Visualization of giant virus particles using BONCAT labeling and STED microscopy

Giant DNA viruses of the phylum Nucleocytoviricota are being increasingly recognized as important regulators of natural protist populations. However, our knowledge of their infection cycles is still very limited due to a lack of cultured virus-host systems and molecular tools to study them. Here, we apply bioorthogonal noncanonical amino acid tagging (BONCAT) to pulse label the marine heterotrophic flagellate Cafeteria burkhardae during infection with the lytic giant virus CroV. In absence of CroV, we report efficient incorporation of the L-methionine analog L-azidohomoalanine (AHA) into newly synthesized proteins of the methionine prototrophic C. burkhardae. During CroV infection, AHA was predominantly found in viral proteins, and single CroV virions were imaged with stimulated emission depletion (STED) super-resolution microscopy. CroV particles incorporated AHA with 95-100% efficiency while retaining their infectivity, which makes BONCAT/STED a powerful tool to study viral replication cycles in this ecologically relevant marine bacterivore. SignificanceGiant DNA viruses are the dominant class of protist-infecting viruses, yet the vast majority of described giant virus-protist systems remain uncultured. One of the better studied cultured systems is composed of the stramenopile Cafeteria burkhardae (previously C. roenbergensis), the giant Cafeteria roenbergensis virus (CroV) and the virophage mavirus. C. burkhardae is a widespread marine phagotrophic protist that plays an important role in regulating bacterial populations. In addition to being grazed upon by larger zooplankton, C. burkhardae populations are controlled by the lytic giant virus CroV. In turn, CroV is parasitized by the virophage mavirus that increases host population survival in the presence of CroV and forms a mutualistic symbiosis with its host. Despite being of fundamental ecological and evolutionary interest, this tripartite host-virus-virophage system suffers from a lack of molecular tools. Here, we show that CroV particles can be fluorescently labeled and imaged by super-resolution microscopy. To achieve this we established robust procedures for analyzing protist and viral populations and implemented the use of bioorthogonal noncanonical amino acid tagging (BONCAT) in a marine unicellular flagellate.