP6), which is broken only by chaotropic agents (Bican et al, 1982). On the other hand, hydrophobic bonds may also play a role, especially in stabilizing the trimeric form of p45K (Gorziglia et al, 1985).…”

“…All experiments were done with the OSU strain of porcine rotavirus (Theil et al, 1977), which was grown and purified as previously described (Gorziglia et al, 1985). Purified rotavirus was studied by the freeze-drying technique, essentially as described by Roseto et al (1979), using a JEOL JEE-4C vacuum evaporator.…”

“…For the human rotavirus, we proposed the existence of an inner capsid composed of morphological subunits arranged as an open mesh, showing a regular pattern of cavities or 'holes', constituting the five-and six-coordinated units of the virion (Esparza & Gil, 1978). Closer examination of the morphological subunits has revealed that they are trimeric structures (Martin et al, 1975;Esparza & Gil, 1978), an observation that has found recent biochemical support (Gorziglia et al, 1985). In our model we suggested the existence of 162 holes formed by the icosahedrat arrangement of 320 subunits, with a pattern characteristic of T : 16.…”

“…This approach has recently been used with rotaviruses (Almeida et al, 1979;Bican et al, 1982;Palmer & Martin, 1982;Gorziglia et al, 1985), and has been extensively used in the past to dissect adenoviruses (Neurath et al, 1968;Stasny et al, 1968) and herpesviruses (McCombs & Williams, 1973;Palmer et al, 1975). In this tPresent address: Microbiology and Immunology Unit, World Health Organization, 1211 Geneva 27, Switzerland.…”