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Recombinant Adenovirus vectors enable highly efficient gene delivery in vitro and in vivo. As a result, they are widely used in gene therapy, vaccination, and anti-cancer applications. We have previously developed the AdZ vector system, which uses recombineering to permit high throughput cloning of transgenes into Adenovirus vectors, simplifies alteration of the vector backbone, and enables rapid recovery of infectious virus, even if a transgene is incompatible with vector replication. Here we adapt this vector system to enable high throughput cloning of sequences for CRISPR/Cas9 editing. Vectors were optimised to ensure efficient cloning, and high editing efficiency using spCas9 and sgRNA sequences in a single vector. Using a multiplicity of infection of 50, knockout efficiencies of up to 80% could be achieved with a single sgRNA. Vectors were further enhanced by altering the spCas9 sequence to match that of SniperCas9, which has reduced off-target activity but maintains on-target efficiency, and by applying modifications to the sgRNA sequence that significantly enhance editing efficiency. Thus, the AdZ-CRISPR vectors offer highly efficient knockout, even in hard to transfect cells, and enables large scale CRISPR/Cas9 projects to be undertaken easily and quickly.

Purpose:Broadly expressed, highly differentiated tumor-associated antigens (TAA) can elicit antitumor immunity. However, vaccines targeting TAAs have demonstrated disappointing clinical results, reflecting poor antigen selection and/or immunosuppressive mechanisms.Experimental Design:Here, a panel of widely expressed, novel colorectal TAAs were identified by performing RNA sequencing of highly purified colorectal tumor cells in comparison with patient-matched colonic epithelial cells; tumor cell purification was essential to reveal these genes. Candidate TAA protein expression was confirmed by IHC, and preexisting T-cell immunogenicity toward these antigens tested.Results:The most promising candidate for further development is DNAJB7 [DnaJ heat shock protein family (Hsp40) member B7], identified here as a novel cancer-testis antigen. It is expressed in many tumors and is strongly immunogenic in patients with cancers originating from a variety of sites. DNAJB7-specific T cells were capable of killing colorectal tumor lines in vitro, and the IFNγ+ response was markedly magnified by control of immunosuppression with cyclophosphamide in patients with cancer.Conclusions:This study highlights how prior methods that sequence whole tumor fractions (i.e., inclusive of alive/dead stromal cells) for antigen identification may have limitations. Through tumor cell purification and sequencing, novel candidate TAAs have been identified for future immunotherapeutic targeting.

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Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV). Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on the titer of the low-passage strain Merlin. We found no obvious difference in expression of several viral proteins between AD169 and Merlin in ZAP knockdown cells, but observed a larger increase in infectious virus in AD169 compared to Merlin in the absence of ZAP, suggesting that ZAP inhibited events late in AD169 replication. In addition, there was no clear difference in the CpG abundance of AD169 and Merlin RNAs, indicating that genomic content of the two virus strains was unlikely to be responsible for differences in their sensitivity to ZAP. Instead, we observed less ZAP expression in Merlin-infected cells late in replication compared to AD169-infected cells, which may be related to different abilities of the two virus strains to regulate interferon signaling. Therefore, there are strain-dependent differences in the sensitivity of HCMV to ZAP, and the ability of low-passage HCMV strain Merlin to evade inhibition by ZAP is likely related to its ability to regulate interferon signaling, not the CpG content of RNAs produced from its genome. IMPORTANCE Determining the function of cellular antiviral factors can inform our understanding of virus replication. The zinc finger antiviral protein (ZAP) can inhibit the replication of diverse viruses. Here, we examined ZAP interaction with the DNA virus human cytomegalovirus (HCMV). We found HCMV strain-dependent differences in the ability of ZAP to influence HCMV replication, which may be related to the interaction of HCMV strains with the type I interferon system. These observations affect our current understanding of how ZAP restricts HCMV and how HCMV interacts with the type I interferon system.

