Hepatitis G virus encodes protease activities which can effect processing of the virus putative nonstructural proteins.

The genome of a recently identified virus, hepatitis G virus (HGV), shows considerable homology to hepatitis C virus (HCV). Two HGV proteases similar to nonstructural proteins NS2 and NS3 of HCV were identified, and their cleavage site specificity was investigated. Amino acids essential for the protease activities were determined by mutation analysis. NS4A of HGV was demonstrated to be a cofactor for NS3-mediated proteolysis, with a region critical for activity residing between Leu1561, and Ala1598.

Crystal structure of SIV matrix antigen and implications for virus assembly.

Simian immunodeficiency virus (SIV) is closely related to human immunodeficiency virus (HIV), their matrix antigens (MAs) sharing some 50% sequence identity. MA is a component of Pr55Gag, the sole protein required for assembly of the virion shell. MA targets Pr55 to the plasma membrane, and facilitates incorporation of the virus envelope protein and assembly of the Pr55Gag shell. Cleavage of Pr55 by the viral protease produces the mature protein of relative molecular mass 17-18K, which underlies the host-derived membrane and is important in both virus entry and nuclear localization of the virion core. Here we report the crystal structure of SIV MA. The molecule forms a trimer consistent with oligomerization in vitro, the observed virion architecture, and various biological properties of MA.

High-level expression of five foreign genes by a single recombinant baculovirus.

Co-infection with several viruses is often used to achieve simultaneous expression of several proteins. From co-infections involving several viruses, the ratio of proteins synthesized in individual cells can be very variable. This is disadvantageous where proteins are needed to interact to provide a maximum yield of a complex product. Multiple-gene-expression vectors offer an alternative to co-infections. They enable reproducible ratios of products to be provided in each infected cell. Until recently, multigene-expression vectors have only been developed to make two proteins simultaneously. Here, we describe the generation of a single recombinant baculovirus synthesising up to five foreign proteins with a fixed ratio comparable to the ratio during the synthesis of native proteins.

Crystallization and preliminary X-ray investigation of recombinant simian immunodeficiency virus matrix protein.

Simian immunodeficiency virus matrix protein has been crystallized from Tris-HCl buffer with polyethylene glycol and isopropanol as precipitants. The crystals belong to space group C2 with unit cell dimensions a = 69.9 A, b = 115.2 A, c = 32.5 A, beta = 108.1 degrees and diffract X rays to a minimum Bragg spacing of 2 A. These crystals appear suitable for a high resolution structure analysis.

Morphogenic capabilities of human immunodeficiency virus type 1 gag and gag-pol proteins in insect cells.

To determine the interaction between the gag precursor and the viral protease and to confirm the role of gag precursor in formation of human immunodeficiency virus type 1 particles, the gag and protease encoding regions of a proviral genome with mutations at the site between p17 and p24 or p24 and p15 were expressed by recombinant baculoviruses under the transcriptional control of the strong polyhedrin promoter. Western blot analyses of the expressed products of p17-p24 mutated viruses revealed that both 41- and 55-kDa proteins were synthesized. However, free p24, p17, and the other smaller cleavage products (p9, p6) could not be detected in infected insect cells. The second recombinant virus (p24-p15) synthesized not only a 55k-Da protein, but also a number of smaller products including a 40k-Da protein, p24, and p17. Examination of the insect cells infected by either of these two recombinant viruses by electron microscopy failed to detect any gag particle formation, although some irregular membrane protrusions and profound distortions of the cell surface were clearly visible in the cells infected with recombinant mutant p17-p24 virus, but not with recombinant p24-p15 mutants. To investigate the morphogenic capability of the gag-pol fusion protein, a mutant gag-pol gene containing an inactive protease as well as a modified gag-pol gene lacking the frameshifting activity were expressed in insect cells. While the inactive protease mutant was capable of forming immature particles that were secreted, the frameshifting mutant synthesized only an aberrant form of gag particles with a large radius of curvature in lieu of spherical particles. However, when this mutant was expressed in insect cells in the presence of a truncated gag protein with M(r) of 46 kDa (lacking only the p6 domain), normal immature particles containing both antigens were formed.

Development of baculovirus triple and quadruple expression vectors: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells.

Baculovirus multiple gene transfer vectors pAcAB3 and pAcAB4 have been developed to facilitate the insertion of three or four foreign genes respectively into the Autographa californica nuclear polyhedrosis virus (AcNPV) genome by a single co-transfection experiment. The pAcAB3 vector contains a polyhedrin promoter and two p10 promoters on either side of the polyhedrin promoter but in opposite orientations. The pAcAB4 vector has an additional polyhedrin promoter in opposite orientation to the first copy that is in juxtaposition to the first p10 promoter. Each of these derived vectors (pAcAB3, pAcAB4) have been used for the simultaneous expression of three or four bluetongue virus (BTV) genes respectively. When Spodoptera frugiperda cells were infected with the recombinant virus (AcBT-3/2/7/5) expressing the four major structural genes of BTV, double-capsid, virus-like particles consisting of VP2, VP3, VP5 and VP7 of BTV were assembled.

Presentation of hepatitis B virus preS2 epitope on bluetongue virus core-like particles.

A chimeric protein containing most of the hepatitis B virus preS2 region (amino acid residues 1-48) upstream to, and colinear with the amino-terminus of bluetongue virus VP7 protein (preS2-VP7) was expressed by a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV). The chimeric protein formed BTV core-like particles (CLPs) in Spodoptera frugiperda cells only when the cells were coinfected with this recombinant virus and a recombinant baculovirus that expresses unmodified VP7 and VP3 of BTV. The ratio of preS2-VP7 incorporated into CLPs was influenced by the relative multiplicities of infection of the two viruses. Immunoelectron microscopy of the chimeric particles indicated that the preS2 epitope was exposed on the surface of the CLPs. When insect cells were coinfected with the preS2-VP7 recombinant virus and a baculovirus vector that synthesized only the VP3 protein, no CLPs were identified.