Human cytomegalovirus UL24 and UL43 products participate in SAMHD1 subcellular localization.

This report has analyzed the potential role of Human Cytomegalovirus (HCMV) UL24 and UL43 products in modulating the subcellular location of a host restriction factor, SAMHD1, in cells of human fibroblast origin. Recent studies have reported that the regulation of SAMHD1 is mediated by the HCMV UL97 product inside the nucleus, and by the CDK pathway when it is located in the cytoplasm of the infected cells but the viral gene products that may involve in cytosolic relocalization remain unknown yet. In the present report, we demonstrate that the HCMV UL24 product interacts with the SAMHD1 protein during infection based on mass spectrometry (MS) data and immunoprecipitation assay. The expression or depletion of the viral UL24 gene product did not affect the subcellular localization of SAMHD1 but when it coexpressed with the viral UL43 gene product, another member of the HCMV US22 family, induced the SAMHD1 cytosolic relocalization. Interestingly, the double deletion of viral UL24 and UL43 gene products impaired the cytosolic translocation and the SAMHD1 was accumulated in the nucleus of the infected cells, especially at the late stage post-infection. Our results provide evidence that the viral UL24 and UL43 gene products play a role in the SAMHD1 subcellular localization during HCMV infection. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00799-3.

Human Cytomegalovirus UL24 and UL43 Cooperate to Modulate the Expression of Immunoregulatory UL16 Binding Protein 1.

The human cytomegalovirus (HCMV) UL24 and UL43 are tegument proteins that have recently been shown to interact with each other in a yeast two-hybrid system. By their overexpression in MRC5 cells, we demonstrate that these viral proteins interact with several important host proteins, especially Dicer and trans-activation response RNA binding protein. As these hots proteins are involved in regulating the production of cellular micro-RNAs, the cytomegalovirus (CMV) proteins could interfere with their actions to favor viral replication directly or through an immune escape mechanism. Double knockout of UL24 and UL43 does not show a remarkable effect on CMV entry or replication, but it significantly downregulates the expression of CMV-encoded miR-UL59, which is thought to regulate the expression of a downstream target UL16 binding protein 1 (ULBP1). Interestingly, the double knockout increases the expression of the ULBP1 recognized by the NKG2D activating receptor of natural killer cells. This study investigates the potential role of several proteins encoded by HCMV in regulating the host cellular environment to favor escape from immunity, and it also provides some basis for the future development of RNA-targeted small molecules to control HCMV infection.

Correction: STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus.

[This corrects the article DOI: 10.1371/journal.ppat.1007416.].

Codelivery of SARS-CoV-2 Prefusion-Spike Protein with CBLB502 by a Dual-Chambered Ferritin Nanocarrier Potentiates Systemic and Mucosal Immunity.

Thousands of breakthrough infections are confirmed after intramuscular (i.m.) injection of the approved vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two major factors might contribute to breakthrough infections. One is the emergence of mutant variants of SARS-CoV-2, and the other is that i.m. injection has an inefficient ability to activate mucosal immunity in the upper respiratory tract. Here, we devised a dual-chambered nanocarrier that can codeliver the adjuvant CBLB502 with prefusion-spike (pre-S) onto a ferritin nanoparticle. This vaccine enabled enhanced systemic and local mucosal immunity in the upper and lower respiratory tract. Further, codelivery of CBLB502 with pre-S induced a Th1/Th2-balanced immunoglobulin G response. Moreover, the codelivery nanoparticle showed a Th1-biased cellular immune response as the release of splenic INF-γ was significantly heightened while the level of IL-4 was elevated to a moderate extent. In general, the developed dual-chambered nanoparticle can trigger multifaceted immune responses and shows great potential for mucosal vaccine development.

Unique binding pattern for a lineage of human antibodies with broad reactivity against influenza A virus.

