Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication

Abstract

Human cytomegalovirus (HCMV) in clinical material cannot replicate efficiently in vitro until it has adapted by mutation. Consequently, wild-type HCMV differ fundamentally from the passaged strains used for research. To generate a genetically intact source of HCMV, we cloned strain Merlin into a self-excising BAC. The Merlin BAC clone had mutations in the RL13 gene and UL128 locus that were acquired during limited replication in vitro prior to cloning. The complete wild-type HCMV gene complement was reconstructed by reference to the original clinical sample. Characterization of viruses generated from repaired BACs revealed that RL13 efficiently repressed HCMV replication in multiple cell types; moreover, RL13 mutants rapidly and reproducibly emerged in transfectants. Virus also acquired mutations in genes UL128, UL130, or UL131A, which inhibited virus growth specifically in fibroblast cells in wild-type form. We further report that RL13 encodes a highly glycosylated virion envelope protein and thus has the potential to modulate tropism. To overcome rapid emergence of mutations in genetically intact HCMV, we developed a system in which RL13 and UL131A were conditionally repressed during virus propagation. This technological advance now permits studies to be undertaken with a clonal, characterized HCMV strain containing the complete wild-type gene complement and promises to enhance the clinical relevance of fundamental research on HCMV.

Stimulatory effects of human cytomegalovirus tegument protein pp71 lead to increased expression of CCL2 (monocyte chemotactic protein-1) during infection

“…Preparation of virus stocks. The bacterial artificial chromosome (BAC) recombinant of CMV clinical strain Merlin (clone pAL1120) containing the full complement of the CMV genome was provided by Richard Stanton (Cardiff University, Cardiff, UK) (Stanton et al, 2010). Merlin-BAC DNA was extracted from bacterial culture using a Nucleobond BAC 100 extraction kit (Macherey-Nagel) and then transfected into MRC-5 fibroblasts using Lipofectamine 2000 (Life Technologies).…”

The susceptibility of primary cultured rhesus macaque kidney epithelial cells to rhesus cytomegalovirus strains

“…In HCMV, clinical strains contain a UL/b¢ region (UL128-UL150) that encodes multiple ORFs involved in cell-type dependent replication, intra-host dissemination, latency and immune evasion (Bradley et al, 2009;Cheung et al, 2005;Goodrum et al, 2007;Hahn et al, 2004;Penfold et al, 1999;Prod'homme et al, 2010;Ryckman et al, 2006Ryckman et al, , 2008Sinzger et al, 2008;Stanton et al, 2010;Tomasec et al, 2005;Umashankar et al, 2011). As viral determinants for HCMV entry of EP, UL128L-encoded proteins, form a pentamer with gH and gL mediating HCMV entry into EP via an endocytotic pathway (Bodaghi et al, 1999;Ryckman et al, 2008;Wang & Shenk, 2005;Wang et al, 2007).…”

“…Notably, the epithelial/endothelial determinant locus (UL128-UL130-UL131A; hereafter UL128L) is especially susceptible to genetic alterations after only a few passages in cultured fibroblasts (Akter et al, 2003;Bradley et al, 2009;Cha et al, 1996;Cunningham et al, 2010;Hahn et al, 2004;Prichard et al, 2001;Ryckman et al, 2006Ryckman et al, , 2008Sinzger et al, 2008;Stanton et al, 2010). Proteins encoded by UL128L, together with glycoprotein H (gH) and L (gL), form a gH-anchored pentamer complex that mediates HCMV entry of EP and endothelial cells via receptor-dependent endocytosis.…”

The immunological underpinnings of vaccinations to prevent cytomegalovirus disease

“…A different 'dial-up' strategy seeks to improve the AD169 LACV by repairing mutations that prevent expression of the gH pentamer complex, 108,109 with the intention of broadening cell tropism and the quality of neutralizing antibody responses. One challenge is the loss of gH pentamer expression during propagation in fibroblasts, 110,111 so the LACV candidate V160 must be propagated on the ARPE19 109,112 epithelial cell line. The V160 LACV shows promise and induces pentamer-specific responses in rabbits and rhesus macaques 113,114 in preclinical studies that support an ongoing phase I evaluation.…”