6/5/2023 0 Comments Protein scaffold mollusk![]() ![]() ![]() We obtained crystals of ORF68 that diffracted X-rays to a maximum resolution of 2.22 Å and used molecular replacement with the initial cryo-EM model to determine the structure of ORF68. However, as ORF68 bears no sequence homology to proteins outside of the Herpesviridae, de novo modeling was challenging. A cryo-EM reconstruction of the pentamer was determined to 3.37 Å, from which an alanine backbone model was built ( Figure 1-figure supplement 1b). In agreement with our prior observation that ORF68 forms a multimer in vitro ( Gardner and Glaunsinger, 2018), negative stain electron microscopy (EM) revealed rings comprised of five subunits ( Figure 1-figure supplement 1a). To structurally characterize KSHV ORF68, we purified the full-length protein from transiently transfected HEK293T cells. We hypothesize that the viral genome is threaded through the ORF68 ring, and that ORF68 acts as a scaffold on which the terminase assembles for genome packaging. The central channel of ORF68 is lined with positively charged residues that are necessary for nucleic acid binding and production of infectious virions. The similarity of these structures, combined with negative stain electron microscopy of homologs from HSV-1 and HCMV, suggests that this topology is conserved across the Herpesviridae. We reveal the structure of KSHV ORF68 and its homolog in EBV (BFLF1), which adopt a novel fold and assemble into homopentameric rings. Here, we applied a combination of structural biology and biochemistry to better define the role of the essential accessory protein ORF68 in KSHV packaging. Phages lack an identifiable homolog of U元2 or ORF68, suggesting that an additional level of complexity exists in herpesvirus packaging. In contrast, despite observations that U元2 and its homologs in HCMV (UL52) and KSHV (ORF68) are essential for production of packaged virions, their function in packaging remains unknown ( Albright et al., 2015 Gardner and Glaunsinger, 2018 Lamberti and Weller, 1998 Borst et al., 2008). UL17 encodes a capsid vertex-specific protein important for stabilizing the capsid ( Gong et al., 2019 Grzesik et al., 2017 Liu et al., 2019). Three of these proteins (UL15/UL28/U元3) form the terminase motor, and the portal protein is composed of a dodecamer of UL6 ( Newcomb et al., 2001 Patel et al., 1996). Packaging minimally requires recognition of the viral genome by the terminase, docking of the terminase-bound genome at the portal of a nascent capsid, translocation of the genome into the capsid by the terminase, and cleavage to release the remaining unpackaged concatemeric genome.Ĭleavage and packaging in the herpesviruses, best studied in HSV-1, requires six conserved proteins in addition to the nascent capsid and concatemeric genome ( Heming et al., 2017): HSV-1 UL6, UL15, UL17, UL28, U元2, and U元3. ![]() The large subunit of the terminase is the most conserved gene across the herpesviruses and possesses sequence and structural similarity to phage terminases, supporting the hypothesis that packaging occurs through an evolutionarily ancient mechanism ( Rixon and Schmid, 2014 Nadal et al., 2010 Selvarajan Sigamani et al., 2013). Furthermore, both depend on a ‘terminase’ motor responsible for packaging and cleavage of the genome. Despite infecting hosts in different kingdoms, both groups of viruses use an icosahedral capsid and an architecturally similar portal protein through which DNA is packaged ( Rixon and Schmid, 2014 Dedeo et al., 2019). DNA packaging in tailed bacteriophages is thought to be mechanistically similar to that of herpesviruses ( Rixon and Schmid, 2014). Cleavage to produce a unit-length genome is intimately tied to packaging and occurs only after that genome is successfully transferred into a capsid. Near the end of the lytic cycle, herpesviruses replicate their genome as a head-to-tail concatemer of linked genomes separated by terminal repeats. The human gamma-herpesviruses Kaposi’s sarcoma-associated virus (KSHV) and Epstein–Barr virus (EBV) are oncogenic viruses, causing cancers such as primary effusion lymphoma and Kaposi’s sarcoma (in the case of KSHV).ĭespite 400 million years of evolution separating the human herpesviruses, several core pathways in replication are conserved ( McGeoch et al., 2006). Human cytomegalovirus (HCMV) is a betaherpesvirus that can cause mononucleosis and congenital birth defects. Herpes simplex virus type 1 (HSV-1) is an alphaherpesvirus that causes cold sores and genital sores. The ability of herpesviruses to efficiently evade the immune system and establish latency, coupled with few available treatments and vaccines, means that nearly all adults in the world harbor at least one of the nine human herpesviruses. Herpesviruses are large double-stranded DNA viruses that cause a variety of diseases in humans. ![]()
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