Most structurally characterized broadly neutralizing antibodies (bnAbs) against influenza A viruses (IAVs) target the conserved conformational epitopes of hemagglutinin (HA). Here, we report a lineage of naturally occurring human antibodies sharing the same germline gene, VH3-48/VK1-12. These antibodies broadly neutralize the major circulating strains of IAV in vitro and in vivo mainly by binding a contiguous epitope of H3N2 HA, but a conformational epitope of H1N1 HA, respectively. Our structural and functional studies of antibody 28-12 revealed that the continuous amino acids in helix A, particularly N49HA2 of H3 HA, are critical to determine the binding feature with 28-12. In contrast, the conformational epitope feature is dependent on the discontinuous segments involving helix A, the fusion peptide, and several HA1 residues within H1N1 HA. We report that this antibody was initially selected by H3 (group 2) viruses and evolved via somatic hypermutation to enhance the reactivity to H3 and acquire cross-neutralization to H1 (group 1) virus. These findings enrich our understanding of different antigenic determinants of heterosubtypic influenza viruses for the recognition of bnAbs and provide a reference for the design of influenza vaccines and more effective antiviral drugs.

Histone Deacetylase Inhibitor SAHA Induces Expression of Fatty Acid-Binding Protein 4 and Inhibits Replication of Human Cytomegalovirus.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor that shows marked efficacy against many types of cancers and is approved to treat severe metastatic cutaneous T-cell lymphomas. In addition to its anticancer activity, SAHA has significant effects on the growth of many viruses. The effect of SAHA on replication of human cytomegalovirus (HCMV) has not, however, been investigated. Here, we showed that the replication of HCMV was significantly suppressed by treatment with SAHA at concentrations that did not show appreciable cytotoxicity. SAHA reduced transcription and protein levels of HCMV immediate early genes, showing that SAHA acts at an early stage in the viral life-cycle. RNA-sequencing data mining showed that numerous pathways and molecules were affected by SAHA. Interferon-mediated immunity was one of the most relevant pathways in the RNA-sequencing data, and we confirmed that SAHA inhibits HCMV-induced IFN-mediated immune responses using quantitative Real-time PCR (qRT-PCR). Fatty acid-binding protein 4 (FABP4), which plays a role in lipid metabolism, was identified by RNA-sequencing. We found that FABP4 expression was reduced by HCMV infection but increased by treatment with SAHA. We then showed that knockdown of FABP4 partially rescued the effect of SAHA on HCMV replication. Our data suggest that FABP4 contributes to the inhibitory effect of SAHA on HCMV replication.

Correction: Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

[This corrects the article DOI: 10.1371/journal.ppat.1006773.].

Development of HBc virus-like particles as modular nanocarrier by intein-mediated trans-splicing.

Hepatitis B virus core protein (HBc) spontaneously assembles as Virus-like particles (VLPs) in Escherichia coli (E. coli) which is extensively used as a nanocarrier to boost antigen immunogenicity. Genetic fusion of cargo protein with HBc occasionally forms inclusion bodies instead of properly assembled VLPs. To this end, we devised HBc VLPs as a modular nanocarrier for antigen delivery by intein-mediated trans-splicing (TS). We introduced split inteinC (intC) to the C-terminus of split HBc N-core to employ intein-mediated TS technology to HBc VLPs. Split HBc with the insertion of intC at N-core C-terminus (designated as HBc N-intC-C) existed in inclusion bodies. Interestingly, introduction of a soluble tag, gb1, to intC C-terminus remarkably improved the solubility of recombinant protein (named HBc N-intC-gb1-C). Moreover, newly designed recombinant spontaneously assembled as VLPs and endowed efficiently coupling two different model antigens onto HBc N-intC-gb1-C VLPs. Furthermore, model antigens delivered by HBc VLPs induced a dramatically enhanced antigen-specific immune responses. Antigen proteins mainly elicited Th2 IgG responses while antigens delivered by HBc VLPs steered Th1/Th2 balanced IgG responses. Taken together, intein-mediated TS was amenable to decorate HBc VLPs with antigens and showed good potential for antigen delivery.

Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation.

