Protein Microarray Vaccinia Virus

A protein microarray relies on high-throughput amplification of each predicted ORF by using gene-specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system. The protein products from the unpurified reactions are printed directly onto nitrocellulose microarrays without further purification [113]. 

This approach was used to determine the complete antigen-specific humoral immune-response profile Ifom infected humans and animals. The vaccinia virus proteome containing 185 individual viral proteins was printed on a chip after cloning and expression. The chips were then used to determine the antibody profile in serum from vaccinia-virus-immunized humans, primates, and mice [113]. 

Once it has been developed and produced, a protein microarray can be a very rapid method (3 days for most of the genes) to comprehensively scan the humoral immune response of vaccinated or infected individuals. 

The generation of a complete proteome is technically challenging. One problem is the amplification of long genes. Furthermore, expression of some proteins in heterologous systems is not efficient. This technique also does not take into account PTM of viral proteins that are expressed in bacteria. Lastly, expression in E. coli might lead to folding problems of the protein. 

One aspect of viral proteomics has been the characterization of virus particles and virally infected cells. Characterization of purified virions has led to the identification of viral proteins that were not originally identified with the virion as well as the identification of cellular proteins associated with the purified virus. For example, analysis of HCMV viral particles identified 12 additional ORFs not previously known to reside in virions as well as the identification of 71 cellular proteins [6]. The importance of these cellular and viral proteins in viral replication or pathogenesis awaits further analysis. Additionally, 12 unique polypeptides were identified that did not correspond to previously identified ORFs [6], illustrating the fact that despite intensive sequence analysis, sequence characteristics of viral promoters and ORFs are still not entirely understood. Analysis of virally infected cells has also led to the characterization of events leading to EBV-induced transformation [9,10,47], identification of cellular proteins induced in HIV-infected 

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