Human cytomegalovirus (HCMV) is the most common vertically transmitted infection worldwide, yet there are no licensed vaccines or therapeutics to prevent congenital HCMV (cCMV) infection. Emerging evidence from studies of natural infection and HCMV vaccine trials indicates that antibody Fc effector functions may defend against HCMV infection. We previously reported that antibody-dependent cellular phagocytosis (ADCP) and IgG activation of FcγRI/FcγRII were associated with reduced risk of cCMV transmission, leading us to hypothesize that other Fc-mediated antibody functions may also contribute to protection. In this same cohort of HCMV transmitting (n = 41) and non-transmitting (n = 40) mother-infant dyads, we found that higher maternal sera antibody-dependent cellular cytotoxicity (ADCC) activation was also associated with decreased risk of cCMV infection. We determined that NK cell-mediated ADCC responses correlated strongly with anti-HCMV IgG FcγRIII/CD16 activation and IgG binding to the HCMV immunoevasin protein UL16. Notably, anti-UL16 IgG binding and engagement of FcγRIII/CD16 were higher in non-transmitting versus transmitting dyads and interacted significantly with ADCC responses. These findings indicate that ADCC-activating antibodies against novel targets such as UL16 may represent an important protective maternal immune response against cCMV infection, which can guide future HCMV correlates studies and vaccine development.

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defences. In exploring the finding that HCMV infection upregulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory anti-viral cytokine TNFa, we discovered 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 itsUL/b’ region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with a 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 (pSignificance statementHuman cytomegalovirus (HCMV) is an important pathogen, being the commonest infectious cause of brain damage to babies and the primary reason for hospital readmissions in transplant recipients. Even though HCMV induces the strongest immune responses by any human pathogen, it evades host defences and persists for life. This study describes a novel immunoregulatory strategy through which HCMV modulates multiple immune pathways simultaneously, by targeting a single host protein. HCMV UL148 and UL148D impair the maturation of the sheddase, A Disintegrin And Metalloproteinase 17, profoundly altering surface expression of numerous immunoregulatory proteins. This is the first description of viral genes targeting this pathway. Our findings may be relevant for future viral therapies and understanding the impact of HCMV in developmental biology.

Viruses such as SARS-CoV-2 can remain viable on solid surfaces for up to one week, hence fomites are a potential route of exposure to infectious virus. Copper has well documented antiviral properties that could limit this problem, however practical deployment of copper surfaces has been limited due to the associated costs and the incompatibility of copper metal in specific environments and conditions. We therefore developed an organic coating containing an intelligent-release Cu2+ pigment based on a cation exchange resin. Organic coatings containing a 50 % weight or higher loading of smart-release pigment were capable of completely inactivating (>6 log reduction in titre) SARS-CoV-2 within 4 h of incubation. Importantly these organic coatings demonstrated a significantly enhanced ability to inactivate SARS-CoV-2 compared to metallic copper and un-pigmented material. Furthermore, the presence of contaminating proteins inhibited the antiviral activity of metallic copper, but the intelligent-release Cu2+ pigment was unaffected. The approach of using a very basic paint system, based on a polymer binder embedded with “smart release” pigment containing an anti-viral agent which is liberated by ion-exchange, holds significant promise as a cost effective and rapidly deployed coating to confer virus inactivating capability to high touch surfaces.

