Paul J. Lehner

Quantitative temporal viromics: an approach to investigate host-pathogen interaction

Summary 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. PaperClip

Viral avoidance and exploitation of the ubiquitin system.

The versatility of ubiquitin in regulating protein function and cell behaviour through post-translational protein modification makes it a particularly attractive target for viruses. Here we review how viruses manipulate the ubiquitin system to favour their propagation by redirecting cellular ubiquitin enzymes or encoding their own ubiquitin components to enable replication, egress and immune evasion. These studies not only reveal the many cellular processes requiring ubiquitin but also illustrate how viruses usurp their host cells.

Efficient Internalization of MHC I Requires Lysine-11 and Lysine-63 Mixed Linkage Polyubiquitin Chains

The downregulation of cell surface receptors by endocytosis is a fundamental requirement for the termination of signalling responses and ubiquitination is a critical regulatory step in receptor regulation. The K5 gene product of Kaposis sarcoma‐associated herpesvirus is an E3 ligase that ubiquitinates and downregulates several cell surface immunoreceptors, including major histocompatibility complex (MHC) class I molecules. Here, we show that K5 targets the membrane proximal lysine of MHC I for conjugation with mixed linkage polyubiquitin chains. Quantitative mass spectrometry revealed an increase in lysine‐11, as well as lysine‐63, linked polyubiquitin chains on MHC I in K5‐expressing cells. Using a combination of mutant ubiquitins and MHC I molecules expressing a single cytosolic lysine residue, we confirm a functional role for lysines‐11 and ‐63 in K5‐mediated MHC I endocytosis. We show that lysine‐11 linkages are important for receptor endocytosis, and that complex mixed linkage polyubiquitin chains are generated in vivo.

Latency-Associated Degradation of the MRP1 Drug Transporter During Latent Human Cytomegalovirus Infection

Hide-and-Seek Virus Human cytomegalovirus (HCMV) establishes latent infection in human progenitor dendritic cells, causing significant morbidity and mortality on reactivation, which may occur in transplantation patients who are immunosuppressed. Neither detection nor selective removal of rare latent HCMV-infected cells has been possible. Weekes et al. (p. 199) have found that the multidrug-resistant ABC transporter, multidrug resistance–associated protein-1 (MRP1) is down-regulated during latent HCMV infection. Consequently, cytotoxic MRP1-specific substrates are not exported from HCMV-infected cells and accumulate—leading to cell death, which could potentially provide a mechanism for eliminating infected cells prior to transplantation. A virally encoded protein eliminates a cell surface drug transporter, promoting latent human cytomegalovirus infection. 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.

HRD1 and UBE2J1 target misfolded MHC class I heavy chains for endoplasmic reticulum-associated degradation.

The assembly of MHC class I molecules is governed by stringent endoplasmic reticulum (ER) quality control mechanisms. MHC class I heavy chains that fail to achieve their native conformation in complex with β2-microglobulin (β2m) and peptide are targeted for ER-associated degradation. This requires ubiquitination of the MHC class I heavy chain and its dislocation from the ER to the cytosol for proteasome-mediated degradation, although the cellular machinery involved in this process is unknown. Using an siRNA functional screen in β2m-depleted cells, we identify an essential role for the E3 ligase HRD1 (Synoviolin) together with the E2 ubiquitin-conjugating enzyme UBE2J1 in the ubiquitination and dislocation of misfolded MHC class I heavy chains. HRD1 is also required for the ubiquitination and degradation of the naturally occurring hemochromatosis-associated HFE-C282Y mutant, which is unable to bind β2m. In the absence of HRD1, misfolded HLA-B27 accumulated in cells with a normal MHC class I assembly pathway, and HRD1 depletion prevented the appearance of low levels of cytosolic unfolded MHC I heavy chains. HRD1 and UBE2J1 associate in a complex together with non-β2m bound MHC class I heavy chains, Derlin 1, and p97 and discriminate misfolded MHC class I from conformational MHC I-β2m-peptide heterotrimers. Together these data support a physiological role for HRD1 and UBE2J1 in the homeostatic regulation of MHC class I assembly and expression.

