Viral pathogens, such as vaccinia virus (VACV), hijack host signalling networks in order to

survive and propagate themselves. VACV is the prototypic member of the Poxviridae, a family of large double-stranded DNA viruses that include variola virus, the causative agent of

smallpox. For many pathogens, including VACV, the molecular mechanisms underlying

successful survival, but also cellular defence and pathogen clearance, are not well understood. Especially, detailed molecular knowledge about protein composition, abundance and nanoscale organisation within the surfaceome of infected cells is sparse. The knowledge about the involved signaling networks and extracellular protein interactions engaged during infection would enable rational engineering anti-viral strategies. Here, we investigated using

spatiotemporal analysis how VACV infection perturbs the host proteotype with specific focus

on longitudinal surfaceome changes.

Using our recently developed, automated and miniaturized Cell Surface Capture (autoCSC)

technology, we took relative quantitative proteotype surfaceome snapshots of VACV infected

Hela CCL2 cells across 24 hours of the infectious life cycle of VACV. AutoCSC, combined

with DIA-based mass spectrometric analysis, enabled the sensitive identification of the pool of cell surface-residing glycoproteins in a time-resolved manner. Subsequently, the longitudinal analysis allowed for the profiling of surfaceome proteotype dynamics during the viral life cycle.

We observed moderate changes within the host surfaceome post-infection, but additionally

detected a set of viral glycoproteins. These VACV proteins are translated and

modified/glycosylated by the host’s cellular machinery. Subsequently, they relocalize into the

plasma membrane. In order to elucidate the functional role of these viral proteins at the host

surfaceome, we investigated their trans-interaction/receptor space applying chemo-proteomic

technologies. Together, we found that the cell’s acute infection state is characterized by a repopulation of the host surfaceome with viral glycoproteins which supports VACV survival and eventually immune evasion.