ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways.

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

Human cytomegalovirus protein RL1 degrades the antiviral factor SLFN11 via recruitment of the CRL4 E3 ubiquitin ligase complex.

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.

Lower Humoral and Cellular Immunity Following Asymptomatic SARS-CoV-2 Infection Compared to Symptomatic Infection in Education (The ACE Cohort).

PURPOSE: Asymptomatic SARS-CoV-2 infections were widely reported during the COVID-19 pandemic, acting as a hidden source of infection. Many existing studies investigating asymptomatic immunity failed to recruit true asymptomatic individuals. Thus, we conducted a longitudinal cohort study to evaluate humoral- and cell-mediated responses to infection and vaccination in well-defined asymptomatic young adults (the Asymptomatic COVID-19 in Education [ACE] cohort). METHODS: Asymptomatic testing services located at three UK universities identified asymptomatic young adults who were subsequently recruited with age- and sex-matched symptomatic and uninfected controls. Blood and saliva samples were collected after SARS-CoV-2 Wuhan infection, and again after vaccination. 51 participant's anti-spike antibody titres, neutralizing antibodies, and spike-specific T-cell responses were measured, against both Wuhan and Omicron B.1.1.529.1. RESULTS: Asymptomatic participants exhibited reduced Wuhan-specific neutralization antibodies pre- and post-vaccination, as well as fewer Omicron-specific neutralization antibodies post-vaccination, compared to symptomatic participants. Lower Wuhan and Omicron-specific IgG titres in asymptomatic individuals were also observed pre- and post-vaccination, compared to symptomatic participants. There were no differences in salivary IgA levels. Conventional flow cytometry analysis and multi-dimensional clustering analysis indicated unvaccinated asymptomatic participants had significantly fewer Wuhan-specific IL-2 secreting CD4+ CD45RA+ T cells and activated CD8+ T cells than symptomatic participants, though these differences dissipated after vaccination. CONCLUSIONS: Asymptomatic infection results in decreased antibody and T cell responses to further exposure to SARS-CoV-2 variants, compared to symptomatic infection. Post-vaccination, antibody responses are still inferior, but T cell immunity increases to match symptomatic subjects, emphasising the importance of vaccination to help protect asymptomatic individuals against future variants.

Killer cell proteases can target viral immediate-early proteins to control human cytomegalovirus infection in a noncytotoxic manner.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and can trigger devastating disease in immune-suppressed patients. Cytotoxic lymphocytes (CD8+ T cells and NK cells) control HCMV infection by releasing interferon-γ and five granzymes (GrA, GrB, GrH, GrK, GrM), which are believed to kill infected host cells through cleavage of intracellular death substrates. However, it has recently been demonstrated that the in vivo killing capacity of cytotoxic T cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we demonstrate that (primary) cytotoxic lymphocytes can block HCMV dissemination independent of host cell death, and interferon-α/ß/γ. Prior to killing, cytotoxic lymphocytes induce the degradation of viral immediate-early (IE) proteins IE1 and IE2 in HCMV-infected cells. Intriguingly, both IE1 and/or IE2 are directly proteolyzed by all human granzymes, with GrB and GrM being most efficient. GrB and GrM cleave IE1 after Asp398 and Leu414, respectively, likely resulting in IE1 aberrant cellular localization, IE1 instability, and functional impairment of IE1 to interfere with the JAK-STAT signaling pathway. Furthermore, GrB and GrM cleave IE2 after Asp184 and Leu173, respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data indicate that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV infection, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic infection.

Human cytomegalovirus degrades DMXL1 to inhibit autophagy, lysosomal acidification, and viral assembly.

Human cytomegalovirus (HCMV) is an important human pathogen that regulates host immunity and hijacks host compartments, including lysosomes, to assemble virions. We combined a quantitative proteomic analysis of HCMV infection with a database of proteins involved in vacuolar acidification, revealing Dmx-like protein-1 (DMXL1) as the only protein that acidifies vacuoles yet is degraded by HCMV. Systematic comparison of viral deletion mutants reveals the uncharacterized 7 kDa US33A protein as necessary and sufficient for DMXL1 degradation, which occurs via recruitment of the E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC). US33A-mediated DMXL1 degradation inhibits lysosome acidification and autophagic cargo degradation. Formation of the virion assembly compartment, which requires lysosomes, occurs significantly later with US33A-expressing virus infection, with reduced viral replication. These data thus identify a viral strategy for cellular remodeling, with the potential to employ US33A in therapies for viral infection or rheumatic conditions, in which inhibition of lysosome acidification can attenuate disease.