The lipid envelope of SARS-CoV2 is an essential component of the virus, however its molecular composition is unknown. Addressing this knowledge gap could support the design of anti-viral agents, and further understanding of viral interaction with extracellular host proteins, infectivity, pathogenicity, and innate immune system clearance. Lipidomics analysis of SARS-CoV2 particles generated from Vero or A549 cells revealed that the virus envelope comprised mainly of phospholipids (PL), primarily phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), with very little cholesterol, sphingolipids or other lipids, indicating significant differences from host membranes. Unlike healthy cellular membranes, procoagulant aminoPL (aPL), specifically PE and phosphatidylserine (PS), were present on the external side at levels far exceeding those seen on activated platelets. As a result, purified virions directly promoted coagulation. To investigate whether these differences enabled the viral envelope to be selectively targeted at relevant sites in vivo, we tested whether non-toxic oral rinses containing lipid disrupting chemicals could reduce viral infectivity. Products containing PL-disrupting surfactant solutions (cetylpyridinium chloride (CPC) or ethyl lauroyl arginate) met EN14476 virucidal standards in vitro, however products containing essential oils, PVP-I, or Chlorhexidine did not, nor did rinses containing components that altered the critical micelle concentration of CPC. This result was recapitulated in vivo, where a 30-second oral rinse with CPC-mouthwash eliminated live virus in the oral cavity of COVID19 patients for at least 1hr, while PVP-Iodine and saline mouthwashes were ineffective. Thus, the SARS-CoV2 lipid envelope is distinct from the host plasma membrane which may enable design of selective anti-viral approaches, it exposes PE and PS which may influence thrombosis, pathogenicity, and inflammation, and can be selectively targeted in vivo by specific oral rinses.

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 novel, orthogonal multiplexed tandem mass tag-based proteomic screens to identify host proteins downregulated 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 characterised, 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.Significance StatementPrevious proteomic analyses of host factors targeted for downregulation by HCMV have focused on early or intermediate stages of infection. Using multiplexed proteomics, we have systematically identified viral factors that target each host protein downregulated during the latest stage of infection, after the onset of viral DNA replication. Schlafen-11 (SLFN11), an interferon-stimulated gene and restriction factor for retroviruses and certain RNA viruses, potently restricted HCMV infection. Our discovery that the late-expressed HCMV protein RL1 targets SLFN11 for proteasomal degradation provides the first evidence for a viral antagonist of this critical cellular protein. We therefore redefine SLFN11 as an important factor that targets DNA viruses as well as RNA viruses, offering novel therapeutic potential via molecules that inhibit RL1-mediated SLFN11 degradation.

Several viruses, including human cytomegalovirus (HCMV), are thought to replicate in the placenta. However, there is little understanding of the molecular mechanisms involved in HCMV replication in this tissue. We investigated replication of HCMV in the extravillous trophoblast cell line SGHPL-4, a commonly used model of HCMV replication in the placenta. We found limited HCMV protein expression and virus replication in SGHPL-4 cells. This was associated with a lack of trophoblast progenitor cell protein markers in SGHPL-4 cells, suggesting a relationship between trophoblast differentiation and limited HCMV replication. We proposed that limited HCMV replication in trophoblast cells is advantageous to vertical transmission of HCMV, as there is a greater opportunity for vertical transmission when the placenta is intact and functional. Furthermore, when we investigated the replication of other vertically transmitted viruses in SGHPL-4 cells we found some limitation to replication of Zika virus, but not herpes simplex virus. Thus, limited replication of some, but not all, vertically transmitted viruses may be a feature of trophoblast cells.

