Search

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of viral immune evasion, targeting intrinsic, innate, and adaptive immunity. We have employed two orthogonal multiplexed tandem mass tag-based proteomic screens to identify host proteins down-regulated by viral factors expressed during the latest phases of viral infection. This approach revealed that the HIV-1 restriction factor Schlafen-11 (SLFN11) was degraded by the poorly characterized, late-expressed HCMV protein RL1, via recruitment of the Cullin4-RING E3 Ubiquitin Ligase (CRL4) complex. SLFN11 potently restricted HCMV infection, inhibiting the formation and spread of viral plaques. Overall, we show that a restriction factor previously thought only to inhibit RNA viruses additionally restricts HCMV. We define the mechanism of viral antagonism and also describe an important resource for revealing additional molecules of importance in antiviral innate immunity and viral immune evasion., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe disease following congenital infection and in immunocompromised individuals. No vaccines are licensed, and there are limited treatment options. We now show that the addition of anti-HCMV antibodies (Abs) can activate NK cells prior to the production of new virions, through Ab-dependent cellular cytotoxicity (ADCC), overcoming viral immune evasins. Quantitative proteomics defined the most abundant HCMV proteins on the cell surface, and we screened these targets to identify the viral antigens responsible for activating ADCC. Surprisingly, these were not structural glycoproteins; instead, the immune evasins US28, RL11, UL5, UL141, and UL16 each individually primed ADCC. We isolated human monoclonal Abs (mAbs) specific for UL16 or UL141 from a seropositive donor and optimized them for ADCC. Cloned Abs targeting a single antigen (UL141) were sufficient to mediate ADCC against HCMV-infected cells, even at low concentrations. Collectively, these findings validated an unbiased methodological approach to the identification of immunodominant viral antigens, providing a pathway toward an immunotherapeutic strategy against HCMV and potentially other pathogens.

Human cytomegalovirus (HCMV) extensively modulates host cells, downregulating >900 human proteins during viral replication and degrading ≥133 proteins shortly after infection. The mechanism of degradation of most host proteins remains unresolved, and the functions of many viral proteins are incompletely characterised. We performed a mass spectrometry-based interactome analysis of 169 tagged, stably-expressed canonical strain Merlin HCMV proteins, and two non-canonical HCMV proteins, in infected cells. This identified a network of >3400 virus-host and >150 virus-virus protein interactions, providing insights into functions for multiple viral genes. Domain analysis predicted binding of the viral UL25 protein to SH3 domains of NCK Adaptor Protein-1. Viral interacting proteins were identified for 31/133 degraded host targets. Finally, the uncharacterised, non-canonical ORFL147C protein was found to interact with elements of the mRNA splicing machinery, and a mutational study suggested its importance in viral replication. The interactome data will be important for future studies of herpesvirus infection., Competing Interests: LN, KN, BR, RA, LS, JN, JD, SS, EW, AD, GW, RS, EH, MW No competing interests declared, (© 2019, Nobre et al.)

Human cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8 + HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors., Competing Interests: The authors declare no conflict of interest.

A systematic quantitative analysis of temporal changes in host and viral proteins throughout the course of a productive infection could provide dynamic insights into virus-host interaction. We developed a proteomic technique called "quantitative temporal viromics" (QTV), which employs multiplexed tandem-mass-tag-based mass spectrometry. Human cytomegalovirus (HCMV) is not only an important pathogen but a paradigm of viral immune evasion. QTV detailed how HCMV orchestrates the expression of >8,000 cellular proteins, including 1,200 cell-surface proteins to manipulate signaling pathways and counterintrinsic, innate, and adaptive immune defenses. QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets. Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined. QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The reactivation of latent human cytomegalovirus (HCMV) infection after transplantation is associated with high morbidity and mortality. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, whose establishment and/or maintenance require expression of the viral transcript UL138. Using stable isotope labeling by amino acids in cell culture-based mass spectrometry, we found a dramatic UL138-mediated loss of cell surface multidrug resistance-associated protein-1 (MRP1) and the reduction of substrate export by this transporter. Latency-associated loss of MRP1 and accumulation of the cytotoxic drug vincristine, an MRP1 substrate, depleted virus from naturally latent CD14(+) and CD34(+) progenitors, all of which are in vivo sites of latency. The UL138-mediated loss of MRP1 provides a marker for detecting latent HCMV infection and a therapeutic target for eliminating latently infected cells before transplantation.

The proportion of human peripheral blood CD8+ T cells that are CD57+ CD28- is low at birth but increases with age and in individuals infected with human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV). These CD57+ CD28- CD8+ T cells contain large oligoclonal T-cell expansions whose antigen specificity is unknown. We identified clonal expansions of virus-specific memory cytotoxic T-lymphocyte precursors (CTLp) in both healthy carriers of HCMV and in asymptomatic HIV-infected subjects. In each subject, from the T-cell receptor (TCR) beta-chain hypervariable sequence of each immunodominant CTL clone, we designed complementary oligonucleotide probes to quantify the size and phenotypic segregation of individual virus-specific CTL clones in highly purified populations of peripheral blood CD8+ T cells. We found large clonal expansions of virus-specific CTL clonotypes in CD57+ CD28- CD8+ T cells. Using limiting dilution analysis, we found functional peptide-specific CTLp at high frequency in CD57+ CD28- cells. Thus, memory CTL specific for persistent viruses account for many oligoclonal expansions within CD57+ CD28- CD8+ T cells.

Human cytomegalovirus (HCMV)-specific CD8(+) cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vbeta segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo.

Cytotoxic T lymphocytes (CTL) appear to play an important role in the control of human cytomegalovirus (HCMV) in the normal virus carrier: previous studies have identified peripheral blood CD8+ CTL specific for the HCMV major immediate-early gene product (IE1) and more recently, by bulk culture and cloning techniques, have identified CTL specific for a structural gene product, the lower matrix protein pp65. In order to determine the relative contributions of CTL which recognize the HCMV proteins IE1, pp65, and glycoprotein B (gB) to the total HCMV-specific CTL response, we have used a limiting-dilution analysis system to quantify HCMV-specific CTL precursors with different specificities, allowing the antigenic specificity of multiple short-term CTL clones to be assessed, in a group of six healthy seropositive donors. All donors showed high frequencies of HCMV-specific major histocompatibility complex-restricted CTL precursors. There was a very high frequency of CTL specific for pp65 (lower matrix protein); IE1-specific CTL were also detectable at lower frequencies in three of five donors, while CTL directed to gB were undetectable. A pp65 gene deletion mutant of HCMV was then used to estimate the contribution of pp65-specific CTL to the total HCMV-specific CTL response; this showed that between 70 and 90% of all CTL recognizing HCMV-infected cells were pp65 specific. Analysis of the peptide specificity of pp65-specific CTL showed that some donors have a highly focused response recognizing a single peptide; the T-cell receptor Vbeta gene usage in these two donors was shown to be remarkably restricted, with over half of the responding CD8+ T cells utilizing a single Vbeta gene rearrangement. Other subjects recognized multiple pp65 peptides: nine new pp65 CTL peptide epitopes were defined, and for five of these the HLA-presenting allele has been identified. All four of the HLA A2 donors tested in this study recognized the same peptide. This apparent domination of the CTL response to HCMV during persistent infection by a single structural protein, irrespective of major histocompatibility complex haplotype, is not clearly described for other persistent virus infections, and the mechanism requires further investigation.