Gammaherpesviruses have evolved various strategies to take advantage of host cellular factors or signaling pathways to establish a lifelong latent infection. Like the human gammaherpesvirus Epstein-Barr virus, murine gammaherpesvirus 68 (MHV68) establishes and maintains latency in the memory B cells during infection of laboratory mice. We have previously shown that MHV68 can immortalize fetal liver-derived B cells that induce lymphomas when injected into immunodeficient mice. Here we identify interleukin 16 (IL16) as a most abundantly expressed cytokine in MHV68-immortalized B cells and show that MHV68 infection elevates IL16 expression. IL16 is not important for MHV68 lytic infection but plays a critical role in MHV68 reactivation from latency. IL16 deficiency increases MHV68 lytic gene expression in MHV68-immortalized B cells and enhances reactivation from splenic latency. Correlatively, IL16 deficiency increases the frequency of MHV68-infected plasma cells that can be attributed to enhanced MHV68 reactivation. Furthermore, similar to TPA-mediated lytic replication of Kaposi's sarcoma-associated herpesvirus, IL16 deficiency markedly induces Tyr705 STAT3 de-phosphorylation and elevates p21 expression, which can be counteracted by the tyrosine phosphatase inhibitor orthovanadate. Importantly, orthovanadate strongly blocks MHV68 lytic gene expression mediated by IL16 deficiency. These data demonstrate that virus-induced IL16 does not directly participate in MHV68 lytic replication, but rather inhibits virus reactivation to facilitate latent infection, in part through the STAT3-p21 axis.

Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity.

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e-/- mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.

Murine Cytomegalovirus Protein pM49 Interacts with pM95 and Is Critical for Viral Late Gene Expression.

Late gene expression of betaherpesviruses and gammaherpesviruses is tightly controlled by virus-encoded transactivation factors (vTFs). We recently proved that the 6 vTFs of murine cytomegalovirus (MCMV) form a complex to regulate late gene transcription. pM49, one of the vTFs that has not been studied before, was identified to be a component of the complex that interacts with pM95. In this study, we began to investigate the potential role of pM49 in viral late gene expression. A recombinant MCMV expressing C-terminal FLAG-tagged pM49 was constructed to study the expression kinetics and localization of pM49. pM49 was expressed at the late time of virus infection. Inhibition of viral DNA synthesis by phosphonate sodium phosphonic acid (PAA) abolished pM49 expression, indicating that it is a late protein. pM49 colocalized with pM44 at the viral replication compartment, similarly to other viral vTFs that have been reported. Mutant virus lacking full-length pM49 expression failed to express viral late genes, leading to nonproductive infection. The expression of immediate early and early genes was not affected, and viral DNA synthesis was only minimally affected during pM49-deficient virus infection. All of these data support the role of pM49 in viral late gene expression. After a series of mutagenesis analyses, two key residues, K325 and C326, were identified as required for pM49-pM95 interaction. Cells expressing pM49 with either single mutation of these two residues failed to rescue the late gene expression and support the replication of pM49-deficient virus. Our results indicated that pM49-pM95 interaction is essential for viral late gene expression.IMPORTANCE Cytomegalovirus (CMV) infections result in morbidity and mortality in immunocompromised individuals, and the virus is also a major cause of birth defects in newborns. Currently, because of the unavailability of vaccines against this virus and restricted antiviral therapies with low toxicity, as well as the emergency of resistant strain of this virus, the understanding of viral late gene regulation may provide clues to study new antiviral drugs or vaccines. In this study, we report that MCMV protein pM49 is critical for viral late gene transcription, based on its interaction with pM95. This finding reveals the important role of pM49-pM95 interaction in the regulation of viral late gene expression and that it could be a future potential target for therapeutic intervention in CMV diseases.

Facile and Modular Pipeline for Protein-Specific Antibody Customization.

Antibodies are fundamental tools for basic science; however, high-quality antibodies suitable for multiple experimental applications are often inaccessible to research laboratories. To this end, a modular and low-cost pipeline for small-scale antibody customization is developed. First, soluble antigens are designed according to the secondary structure of a desired protein. Then, the antigens are efficiently displayed on a modular nanoplatform by intein-mediated trans-splicing (TS) that enables elicitation of high titers of protein-specific antibodies. After that, target antibodies are obtained by a modular HaloLink resin platform with antigens as the ligand that is devised by intein-mediated TS. Finally, purified antibodies show excellent properties in immunofluorescence, immunoprecipitation, and western blotting assays. Overall, these results suggest that the proposed pipeline is amenable to the generation of high-quality, research-grade antibodies and to aid in protein functional studies.