Long noncoding RNAs (lncRNAs) are frequently associated with broad modulation of gene expression and thus provide the cell with the ability to synchronize entire metabolic processes. We used transcriptomic approaches to investigate whether the most abundant human cytomegalovirus-encoded lncRNA, RNA2.7, has this characteristic. By comparing cells infected with wild-type virus (WT) to cells infected with RNA2.7 deletion mutants, RNA2.7 was implicated in regulating a large number of cellular genes late in lytic infection. Pathway analysis indicated that >100 of these genes are associated with promoting cell movement, and the 10 most highly regulated of these were validated in further experiments. Morphological analysis and live cell tracking of WT- and RNA2.7 mutant-infected cells indicated that RNA2.7 is involved in promoting the movement and detachment of infected cells late in infection, and plaque assays using sparse cell monolayers indicated that RNA2.7 is also involved in promoting cell-to-cell spread of virus. Consistent with the observation that upregulated mRNAs are relatively A+U-rich, which is a trait associated with transcript instability, and that they are also enriched in motifs associated with mRNA instability, transcriptional inhibition experiments on WT- and RNA2.7 mutant-infected cells showed that four upregulated transcripts lived longer in the presence of RNA2.7. These findings demonstrate that RNA2.7 is required for promoting cell movement and viral spread late in infection and suggest that this may be due to general stabilization of A+U-rich transcripts. IMPORTANCE In addition to messenger RNAs (mRNAs), the human genome encodes a large number of long noncoding RNAs (lncRNAs). Many lncRNAs that have been studied in detail are associated with broad modulation of gene expression and have important biological roles. Human cytomegalovirus, which is a large, clinically important DNA virus, specifies four lncRNAs, one of which (RNA2.7) is expressed at remarkably high levels during lytic infection. Our studies show that RNA2.7 is required for upregulating a large number of human genes, about 100 of which are associated with cell movement, and for promoting the movement of infected cells and the spread of virus from one cell to another. Further bioinformatic and experimental analyses indicated that RNA2.7 may exert these effects by stabilizing mRNAs that are relatively rich in A and U nucleotides. These findings increase our knowledge of how human cytomegalovirus regulates the infected cell to promote its own success.

Funder: Ser Cymru, The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This occurred at the level of protein synthesis, could be mediated by Nsp1 and Nsp14, and correlated with a reduction in NK cell activation. This identifies a novel mechanism by which SARS-CoV-2 host-shutoff antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.

SummaryInterferon induced transmembrane protein 3 (IFITM3) is an important viral restriction factor in viral pathogenesis that also exhibits poorly understood immune regulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 regulates MyD88-dependent TLR-mediated cytokine production following dendritic cell exposure to cytomegalovirus (CMV), and this process limits viral pathogenesisin vivo. IFITM3 also restricted pro-inflammatory (IL-6) cytokine production in response to influenza. IFITM3 bound to and promoted ubiquitination and proteasomal degradation of the reticulon 4 isoform Nogo-B. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesisin vivo, and this process involved alteration of TLR dynamics. The anti-inflammatory function of IFITM3 was intrinsically linked to its ability to regulate Nogo-B. Thus, we uncover Nogo-B as an unappreciated driver of viral pathogenesis and highlight a novel immune regulatory pathway where IFITM3 fine-tunes TLR responsiveness of myeloid cells to viral stimulation.

Cell autonomous antiviral defenses can inhibit the replication of viruses and reduce transmission and disease severity. To better understand the antiviral response to SARS-CoV-2, we used interferon-stimulated gene (ISG) expression screening to reveal that OAS1, through RNase L, potently inhibits SARS-CoV-2. We show that while some people can express a prenylated OAS1 variant, that is membrane-associated and blocks SARS-CoV-2 infection, other people express a cytosolic, nonprenylated OAS1 variant which does not detect SARS-CoV-2 (determined by the splice-acceptor SNP Rs10774671). Alleles encoding nonprenylated OAS1 predominate except in people of African descent. Importantly, in hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting this antiviral defense is a major component of a protective antiviral response. Remarkably, approximately 55 million years ago, retrotransposition ablated the OAS1 prenylation signal in horseshoe bats (the presumed source of SARS-CoV-2). Thus, SARS-CoV-2 never had to adapt to evade this defense. As prenylated OAS1 is widespread in animals, the billions of people that lack a prenylated OAS1 could make humans particularly vulnerable to the spillover of coronaviruses from horseshoe bats.

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.

