Improving the physiological relevance of HIV CLEM studies via duck circovirus HUH-tagging of Gag
Correlative light and electron microscopy (CLEM) is a powerful multi-method approach that allows for rapid identification and location of fluorescently-tagged constructs before cryo-electron tomography, reducing time required for tomographic imaging in the cellular context and providing molecular confirmation of the imaged subject. Fluorescent labeling of viral structural proteins, such as the HIV Gag polyprotein used in this study, suffers from perturbations to proper virion formation due to steric hindrance caused by the fluorescent tag, reducing the physiological relevance of such constructs. HIV Gag tagged with fluorescent proteins requires recovery with an excess of wild-type Gag to properly form HIV virions, but the GFP-tagged Gag is excluded from the Gag lattice in the immature and mature virions, preventing the use of CLEM. HUH-tags are small endonucleases (11.3-36.4 kD), typically involved in rolling-circle replication of viruses, that exhibit specific cleavage of ssDNA by forming a covalent phosphotyrosine linkage with a nucleotide within their recognition sequence. When used with quencher-fluorophore conjugated oligonucleotides, HUH endonucleases can act as molecular tags. This study utilizes the duck circovirus (DCV) HUH-tag to replace GFP as an internal tag of the Gag polyprotein via molecular cloning in order to eliminate the need for wild-type recovery and generate a fluorescently-tagged functional Gag lattice. CLEM studies utilizing this construct will exhibit greater physiological accuracy, expanding the arsenal of molecular tools for investigations of HIV assembly and maturation.