Identification of viral SIM-SUMO2-interaction inhibitors for treating primary effusion lymphoma.

Primary effusion lymphoma (PEL) is an aggressive B-cell malignancy without effective treatment, and caused by the infection of Kaposi's sarcoma-associated herpesvirus (KSHV), predominantly in its latent form. Previously we showed that the SUMO2-interacting motif within the viral latency-associated nuclear antigen (LANASIM) is essential for establishment and maintenance of KSHV latency. Here, we developed a luciferase based live-cell reporter system to screen inhibitors selectively targeting the interaction between LANASIM and SUMO2. Cambogin, a bioactive natural product isolated from the Garcinia genus (a traditional herbal medicine used for cancer treatment), was obtained from the reporter system screening to efficiently inhibit the association of SUMO2 with LANASIM, in turn reducing the viral episome DNA copy number for establishment and maintenance of KSHV latent infection at a low concentration (nM). Importantly, Cambogin treatments not only specifically inhibited proliferation of KSHV-latently infected cells in vitro, but also induced regression of PEL tumors in a xenograft mouse model. This study has identified Cambogin as a novel therapeutic agent for treating PEL as well as eliminating persistent infection of oncogenic herpesvirus.

Dissecting the Role of DDX21 in Regulating Human Cytomegalovirus Replication.

DDX21 regulates the biogenesis of rRNA and transcription of ribonucleoprotein genes. Recently, it has been reported that DDX21 regulates the growth of some RNA viruses through various mechanisms, such as inhibiting viral genome replication, suppressing virion assembly and release, and modulating antiviral immune responses (Chen et al., Cell Host Microbe 15:484-493, 2014, https://doi.org/10.1016/j.chom.2014.03.002; Dong et al., Biophys Res Commun, 473:648-653, 2016, https://doi.org/10.1016/j.bbrc.2016.03.120; and Watanabe et al., PLoS Pathog 5:e1000654, 2009, https://doi.org/10.1371/journal.ppat.1000654). The relationship between DDX21 and DNA viruses has not yet been explored. In this study, we used human cytomegalovirus (HCMV), a large human DNA virus, to investigate the potential role of DDX21 in DNA virus replication. We found that HCMV infection prevented the repression of DDX21 at protein and mRNA levels. Knockdown of DDX21 inhibited HCMV growth in human fibroblast cells (MRC5). Immunofluorescence and quantitative PCR (qPCR) results showed that knockdown of DDX21 did not affect viral DNA replication or the formation of the viral replication compartment but did significantly inhibit viral late gene transcription. Some studies have reported that DDX21 knockdown promotes the accumulation of R-loops that could restrain RNA polymerase II elongation and inhibit the transcription of certain genes. Thus, we used the DNA-RNA hybrid-specific S9.6 antibody to stain R-loops and observed that more R-loops formed in DDX21-knockdown cells than in control cells. Moreover, an DNA-RNA immunoprecipitation assay showed that more R-loops accumulated on a viral late gene in DDX21-knockdown cells. Altogether, these results suggest that DDX21 knockdown promotes the accumulation of R-loops, which prevents viral late gene transcription and consequently results in the suppression of HCMV growth. This finding provides new insight into the relationship between DDX21 and DNA virus replication.IMPORTANCE Previous studies have confirmed that DDX21 is vital for the regulation of various aspects of RNA virus replication. Our research is the first report on the role of DDX21 in HCMV DNA virus replication. We identified that DDX21 knockdown affected HCMV growth and viral late gene transcription. In order to elucidate how DDX21 regulated this transcription, we applied DNA-RNA immunoprecipitation by using the DNA-RNA hybrid-specific S9.6 antibody to test whether more R-loops accumulated on the viral late gene. Consistent with our expectation, more R-loops were detected on the viral late gene at late HCMV infection time points, which demonstrated that the accumulation of R-loops caused by DDX21 knockdown prevented viral late gene transcription and consequently impaired HCMV replication. These results reveal that DDX21 plays an important role in regulating HCMV replication and also provide a basis for investigating the role of DDX21 in regulating other DNA viruses.

Versatile Functionalization of Ferritin Nanoparticles by Intein-Mediated Trans-Splicing for Antigen/Adjuvant Co-delivery.

