Interrogating marine virus‐host interactions and elemental transfer with BONCAT and nanoSIMS‐based methods

Abstract

Summary While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus‐host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single‐virus level. We describe two new complementary approaches – stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence‐based biorthogonal non‐canonical amino acid tagging (BONCAT) – for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207, Synechococcus sp. WH8101/Syn1 and Escherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labelling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean.

Visualization of giant virus particles using BONCAT labeling and STED microscopy

“…The highly efficient AHA labeling of CroV particles (>95%) is in concordance with results from other virus-host systems, for instance, an 87-94% labeling efficiency was reported for EhV207 (Pasulka et al , 2018), >99% for herpes simplex virus (Serwa et al , 2019) and 90% for vaccinia virus (Huang et al , 2017). BONCAT can therefore be applied to a wide range of viruses.…”

“…BONCAT labeling with AHA exploits this hallmark property of viral infection by preferentially labeling viral proteins during a specific phase of infection and in combination with STED microscopy, we show that visualization of individual CroV particles is possible. Although BONCAT has been applied to viruses such adenovirus (Banerjee et al , 2011), vaccinia virus (Huang et al , 2017) and Emiliania huxleyi virus (EhV207) (Pasulka et al , 2018), we image individual giant virus particles for the first time using BONCAT in combination with super-resolution microscopy.…”

“…Although AHA and its alkyne analog L-homopropargylglycine (HPG) have been mostly used in methionine auxotrophic organisms to increase the efficiency of incorporation (Wisse et al , 2017;Zhang et al , 2017;Saleh et al , 2019), its use has recently been extended to the analysis of natural bacterial communities, resulting in good incorporation in active methionine prototrophic bacteria (Hatzenpichler et al , 2014;Leizeaga et al , 2017;Couradeau et al , 2019). In addition, four wild-type prototrophic eukaryotic organisms were reported to incorporate AHA or HPG: Danio rerio (Hinz et al , 2012), Caenorhabditis elegans (Ullrich et al , 2014), Arabidiopsis thaliana (Glenn et al , 2017) and the coccolithophore Emiliania huxleyi (Pasulka et al , 2018).…”

Nitrogen sourcing during viral infection of marine cyanobacteria

“…Pasulka et al (46) recently documented enhanced incorporation of 15 N into Syn1 viral particles using NanoSIMS during a multiday infection experiment using Synechococcus WH8101 as a host but with the extended duration of that experiment and the inability to measure isotopic compositions of specific host-and virus-derived biomolecules that study could not differentiate between concurrent labeling of host and phage proteins and direct incorporation of the exogenous nitrogen into viral particles. We have shown that such labeling of viral biomolecules by an extracellular isotope tracer can occur in the absence of detectable incorporation of the label into host biomass, clearly demonstrating direct transfer of dissolved nutrients to viral particles.…”

Dynamic marine viral infections and major contribution to photosynthetic processes shown by spatiotemporal picoplankton metatranscriptomes

“…The rise of next-generation sequencing and metagenomics made the study of environmental phages more accessible 6,26,[30][31][32] . In recent years new techniques for the study of viruses, such as vSAGs, polonies, and viral-BONCAT, were introduced, and promise to move the field further into the realm of culture-independent research 31,[33][34][35] .…”