Human cytomegalovirus (HCMV) is a large double-stranded DNA virus and member of the β-herpesvirus family. HCMV is ubiquitous in the human population and causes lifelong infections. HCMV infection is associated with high morbidity and mortality in immunocompromised individuals and the virus is a major cause of virus-mediated congenital disease. There have been a number of HCMV entry receptors identified that use one of two viral receptor binding complexes, including the gH/gL/gO complex and the pentamer made up of gH/gL/UL128/UL130/UL131a. Cytomegaloviruses (CMVs) are typically host-restricted requiring the use of species-specific modeling and culture conditions. We use rat CMV (RCMV) to study CMV-accelerated vascular disease and chronic allograft rejection. RCMV encodes homologous versions of the entry complex proteins but their incorporation and copy number per virion are still unknown. In this methods article, we describe a novel approach of HiBiT tagging viral proteins in order to detect and quantify protein incorporation into particles. This method is independent of protein-specific antibodies and can be standardized using a commercially available HiBiT protein standard. Using bacterial artificial chromosome (BAC) recombineering, we have constructed two individual viruses containing a HiBiT tag fused to the C'-terminus of either the UL128 homolog (R129) or the UL130 homolog (R131). Viruses containing these mutations were rescued, purified and analyzed. Our data demonstrate that R129 and R131 are both incorporated into RCMV virions at equimolar ratios relative to genome copy number, supporting this antibody-free approach for quantifying viral protein incorporation and its application toward the identification of domains required for incorporation.
Keywords: Cytomegalovirus; HiBiT; Pentameric entry complex; Viral entry; nanoLuc.