The ability of widely-available mouthwashes to inactivate SARS-CoV-2 in vitro was tested using a protocol capable of detecting a 5-log10 reduction in infectivity, under conditions mimicking the naso/oropharynx. During a 30 second exposure, two rinses containing cetylpyridinium-chloride and a third with ethanol/ethyl lauroyl arginate eliminated live virus to EN14476 standards (>4-log10 reduction), while others with ethanol/essential oils and povidone-iodine (PVP-I) eliminated virus by 2-3-log10. Chlorhexidine or ethanol alone displayed little or no ability to inactivate virus. Studies are warranted to determine whether these formulations can inactivate virus in the human oropharynx in vivo, and whether this might impact transmission risk.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68–1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68–1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68–1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68–1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68–1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques., Author summary Human cytomegalovirus (HCMV) infections are generally asymptomatic in healthy immunocompetent individuals, but HCMV can cause serious disease after congenital infection and in individuals with immunocompromised immune systems. Since HCMV is highly species specific and cannot productively infect immunocompetent laboratory animals, experimental infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a closely related animal model for HCMV. By employing the unique ability of CMV to elicit robust and lasting cellular immunity, this model has also been instrumental in developing novel CMV-based vaccines against chronic and recurring infections with pathogens such as the human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (Mtb). However, most of this work was conducted with derivatives of the 68–1 strain of RhCMV which has acquired multiple genomic alterations in tissue culture. To model pathogenesis and immunology of clinical HCMV isolates we generated a full-length (FL) RhCMV clone representative of low passage isolates. Infection of RhCMV-naïve RM with FL-RhCMV demonstrated viremia and tissue dissemination that was comparable to that of non-clonal low passage isolates. We further demonstrate that FL-RhCMV is strongly attenuated upon deletion of gene regions absent in 68–1 thus demonstrating the usefulness of FL-RhCMV to study RhCMV pathogenesis.

Significance Cell death is a key defense against viral infection, preventing spread from infected to uninfected cells. Correspondingly, certain viruses encode inhibitors of apoptotic and necroptotic cell death pathways in order to facilitate their persistence. Human cytomegalovirus (HCMV) is an important human pathogen that can block apoptosis, but hitherto it has been unclear whether or how the virus blocks necroptosis. Here, we used a proteomic screen to identify human proteins targeted for destruction by HCMV, finding that the key necroptosis mediator MLKL is degraded throughout infection. MLKL is targeted for degradation by HCMV protein pUL36, which is also instrumental in inhibiting apoptosis. Thus, pUL36 is a dual cell death pathway inhibitor, and may represent an important therapeutic target., Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of intrinsic, innate, and adaptive viral immune evasion. Here, we employed multiplexed tandem mass tag-based proteomics to characterize host proteins targeted for degradation late during HCMV infection. This approach revealed that mixed lineage kinase domain-like protein (MLKL), a key terminal mediator of cellular necroptosis, was rapidly and persistently degraded by the minimally passaged HCMV strain Merlin but not the extensively passaged strain AD169. The strain Merlin viral inhibitor of apoptosis pUL36 was necessary and sufficient both to degrade MLKL and to inhibit necroptosis. Furthermore, mutation of pUL36 Cys131 abrogated MLKL degradation and restored necroptosis. As the same residue is also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of procaspase-8, we define pUL36 as a multifunctional inhibitor of both apoptotic and necroptotic cell death.

Molluscum contagiosum virus (MCV) is a common cause of benign skin lesions in young children and currently the only endemic human poxvirus. Following the infection of primary keratinocytes in the epidermis, MCV induces the proliferation of infected cells and this results in the production of wart-like growths. Full productive infection is observed only after the infected cells differentiate. During this prolonged replication cycle the virus must avoid elimination by the host immune system. We therefore sought to investigate the function of the two major histocompatibility complex class-I-related genes encoded by the MCV genes mc033 and mc080. Following insertion into a replication-deficient adenovirus vector, codon-optimized versions of mc033 and mc080 were expressed as endoglycosidase-sensitive glycoproteins that localized primarily in the endoplasmic reticulum. MC080, but not MC033, downregulated cell-surface expression of endogenous classical human leucocyte antigen (HLA) class I and non-classical HLA-E by a transporter associated with antigen processing (TAP)-independent mechanism. MC080 exhibited a capacity to inhibit or activate NK cells in autologous assays in a donor-specific manner. MC080 consistently inhibited antigen-specific T cells being activated by peptide-pulsed targets. We therefore propose that MC080 acts to promote evasion of HLA-I-restricted cytotoxic T cells.