Self-assembling protein nanoparticles are extensively and increasingly engineered to integrate adjuvants with antigens to elicit potent and long-term immunity due to uniform architecture, inherent biocompatibility, and excellent plasticity. However, functionalization of nanoparticles by surface tailoring has two common problems: (1) disassembly caused by loaded cargoes; and (2) an adjuvant that is inconvenient to co-deliver with an antigen by genetic fusion. Here, we report an intein-mediated trans-splicing approach that overcomes the detrimental effects of loaded proteins on ferritin nanoparticle stability and allows concurrent display of antigen and adjuvant in a facile, efficient, and site-specific manner. An immunization study with an epitope-based model antigen reveals that antigen and adjuvant co-delivery nanoparticles induce a more potent protective immunity than other formulations do. Our results demonstrate that protein engineering represents an intriguing approach for antigen/adjuvant co-delivery to potentiate antigen-associated immune responses.

STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus.

Aberrations in STAT6-mediated signaling are linked to the development of multiple cancer types. Increasing evidence has shown that activation of human oncogenic herpesvirus lytic replication is crucial for viral tumorigenesis. However, the role of STAT6 in herpesvirus lytic replication remains elusive. Here, by using Kaposi's sarcoma-associated herpesvirus (KSHV) as a model, we revealed that RTA, the master regulator of lytic replication, interacts with STAT6 and promotes lysine 48 (K48) and K63-linked ubiquitylation of STAT6 for degradation via the proteasome and lysosome systems. Moreover, degradation of STAT6 is dramatically associated with the increased ubiquitylated form of tripartite motif family like 2 (TRIML2, a tumor suppressor) for prolonged cell survival and virion production, which is also commonly observed in lytic activation of Epstein-Barr virus, herpes simplex virus 1 and cytomegalovirus. These results suggest that degradation of STAT6 is important for the lytic activation of KSHV and as such, may be an attractive therapeutic target.

Activation of E2F-dependent transcription by the mouse cytomegalovirus M117 protein affects the viral host range.

Cytomegaloviruses (CMVs) have a highly restricted host range as they replicate only in cells of their own or closely related species. To date, the molecular mechanisms underlying the CMV host restriction remain poorly understood. However, it has been shown that mouse cytomegalovirus (MCMV) can be adapted to human cells and that adaptation goes along with adaptive mutations in several viral genes. In this study, we identify MCMV M117 as a novel host range determinant. Mutations in this gene enable the virus to cross the species barrier and replicate in human RPE-1 cells. We show that the M117 protein is expressed with early kinetics, localizes to viral replication compartments, and contributes to the inhibition of cellular DNA synthesis. Mechanistically, M117 interacts with members of the E2F transcription factor family and induces E2F target gene expression in murine and human cells. While the N-terminal part of M117 mediates E2F interaction, the C-terminal part mediates self-interaction. Both parts are required for the activation of E2F-dependent transcription. We further show that M117 is dispensable for viral replication in cultured mouse fibroblasts and endothelial cells, but is required for colonization of mouse salivary glands in vivo. Conversely, inactivation of M117 or pharmacological inhibition of E2F facilitates MCMV replication in human RPE-1 cells, whereas replacement of M117 by adenovirus E4orf6/7, a known E2F activator, prevents it. These results indicate that E2F activation is detrimental for MCMV replication in human cells. In summary, this study identifies MCMV M117 as a novel E2F activator that functions as a host range determinant by precluding MCMV replication in human cells.

Murine Cytomegalovirus Protein pM91 Interacts with pM79 and Is Critical for Viral Late Gene Expression.