Proteomic analysis of circulating immune cells identifies cellular phenotypes associated with COVID-19 severity.

Certain serum proteins, including C-reactive protein (CRP) and D-dimer, have prognostic value in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nonetheless, these factors are non-specific, providing limited mechanistic insight into the peripheral blood mononuclear cell (PBMC) populations that drive the pathogenesis of severe COVID-19. To identify cellular phenotypes associated with disease, we performed a comprehensive, unbiased analysis of total and plasma-membrane PBMC proteomes from 40 unvaccinated individuals with SARS-CoV-2, spanning the whole disease spectrum. Combined with RNA sequencing (RNA-seq) and flow cytometry from the same donors, we define a comprehensive multi-omic profile for each severity level, revealing that immune-cell dysregulation progresses with increasing disease. The cell-surface proteins CEACAMs1, 6, and 8, CD177, CD63, and CD89 are strongly associated with severe COVID-19, corresponding to the emergence of atypical CD3+CD4+CEACAM1/6/8+CD177+CD63+CD89+ and CD16+CEACAM1/6/8+ mononuclear cells. Utilization of these markers may facilitate real-time patient assessment by flow cytometry and identify immune populations that could be targeted to ameliorate immunopathology.

Atypical B cells and impaired SARS-CoV-2 neutralization following heterologous vaccination in the elderly.

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response.

Comparative Cell Surface Proteomic Analysis of the Primary Human T Cell and Monocyte Responses to Type I Interferon.

The cellular response to interferon (IFN) is essential for antiviral immunity, IFN-based therapy and IFN-related disease. The plasma membrane (PM) provides a critical interface between the cell and its environment, and is the initial portal of entry for viruses. Nonetheless, the effect of IFN on PM proteins is surprisingly poorly understood, and has not been systematically investigated in primary immune cells. Here, we use multiplexed proteomics to quantify IFNα2a-stimulated PM protein changes in primary human CD14+ monocytes and CD4+ T cells from five donors, quantifying 606 and 482 PM proteins respectively. Comparison of cell surface proteomes revealed a remarkable invariance between donors in the overall composition of the cell surface from each cell type, but a marked donor-to-donor variability in the effects of IFNα2a. Furthermore, whereas only 2.7% of quantified proteins were consistently upregulated by IFNα2a at the surface of CD4+ T cells, 6.8% of proteins were consistently upregulated in primary monocytes, suggesting that the magnitude of the IFNα2a response varies according to cell type. Among these differentially regulated proteins, we found the viral target Endothelin-converting enzyme 1 (ECE1) to be an IFNα2a-stimulated protein exclusively upregulated at the surface of CD4+ T cells. We therefore provide a comprehensive map of the cell surface of IFNα2a-stimulated primary human immune cells, including previously uncharacterized interferon stimulated genes (ISGs) and candidate antiviral factors.

Nuclear phosphorylated Dicer processes double-stranded RNA in response to DNA damage.

The endoribonuclease Dicer is a key component of the human RNA interference pathway and is known for its role in cytoplasmic microRNA production. Recent findings suggest that noncanonical Dicer generates small noncoding RNA to mediate the DNA damage response (DDR). Here, we show that human Dicer is phosphorylated in the platform-Piwi/Argonaute/Zwille-connector helix cassette (S1016) upon induction of DNA damage. Phosphorylated Dicer (p-Dicer) accumulates in the nucleus and is recruited to DNA double-strand breaks. We further demonstrate that turnover of damage-induced nuclear, double-stranded (ds) RNA requires additional phosphorylation of carboxy-terminal Dicer residues (S1728 and S1852). DNA damage-induced nuclear Dicer accumulation is conserved in mammals. Dicer depletion causes endogenous DNA damage and delays the DDR by impaired recruitment of repair factors MDC1 and 53BP1. Collectively, we place Dicer within the context of the DDR by demonstrating a DNA damage-inducible phosphoswitch that causes localized processing of nuclear dsRNA by p-Dicer to promote DNA repair.