Immunomodulation of Cowpox Virus

Cowpox virus (CPXV) is a double stranded DNA virus belonging to the family Poxviridae, genus Orthopoxvirus. Poxviruses carry a large number of genes encoding immunomodulatory proteins which are used to escape the defense mechanisms of the host’s immune system. Targeting various mechanisms of innate and acquired immunity, poxviruses are experts of mimicry, interaction and escape. To avoid the disadvantages of classical mutant virus production protocols, which include long generation times, high costs and remaining marker sequences, the complete genome of CPXV was cloned as a Bacterial Artificial Chromosome (BAC) in E. coli. One BAC clone contains mini-F sequences including the gfp gene (pBRf) that are maintained after virus reconstitution, a fact that allows the tracking of virus via fluorescence detection. A second clone was designed to make the gfp gene “self-excisable”: A duplication of sequences enabled a homologous recombination upon reconstitution in mammalian cells, which resulted in the excision of all non-CPXV sequences (pBR). Reconstituted virus vBR is equivalent to wild-type virus and does not carry any marker sequences. In addition, mutant viruses can be generated with markerless recombination techniques in the BAC system, giving rise to mutant viruses with alterations of the virus sequence at the desired site only. As an example for viral proteins interacting with the host’s immune system, the 38 kDa protein cytokine response modifier A (CrmA), also named SPI-2, was chosen for further studies. The serine protease inhibitor (serpin) has anti-apoptotic and anti-inflammatory functions and was detected early on as the protein responsible for the red hemorrhagic phenotype of pocks on chicken chorioallantoic membranes (CAMs). Few poxviruses, namely CPXV, monkeypox virus (MPXV), and rabbitpox virus (RPXV) cause red lesions on CAMs whereas white pocks, mainly characterized by the influx of inflammatory cells, can be seen in other poxvirus infections. Methionine deletion mutants of two putative start codons were generated introducing markerless minimal modifications. Our investigations revealed that only the first methionine is used for the protein synthesis of crmA. Poxviral antigen was shown to accumulate in endothelial cells of CAMs after infection only after infection with CrmA-positive viruses, indicating that CrmA mediates endotheliotropism. A mutant virus with CrmA expressed in the background of the highly attenuated vaccine strain modified vaccinia virus Ankara (MVA) caused white and not red pocks on CAMs despite confirmation of CrmA expression by western blot analyses. We concluded that CrmA alone is not sufficient for the development of red hemorrhagic pocks and the accumulation of virus in endothelial cells and that at least one other viral factor is necessary for the phenomenon.