Ulrike Boege

Establishment and analysis of a system which allows assembly and disassembly of alphavirus core-like particles under physiological conditions in vitro.

Core-like (CL) particles which closely resemble alphavirus cores in size, shape, and relative amount of nucleic acid and protein have been assembled in vitro from Sindbis (SIN) virus core (C) protein and single-stranded nucleic acids in buffer containing 1 M urea [G. Wengler, U. Boege, G. Wengler, H. Bischoff, and K. Wahn (1982) Virology 118, 401-410]. We have now analyzed the interaction of SIN virus C protein and nucleic acids in vitro under conditions designed to resemble those present in the cell during core assembly. In buffer containing 100 mM K-acetate, 1.7 mM Mg-acetate, pH 7.4, CL particles are efficiently assembled from all single-stranded nucleic acids analyzed, and even heparin and polyvinylsulfate are incorporated into such particles. A reticulocyte lysate translates SIN virus-specific mRNA into C protein under these ionic conditions. Interactions of C protein with nucleic acids and ribosomes in a reticulocyte lysate have also been analyzed. The following conclusions can be drawn from these analyses: (1) In accordance with earlier findings [N. Glanville and I. Ulmanen (1976) Biochem. Biophys. Res. Commun. 71, 393-399] the C protein translated in vitro efficiently binds to ribosomes. (2) Exogenously added C protein binds to the large subunit of the ribosomes in the lysate. (3) CL particles can be assembled in the lysate from exogenous added 42 S genome RNA and exogenous added C protein if both components are present at sufficiently high concentrations. (4) The C protein translated from viral mRNA in the lysate is transferred from the ribosomes into preassembled CL particles containing 42 S RNA in the lysate. (5) If only small amounts of CL particles are added into a lysate these particles disaggregate and core protein molecules are transferred from the particles to the large subunit of the ribosomes. The results on the assembly of CL particles in vitro allow the formulation of some hypotheses concerning the assembly and disassembly of core particles in vivo.

Mengo virus maturation is accompanied by C-terminal modification of capsid protein VP1

We have demonstrated that the C-terminal ends of the VP1 proteins of Mengo virus undergo a postassembly trimming of three amino acid residues. A variable proportion of the VP1 molecules isolated from purified virions terminate at Glu 277, which corresponds to the initial viral protease 3C cleavage site. The remainder of the VP1 molecules terminates at Leu 274. When chymotrypsin treatment is included in the virus purification procedure, Leu 274 is the exclusive C-terminal amino acid of the VP1 molecules. The trimming process was found to influence the pH-mediated dissociability of virions in vitro: this was demonstrated by sucrose gradient analysis and electron microscopy. Since long-term incubation of purified virions did not alter the C-terminal Glu 277/Leu 274 ratio, it appears that the trimming process is not autocatalytic.

Picornaviruses of two different genera have similar structures

Crystals of Mengo virus were used to collect three-dimensional X-ray diffraction data to 7 A resolution. A self-rotation function showed the precise orientation of the Mengo particles in the crystal unit cell. A cross-rotation function against similar data of cubic rhinovirus crystals showed a peak when the orientations of these two icosahedral viruses were superimposed. This demonstrates similarity of capsid construction between two picornaviruses of different taxonomic genera.

Sindbis virus core protein crystals.

The core protein of Sindbis virus has been crystallized. Three different crystal forms have been observed. They diffract variously from 2.5 A to 3.5 A resolution.

The synthesis of a particle-forming cellular protein is enhanced by Mengo virus infection

We have detected a cellular protein which not only escapes the shutoff of host translation induced by Mengo virus, but is synthesized in increasing amounts during Mengo virus infection. The protein has an apparent molecular weight of 20,000 and is contained in a remarkably stable cytoplasmic particle with a sedimentation coefficient of approximately 16 S in sucrose gradients. In the electron microscope this particle appears as a sphere of approximately 12 nm diameter. The synthesis of the protein is stimulated in mouse L cells and in HeLa cells infected with Mengo virus. Its synthesis cannot be induced, however, by stress (heat) or by infection with reovirus.

Structure of the mengo virion. V11. Crystallization and preliminary X-ray diffraction analysis

Crystals of Mengo virions have been grown reproducibly and analyzed by X-ray diffraction. These crystals diffract to a resolution of 7.0 A. The unit cell exhibits cubic symmetry with a = 422 A. The space group is P23, with four virus particles situated on crystallographic threefold axes. Picornavirions from three of the four recognized genera (Study Group on Picornaviridae, Intervirology 10, 165-180, 1978) have now been examined at low resolution by X-ray diffraction: poliovirus type 1 (J. T. Finch and A. Klug, Nature (London) 183, 1709-1714, 1959; J. M. Hogle, J. Mol. Biol. 160, 663-668, 1982); human rhinovirus 14 (J. W. Erickson, E. A. Frankenberger, M. G. Rossmann, G. S. Fout, K. C. Medappa, and R. R. Rueckert, Proc. Natl. Acad. Sci. USA 80, 931-934, 1983); and Mengo virus.

Viral Particles at Atomic Resolution

Crick and Watson (ref. 1) first recognized that spherical viruses had to be regular polyhedra. Of these, the icosahedron has the largest number (60) of asymmetric units and was subsequently found to be the preferred envelope. The coding capacity of the enclosed genetic material could therefore be limited to coding only a relatively limited structural protein(s) for one-sixth of the virion shell. The assembly of viral particles from smaller, repeated subunit s presents several defined advantages: such strategy of replication reduces considerably the amount of genetic information needed to code for the structural protein(s), and minimizes the risks of incurring in fatal errors, as faulty subunits inaccurately synthesized can be discarded at assembly time. The entire replication cycle of a virus, therefore, can be visualized as a two-step process: a) the synthesis of viral components (nucleic acid and proteins), i.e: a template dependent, energy consuming process of polymerization of preformed blocks (nucleotides or amino acids), and b) the self-assembly of the subunits into more complex structures, a process that does not involve the formation of stable chemical bonds but brings the the subunits (or the intermediate structures) to a thermodynamically stable configuration.