Molluscum contagiosum virus (MCV) is a common cause of benign skin lesions in young children and currently the only endemic human poxvirus. Following the infection of primary keratinocytes in the epidermis, MCV induces the proliferation of infected cells and this results in the production of wart-like growths. Full productive infection is observed only after the infected cells differentiate. During this prolonged replication cycle the virus must avoid elimination by the host immune system. We therefore sought to investigate the function of the two major histocompatibility complex class-I-related genes encoded by the MCV genes mc033 and mc080. Following insertion into a replication-deficient adenovirus vector, codon-optimized versions of mc033 and mc080 were expressed as endoglycosidase-sensitive glycoproteins that localized primarily in the endoplasmic reticulum. MC080, but not MC033, downregulated cell-surface expression of endogenous classical human leucocyte antigen (HLA) class I and non-classical HLA-E by a transporter associated with antigen processing (TAP)-independent mechanism. MC080 exhibited a capacity to inhibit or activate NK cells in autologous assays in a donor-specific manner. MC080 consistently inhibited antigen-specific T cells being activated by peptide-pulsed targets. We therefore propose that MC080 acts to promote evasion of HLA-I-restricted cytotoxic T cells.

Emerging studies increasingly demonstrate the importance of the throat and salivary glands as sites of virus replication and transmission in early COVID-19 disease. SARS-CoV-2 is an enveloped virus, characterized by an outer lipid membrane derived from the host cell from which it buds. While it is highly sensitive to agents that disrupt lipid biomembranes, there has been no discussion about the potential role of oral rinsing in preventing transmission. Here, we review known mechanisms of viral lipid membrane disruption by widely available dental mouthwash components that include ethanol, chlorhexidine, cetylpyridinium chloride, hydrogen peroxide, and povidone-iodine. We also assess existing formulations for their potential ability to disrupt the SARS-CoV-2 lipid envelope, based on their concentrations of these agents, and conclude that several deserve clinical evaluation. We highlight that already published research on other enveloped viruses, including coronaviruses, directly supports the idea that oral rinsing should be considered as a potential way to reduce transmission of SARS-CoV-2. Research to test this could include evaluating existing or specifically tailored new formulations in well-designed viral inactivation assays, then in clinical trials. Population-based interventions could be undertaken with available mouthwashes, with active monitoring of outcome to determine efficacy. This is an under-researched area of major clinical need.

Long non-coding RNAs (lncRNAs) are transcripts of >200 nucleotides that are not translated into functional proteins. Cellular lncRNAs have been shown to act as regulators by interacting with target nucleic acids or proteins and modulating their activities. We investigated the role of RNA1.2, which is one of four major lncRNAs expressed by human cytomegalovirus (HCMV), by comparing the properties of parental virus in vitro with those of deletion mutants lacking either most of the RNA1.2 gene or only the TATA element of the promoter. In comparison with parental virus, these mutants exhibited no growth defects and minimal differences in viral gene expression in human fibroblasts. In contrast, 76 cellular genes were consistently up- or down-regulated by the mutants at both the RNA and protein levels at 72 hours after infection. Differential expression of the gene most highly upregulated by the mutants (Tumor protein p63-regulated gene 1-like protein; TPRG1L) was confirmed at both levels by RT-PCR and immunoblotting. Consistent with the known ability of TPRG1L to upregulate IL-6 expression via NF-kappaB stimulation, RNA1.2 mutant-infected fibroblasts were observed to upregulate IL-6 in addition to TPRG1L. Comparable surface expression of TNF receptors and responsiveness to TNF-α in cells infected by the parental and mutant viruses indicated that activation of signalling by TNF-α is not involved in upregulation of IL-6 by the mutants. In contrast, inhibition of NF-kappaB activity and knockdown of TPRG1L expression reduced the extracellular release of IL-6 by RNA1.2 mutant-infected cells, thus demonstrating that upregulation of TPRG1L activates NF-kappaB. The levels of MCP-1 and CXCL1 transcripts were also increased in RNA1.2 mutant-infected cells, further demonstrating the presence of active NF-kappaB signalling. These results suggest that RNA1.2 plays a role in manipulating intrinsic NF-kappaB-dependent cytokine and chemokine release during HCMV infection, thereby impacting downstream immune responses.

