David C. Ansardi

Recombinant Viruses as Vectors for Mucosal Immunity

The development and characterization of viral based vaccine vectors is extremely active research field. Much of this work has been facilitated by developments in molecular biology that allow work with large plasmid-based vectors, as well as the use of PCR. Several different vector systems are now available using RNA viruses and DNA viruses. Each vector system has its own strengths and weaknesses. Due to the differences and diversity between the viruses used as vectors, it is doubtful that a single system will be useful for all desired vaccines. However, the further development of existing, as well as potentially new systems, will provide a repertoire for vaccinologists to design the recombinant vaccine which will generate an optimal humoral and immune response for protection against infection or disease caused by pathogens that infect via mucosal surfaces.

Expression of poliovirus P3 proteins using a recombinant vaccinia virus results in proteolytically active 3CD precursor protein without further processing to 3Cpro and 3Dpol

The expression of the poliovirus genome occurs by the translation of a single open reading frame to generate a long polyprotein which is subsequently processed by viral encoded proteases. The initial proteolytic cleavages result in the production of a P1 polyprotein which contains the capsid proteins, and the P2 and P3 polyproteins which contain proteins required for replication. The P3 polyprotein consists of the 3AB protein (containing the viral genome-linked protein, VPg), the viral protease, 3Cpro, and RNA polymerase, 3Dpol. To further study the expression and proteolytic processing of poliovirus P3 proteins in vivo, we have utilized recombinant vaccinia virus vectors to express nucleotides 5240-7400 containing the P3 region proteins of poliovirus. The P3 protein expressed from the recombinant vaccinia virus VV-P3 exhibited in vivo proteolytic activity as evident by processing of the polyprotein to generate the 3CD protein, consisting of a fusion between the 3Cpro and 3Dpol proteins. Further processing of the 3CD protein to 3Cpro and 3Dpol, however, was not detected in cells infected with VV-P3. Subcellular fractionation of VV-P3-infected cells demonstrated that the 3CD protein was present in both the soluble and membrane fractions. Finally, the 3CD protein expressed from VV-P3 was stable in cells co-infected with VV-P3 and poliovirus and no further processing to 3Dpol was detected. These results are discussed with regards to in vivo studies which suggest that the 3CD polyprotein is not a precursor to 3Dpol in poliovirus-infected cells.

[19] New methods to study poliovirus assembly and encapsidation of genomic RNA

Publisher Summary This chapter describes new methods to study poliovirus assembly and the encapsidation of genomic RNA. Poliovirus, a member of the Picornaviridae family, is a small nonenveloped icosahedral virus that encapsidates a single genome molecule of the positive sense. The capsid shell that encloses the viral RNA genome is composed of 60 copies of each of four structural proteins: VP1 (33 kDa), VP2 (30 kDa), VP3 (26 kDa), and VP4 (7.5 kDa). The availability of an infectious clone for poliovirus has made it possible to use molecular genetics for dissecting the assembly and encapsidation process. This chapter describes the growth and characterization of the recombinant vaccinia viruses that express the poliovirus capsid precursor protein and the 3CD proteinase and the use of these recombinant vectors to study P1 cleavage and the assembly of subviral particles. The chapter discusses the use of the recombinant vaccinia virus expressing the P1 capsid precursor in conjunction with the defective poliovirus genome to study the individual steps of poliovirus assembly and encapsidation. The derivation of recombinant vaccinia viruses VV-P1, which expresses the poliovirus P1 capsid precursor protein, and VV-P3, which expresses the poliovirus 3CD proteinase, is described in the chapter.