The RNA‐binding E3 ubiquitin ligase MEX‐3C links ubiquitination with MHC‐I mRNA degradation

RNA‐binding E3 ubiquitin ligases were recently identified, though their function remains unclear. While studying the regulation of the MHC class I (MHC‐I) pathway, we here characterize a novel role for ubiquitin in mRNA degradation. MHC‐I molecules provide ligands for both cytotoxic T‐lymphocytes as well as natural killer (NK) cells, and play a central role in innate and adaptive immunity. MHC‐I cell‐surface expression is closely monitored by NK cells, whose killer immunoglobulin‐like receptors encode MHC‐I‐specific activatory and inhibitory receptors, implying that MHC‐I expression needs to be tightly regulated. In a functional siRNA ubiquitome screen we identified MEX‐3C, a novel RNA‐binding ubiquitin E3 ligase, as responsible for the post‐transcriptional, allotype‐specific regulation of MHC‐I. MEX‐3C binds the 3′UTR of HLA‐A2 mRNA, inducing its RING‐dependent degradation. The RING domain of MEX‐3C is not required for HLA‐A2 cell‐surface downregulation, but regulates the degradation of HLA‐A2 mRNA. We have therefore uncovered a novel post‐transcriptional pathway for regulation of HLA‐A allotypes and provide a link between ubiquitination and mRNA degradation.

RNA-binding E3 ubiquitin ligases: novel players in nucleic acid regulation.

Non-coding RNAs and their interaction with RNA-binding proteins regulate mRNA levels in key cellular processes. This has intensified interest in post-transcriptional regulation. Recent studies on the turnover of AU-rich cytokine mRNAs have linked mRNA metabolism with ubiquitination. Ubiquitin is well recognized for its role in protein regulation/degradation. In the present paper, we describe a new group of RNA-binding E3 ubiquitin ligases which are predicted to bind and regulate RNA stability. Although much effort has been focused on understanding the role of these proteins as key regulators of mRNA turnover, the requirement for E3 ligase activity in mRNA decay remains unclear. It is remarkable that the ubiquitin system is involved, either directly or indirectly, in both the degradation of nucleic acids as well as proteins. These new RNA-binding E3 ligases are potential candidates which link two important cellular regulatory pathways: the regulation of both protein and mRNA stability.

Tapasin-related protein TAPBPR is an additional component of the MHC class I presentation pathway

Tapasin is an integral component of the peptide-loading complex (PLC) important for efficient peptide loading onto MHC class I molecules. We investigated the function of the tapasin-related protein, TAPBPR. Like tapasin, TAPBPR is widely expressed, IFN-γ–inducible, and binds to MHC class I coupled with β2-microglobulin in the endoplasmic reticulum. In contrast to tapasin, TAPBPR does not bind ERp57 or calreticulin and is not an integral component of the PLC. β2-microglobulin is essential for the association between TAPBPR and MHC class I. However, the association between TAPBPR and MHC class I occurs in the absence of a functional PLC, suggesting peptide is not required. Expression of TAPBPR decreases the rate of MHC class I maturation through the secretory pathway and prolongs the association of MHC class I on the PLC. The TAPBPR:MHC class I complex trafficks through the Golgi apparatus, demonstrating a function of TAPBPR beyond the endoplasmic reticulum/cis-Golgi. The identification of TAPBPR as an additional component of the MHC class I antigen-presentation pathway demonstrates that mechanisms controlling MHC class I expression remain incompletely understood.

What Has the Study of the K3 and K5 Viral Ubiquitin E3 Ligases Taught Us about Ubiquitin-Mediated Receptor Regulation?

Cells communicate with each other and the outside world through surface receptors, which need to be tightly regulated to prevent both overstimulation and receptor desensitization. Understanding the processes involved in the homeostatic control of cell surface receptors is essential, but we are not alone in trying to regulate these receptors. Viruses, as the ultimate host pathogens, have co-evolved over millions of years and have both pirated and adapted host genes to enable viral pathogenesis. K3 and K5 (also known as MIR1 and MIR2) are viral ubiquitin E3 ligases from Kaposi’s Sarcoma Associated Herpesvirus (KSHV) which decrease expression of a number of cell surface receptors and have been used to interrogate cellular processes and improve our understanding of ubiquitin-mediated receptor endocytosis and degradation. In this review, we summarize what has been learned from the study of these viral genes and emphasize their role in elucidating the complexity of ubiquitin in receptor regulation.

Fluorescence-based phenotypic selection allows forward genetic screens in haploid human cells.

The isolation of haploid cell lines has recently allowed the power of forward genetic screens to be applied to mammalian cells. The interest in applying this powerful genetic approach to a mammalian system is only tempered by the limited utility of these screens, if confined to lethal phenotypes. Here we expand the scope of these approaches beyond live/dead screens and show that selection for a cell surface phenotype via fluorescence-activated cell sorting can identify the key molecules in an intracellular pathway, in this case MHC class I antigen presentation. Non-lethal haploid genetic screens are widely applicable to identify genes involved in essentially any cellular pathway.