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.

Purpose: Broadly expressed, highly differentiated tumor-associated antigens (TAA) can elicit antitumor immunity. However, vaccines targeting TAAs have demonstrated disappointing clinical results, reflecting poor antigen selection and/or immunosuppressive mechanisms. Experimental Design: Here, a panel of widely expressed, novel colorectal TAAs were identified by performing RNA sequencing of highly purified colorectal tumor cells in comparison with patient-matched colonic epithelial cells; tumor cell purification was essential to reveal these genes. Candidate TAA protein expression was confirmed by IHC, and preexisting T-cell immunogenicity toward these antigens tested. Results: The most promising candidate for further development is DNAJB7 [DnaJ heat shock protein family (Hsp40) member B7], identified here as a novel cancer-testis antigen. It is expressed in many tumors and is strongly immunogenic in patients with cancers originating from a variety of sites. DNAJB7-specific T cells were capable of killing colorectal tumor lines in vitro, and the IFNγ+ response was markedly magnified by control of immunosuppression with cyclophosphamide in patients with cancer. Conclusions: This study highlights how prior methods that sequence whole tumor fractions (i.e., inclusive of alive/dead stromal cells) for antigen identification may have limitations. Through tumor cell purification and sequencing, novel candidate TAAs have been identified for future immunotherapeutic targeting.

Human Cytomegalovirus (HCMV) is a master immune regulator, encoding multiple proteins that modulate a variety of immune signalling pathways. We previously performed a systematic proteomic analysis of temporal changes in host and viral proteins throughout the course of infection and determined that HCMV downregulates >900 host proteins. HCMV is the largest human herpesvirus, potentially encoding hundreds of ORFs. Identification of which individual gene targets a given cellular factor can therefore be challenging. To facilitate the mapping of viral gene functions, we employed a panel of HCMV mutants, each deleted in contiguous gene blocks dispensable for virus replication in vitro. Three proteomic screens of these mutants were performed, with each mutant represented in at least duplicate. From these data we have defined the genetic loci responsible for targeting >250 host proteins. Bioinformatic enrichment analysis on the targets of each mutant virus enabled attribution of novel functions to blocks of uncharacterised genes. Our approach was validated from analysis of the US1-11 genetic locus, which confirmed that the major function of US1-11 genes is the regulation of MHC class I molecules and other cell surface receptors. The data also suggests that the major functions of the poorly characterised blocks RL1-6 and US29-34A are the regulation of secreted proteins and the regulation of a family of cell surface adhesion molecules respectively. Overall this approach can be used to gain global insights into HCMV gene function, the study of which has previously been only been possible on a single gene basis.

Human cytomegalovirus (HCMV) rapidly mutates during in vitro passage, and this strongly alters the way the virus spreads. In vivo HCMV spreads by direct cell-cell contact, as do recent clinical isolates. In contrast, passaged strains spread via cell-free virions. Because of this, cell-cell spread remains largely uncharacterised. We have developed a strain (Merlin) that retains a full length, wildtype genome. As a result, it mimics clinical HCMV and spreads by direct cell-cell contact, a method of spread that is more resistant to neutralising antibodies, and innate and intrinsic immunity. We now show that each cell-cell transfer is equivalent to an extremely high MOI infection, with up to 300 genomes delivered to each cell, potentially providing an explanation for the ‘immune-evasive’ properties of cell-cell transfer. Furthermore, infectious virions accumulate at cell-cell contacts between cells. This may represent a ‘virological synapse’ that protects virions from neutralising antibodies. Not only does Merlin enable us to characterise cell-cell spread, but it enables us to infect a wide range of clinically relevant cells with a virus expressing the complete complement of virus genes. In vivo, HCMV infects dendritic cells (DCs), but is never cleared, implying that it is able to subvert DC function. Therefore, we performed quantitative proteomic analysis of infected primary immature DCs, following cell-cell transfer. This quantified 7992 intracellular proteins, and 703 plasma membrane proteins. Over 99 proteins were downregulated following infection. Many of these are DC-specific, and have roles in regulating adaptive immunity. These viral-manipulations may therefore dramatically impact DC function.

