Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

Abstract

Orthopoxviruses are among the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, two orthopoxviruses with different pathogenic potentials, human monkeypox virus and vaccinia virus, were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest™ surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by high-resolution reversed-phase nano-LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST® and X! Tandem resulted in the confident identification of hundreds of monkeypox, vaccinia, and copurified host-cell proteins. The unfractionated samples were additionally analyzed by LC-MS on an LTQ-Orbitrap™, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially abundant Orthopoxvirus proteins are discussed. Data, processed results, and protocols are available at http://www.proteomicsresource.org/.

Cowpox Virus Expresses a Novel Ankyrin Repeat NF-κB Inhibitor That Controls Inflammatory Cell Influx into Virus-Infected Tissues and Is Critical for Virus Pathogenesis

“…Although the proteomic components of the CPXV virion have not yet been reported, MPXV-003, the MPXV homolog of VARV G1R and CPXV-006, was recently identified as being virion associated in a comparative proteomic analysis of MPXV virion particles using liquid chromatography coupled with (tandem) mass spectrometry (25). Given both sequence and functional similarities between MPXV-003 and CPXV-006 (27), we suggest that CPXV-006 is thus likely to be virion associated.…”

Poxvirus Proteomics and Virus-Host Protein Interactions

“…Due to renewed interest in studies of poxvirus pathogenesis, the proteomes of two orthopoxviruses with differing pathogenic potentials, monkeypox virus (MPXV) and VACV, were compared with the goal of identifying proteins that might influence pathogenesis (98). In that study, both MV and EV infectious particles from MPXV and VACV were produced in HeLa cells and purified using sucrose gradient ultracentrifugation.…”

“…These virions were determined to be Ͼ98% pure. MVs and EVs were analyzed by LC-MS/MS, in contrast to previous studies that analyzed only VACV MVs (30,78,134,163,179), resulting in the identification of a total of 164 viral proteins, from both MPXV and VACV, as being virion associated (i.e., proteins bound to or contained within the virion) (98). Due to a larger number of samples for MPXV (seven MPXV samples versus three VACV samples), more MPXV peptides were identified than VACV peptides.…”

Accelerated immune senescence and reduced response to vaccination in ovariectomized female rhesus macaques

“…MVA virus was propagated in chicken embryonic fibroblast cells (CEF; Charles River Avian Services) and purified by ultracentrifugation over a 36% sucrose gradient as described for other orthopox viruses (Manes et al 2008). Virus titers were determined by standard plaque assay on CEF cells.…”