Viral gene expression is tightly regulated during cytomegalovirus (CMV) lytic replication, but the detailed mechanism of late gene transcription remains to be fully understood. Previous studies reported that six viral proteins (named viral transactivation factors [vTFs]) supporting late gene expression were conserved in beta- and gammaherpesviruses but not in alphaherpesviruses. Here, we performed coimmunoprecipitation experiments to elucidate the organization of these six proteins in murine CMV. Our results showed that these proteins formed a complex by both direct and indirect interactions. Specifically, pM91 strongly bound to pM79 even in the absence of other vTFs. Similar to pM79, pM91 exhibited early-late expression kinetics and localized within nuclear viral replication compartments during infection. Functional analysis was also performed using the pM91-deficient virus. Real-time PCR results revealed that abrogation of M91 expression markedly reduced viral late gene expression and progeny virus production without affecting viral DNA synthesis. Using mutagenesis, we found that residues E61, D62, D89, and D96 in pM91 were required for the pM91-pM79 interaction. Disruption of the interaction via E61A/D62A or D89A/D96A double mutation in the context of virus infection inhibited progeny virus production. Our data indicate that pM91 is a component of the viral late gene transcription factor complex and that the pM91-pM79 interaction is essential for viral late gene expression.IMPORTANCE Cytomegalovirus (CMV) infection is the leading cause of birth defects and causes morbidity and mortality in immunocompromised patients. The regulation of viral late gene transcription is not well elucidated, and understanding of this process benefits the development of novel therapeutics against CMV infection. This study (i) identified that six viral transactivation factors encoded by murine CMV form a complex, (ii) demonstrated that pM91 interacts with pM79 and that pM91 and pM79 colocalize in the nuclear viral replication compartments, (iii) confirmed that pM91 is critical for viral late gene expression but dispensable for viral DNA replication, and (iv) revealed that the pM91-pM79 interaction is required for progeny virus production. These findings give an explanation of how CMV regulates late gene expression and have important implications for the design of antiviral strategies.

Correction: Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

[This corrects the article DOI: 10.1371/journal.ppat.1006773.].

Human Cytomegalovirus Protein pUL38 Prevents Premature Cell Death by Binding to Ubiquitin-Specific Protease 24 and Regulating Iron Metabolism.

Human cytomegalovirus (HCMV) protein pUL38 has been shown to prevent premature cell death by antagonizing cellular stress responses; however, the underlying mechanism remains unknown. In this study, we identified the host protein ubiquitin-specific protease 24 (USP24) as an interaction partner of pUL38. Mutagenesis analysis of pUL38 revealed that amino acids TFV at positions 227 to 230 were critical for its interaction with USP24. Mutant pUL38 TFV/AAA protein did not bind to USP24 and failed to prevent cell death induced by pUL38-deficient HCMV infection. Knockdown of USP24 suppressed the cell death during pUL38-deficient HCMV infection, suggesting that pUL38 achieved its function by antagonizing the function of USP24. We investigated the cellular pathways regulated by USP24 that might be involved in the cell death phenotype by testing several small-molecule compounds known to have a protective effect during stress-induced cell death. The iron chelators ciclopirox olamine and Tiron specifically protected cells from pUL38-deficient HCMV infection-induced cell death, thus identifying deregulated iron homeostasis as a potential mechanism. Protein levels of nuclear receptor coactivator 4 (NCOA4) and lysosomal ferritin degradation, a process called ferritinophagy, were also regulated by pUL38 and USP24 during HCMV infection. Knockdown of USP24 decreased NCOA4 protein stability and ferritin heavy chain degradation in lysosomes. Blockage of ferritinophagy by genetic inhibition of NCOA4 or Atg5/Atg7 prevented pUL38-deficient HCMV infection-induced cell death. Overall, these results support the hypothesis that pUL38 binds to USP24 to reduce ferritinophagy, which may then protect cells from lysosome dysfunction-induced cell death.IMPORTANCE Premature cell death is considered a first line of defense against various pathogens. Human cytomegalovirus (HCMV) is a slow-replicating virus that encodes several cell death inhibitors, such as pUL36 and pUL37x1, which allow it to overcome both extrinsic and intrinsic mitochondrion-mediated apoptosis. We previously identified HCMV protein pUL38 as another virus-encoded cell death inhibitor. In this study, we demonstrated that pUL38 achieved its activity by interacting with and antagonizing the function of the host protein ubiquitin-specific protease 24 (USP24). pUL38 blocked USP24-mediated ferritin degradation in lysosomes, which could otherwise be detrimental to the lysosome and initiate cell death. These novel findings suggest that iron metabolism is finely tuned during HCMV infection to avoid cellular toxicity. The results also provide a solid basis for further investigations of the role of USP24 in regulating iron metabolism during infection and other diseases.