The tropism of human cytomegalovirus (HCMV) is influenced by the envelope glycoprotein complexes gH/gL/gO and gH/gL/UL128-131. During virion assembly, gO and the UL128-131 proteins compete for binding to gH/gL in the endoplasmic reticulum (ER). This assembly process clearly differs among strains, since Merlin (ME) virions contain abundant gH/gL/UL128-131 and little gH/gL/gO, whereas TR contains much higher levels of total gH/gL, mostly in the form of gH/gL/gO, but much lower levels of gH/gL/UL128-131 than ME. Remaining questions include (i) what are the mechanisms behind these assembly differences, and (ii) do differences reflect in vitro culture adaptations or natural genetic variations? Since the UL74(gO) open reading frame (ORF) differs in 25% of amino acids between TR and ME, we analyzed recombinant viruses in which the UL74(gO) ORF was swapped. TR virions were >40-fold more infectious than ME. Transcriptional repression of UL128-131 enhanced the infectivity of ME to the level of TR, despite still far lower levels of gH/gL/gO. Swapping the UL74(gO) ORF had no effect on either TR or ME. A quantitative immunoprecipitation approach revealed that gH/gL expression levels were within 4-fold between TR and ME, but the gO expression level was 20-fold lower for ME, which suggested differences in mRNA transcription, translation, or rapid ER-associated degradation of gO. trans -Complementation of gO expression during ME replication gave a 6-fold enhancement of infectivity beyond the 40-fold effect of UL128-131 repression alone. Overall, strain variations in the assembly of gH/gL complexes result from differences in the expression of gO and UL128-131, and selective advantages for reduced UL128-131 expression during fibroblast propagation are much stronger than those for higher gO expression. IMPORTANCE Specific genetic differences between independently isolated HCMV strains may result from purifying selection on de novo mutations arising during propagation in culture or random sampling among the diversity of genotypes present in clinical specimens. Results presented indicate that while reduced UL128-131 expression may confer a powerful selective advantage during cell-free propagation of HCMV in fibroblast cultures, selective pressures for increased gO expression are much weaker. Thus, variation in gO expression among independent strains may represent natural genotype variability present in vivo . This may have important implications for virus-host interactions, such as immune recognition, and underscores the value of studying molecular mechanisms of replication using multiple HCMV strains.

Candidate vaccines designed to generate T cell–based immunity are typically vectored by nonpersistent viruses, which largely fail to elicit durable effector memory T cell responses. This limitation can be overcome using recombinant strains of CMV. Proof-of-principle studies have demonstrated the potential benefits of this approach, most notably in the SIV model, but safety concerns require the development of nonreplicating alternatives with comparable immunogenicity. In this study, we show that IL-33 promotes the accumulation and recall kinetics of circulating and tissue-resident memory T cells in mice infected with murine CMV. Using a replication-deficient murine CMV vector, we further show that exogenous IL-33 boosts vaccine-induced memory T cell responses, which protect against subsequent heterologous viral challenge. These data suggest that IL-33 could serve as a useful adjuvant to improve the efficacy of vaccines based on attenuated derivatives of CMV.

Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell–cell transmission. This process of cell–cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128–131A complex, a feature of WT but not passaged strains of HCMV.