Abstract
The human cytomegalovirus (HCMV) protein RL13 has recently been described to be present in all primary isolates but rapidly mutated in culture adapted viruses. Although these data suggest a crucial role for this gene product in HCMV primary infection, no function has so far been assigned to this protein. Working with RL13 expressed in isolation in transfected human epithelial cells, we demonstrated that recombinant RL13 from the clinical HCMV isolates TR and Merlin have selective human immunoglobulin (Ig)-binding properties towards IgG1 and IgG2 subtypes. An additional Fc binding protein, RL12, was also identified as an IgG1 and IgG2 binding protein but not further characterized. The glycoprotein RL13 trafficked to the plasma membrane where it bound and internalized exogenous IgG or its constant fragment (Fcγ). Analysis of RL13 ectodomain mutants suggested that the RL13 Ig-like domain is responsible for the Fc binding activity. Ligand-dependent internalization relied on a YxxL endocytic motif located in the C-terminal tail of RL13. Additionally, we showed that the tyrosine residue could be replaced by phenylalanine but not by alanine, indicating that the internalization signal was independent from phosphorylation events. In sum, RL13 binds human IgG and may contribute to HCMV immune evasion in the infected host, but this function does not readily explain the instability of the RL13 gene during viral propagation in cultured cells.
Highlights
Human cytomegalovirus (HCMV) infection is common and, typically subclinical in the healthy population, it can cause severe disease in congenitally infected infants and in individuals with suppressed immunity [1]
HEK293T cells were transfected with expression plasmids for myc-His tagged gB, RL10, RL11, RL12, RL13. 48 h after transfection, flow cytometry analysis was performed with DyLight 649-conjugated human Fcc and FITCconjugated anti-myc
Expression of the different proteins was equal as demonstrated by comparable percentage of FITC positive populations and their mean fluorescence intensity. These data suggested that RL12 and RL13 are two previously unidentified human Fcc binding proteins encoded by HCMV
Summary
Human cytomegalovirus (HCMV) infection is common and, typically subclinical in the healthy population, it can cause severe disease in congenitally infected infants and in individuals with suppressed immunity [1]. Comparative sequencing analysis of unpassaged clinical isolates versus cell culture adapted viruses allowed a more refined identification of the genetic changes corresponding to functions that are lost in in vitro cultures This is the case of RL13 and UL128 gene products, both possessing a suppressive phenotype on tissue culture adapted viruses [4,5]. For RL13 in particular, Stanton et al recently reported rapidly emerging genetic mutations following a few passages of BACderived Merlin strain virus in cultures of human cells of different origins [6] These authors, using an elegant BAC system of conditional gene repression during virus propagation, were able to show that virus with reconstructed wild type RL13 repressed cell culture growth while the emerging deletion mutants allowed the virus to adapt to cell culture growth [6]. Providing that a functional RL13 gene appears to be carried by all sequenced clinical isolates, the authors hypothesized that this protein is critical for productive HCMV replication in vivo, perhaps increasing the repertoire of HCMV cell tropism [6]
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