Deletion of the Human Cytomegalovirus US17 Gene Increases the Ratio of Genomes per Infectious Unit and Alters Regulation of Immune and Endoplasmic Reticulum Stress Response Genes at Early and Late Times after Infection

Abstract

Human cytomegalovirus (HCMV) employs numerous strategies to combat, subvert, or co-opt host immunity. One evolutionary strategy for this involves capture of a host gene and then its successive duplication and divergence, forming a family of genes, many of which have immunomodulatory activities. The HCMV US12 family consists of 10 tandemly arranged sequence-related genes in the unique short (US) region of the HCMV genome (US12 to US21). Each gene encodes a protein possessing seven predicted transmembrane domains, patches of sequence similarity with cellular G-protein-coupled receptors, and the Bax inhibitor 1 family of antiapoptotic proteins. We show that one member, US17, plays an important role during virion maturation. Microarray analysis of cells infected with a recombinant HCMV isolate with a US17 deletion (the ⌬US17 mutant virus) revealed blunted host innate and interferon responses at early times after infection (12 h postinfection [hpi]), a pattern opposite that previously seen in the absence of the immunomodulatory tegument protein pp65 (pUL83). Although the ⌬US17 mutant virus produced numbers of infectious particles in fibroblasts equal to the numbers produced by the parental virus, it produced >3-fold more genome-containing noninfectious viral particles and delivered increased amounts of pp65 to newly infected cells. These results suggest that US17 has evolved to control virion composition, to elicit an appropriately balanced host immune response. At later time points (96 hpi), ⌬US17 mutant-infected cells displayed aberrant expression of several host endoplasmic reticulum stress response genes and chaperones, some of which are important for the final stages of virion assembly and egress. Our results suggest that US17 modulates host pathways to enable production of virions that elicit an appropriately balanced host immune response.

Inactivation of the Human Cytomegalovirus US20 Gene Hampers Productive Viral Replication in Endothelial Cells

“…In this regard, the intracellular localization of the US14, US16, US17, and US18 proteins was determined by immunofluorescence analyses that revealed an association with the cytoplasmic virion assembly compartment (cVAC), thus suggesting that their functions may be linked to virion maturation and egress (13,14). In support of this hypothesis, it was observed that inactivation of the US17 gene in producer fibroblasts results in increased production of noninfectious viral particles that can, in turn, deliver augmented amounts of the pp65 immunomodulatory tegument protein to newly infected cells, thus altering the regulation of both intrinsic and innate responses of cells infected with the US17-deficient virus (15). These data suggest a role of US17 in regulating adequate virion composition during HCMV maturation (15).…”

“…In support of this hypothesis, it was observed that inactivation of the US17 gene in producer fibroblasts results in increased production of noninfectious viral particles that can, in turn, deliver augmented amounts of the pp65 immunomodulatory tegument protein to newly infected cells, thus altering the regulation of both intrinsic and innate responses of cells infected with the US17-deficient virus (15). These data suggest a role of US17 in regulating adequate virion composition during HCMV maturation (15). Interestingly, two other US12 family members, US18 and US20, were recently shown to affect in fibroblasts the expression of the major histocompatibility complex class I (MHC-I) chain-related molecule (MICA), an NKG2D ligand induced by HCMV infection (16).…”

“…In fact, inactivation of different members may affect the normal virion composition (15) and the virus's ability to modulate the host immune response (16) or, as we reported for US16 and US20, may impact viral replication in specific cell types. Our current knowledge about the functions of pUS12 proteins is therefore coherent with the hypothesis that individual US12 gene family members have evolved the ability to fulfill different and specific functions by adapting a host-derived common and flexible structural protein scaffold, one that is intimately associated with various cellular membranes, to new purposes (11,51).…”

Loss of the Human Cytomegalovirus US16 Protein Abrogates Virus Entry into Endothelial and Epithelial Cells by Reducing the Virion Content of the Pentamer

“…In fact, inactivation of some US12 gene members affects virusmediated manipulation of the host innate immune response (37,38) or, as we reported for US16 (12) and US20 (39), HCMV cell tropism. However, irrespective of the final outcome for virus-host cell interactions, the functions of several US12 proteins appear to be related to the late stages of HCMV maturation and the fine-tuning of the most appropriate protein composition of virions (38; this study).…”

High-Throughput Analysis of Human Cytomegalovirus Genome Diversity Highlights the Widespread Occurrence of Gene-Disrupting Mutations and Pervasive Recombination

“…The natural occurrence of mutants in US6, US7, and US9 may suggest some functional redundancy, as the US2, US3, US10, and US11 genes all target MHC-I antigen presentation (110). The US12 gene family encodes 10 seven-transmembrane proteins, with some members recently being associated with immunomodulation (111,112) and cell tropism for (1) a n ϭ 128, unless stated otherwise (mutations that are certain to be artifacts of culture passage are omitted). b del, deletion; ins, insertion; sub, substitution; cod, codon (codon refers to the actual codon position in the specific strain that contains the mutation).…”