Discovery of a Novel Antiviral Effect of the Restriction Factor SPOC1 against Human Cytomegalovirus.

The chromatin-remodeler SPOC1 (PHF13) is a transcriptional co-regulator and has been identified as a restriction factor against various viruses, including human cytomegalovirus (HCMV). For HCMV, SPOC1 was shown to block the onset of immediate-early (IE) gene expression under low multiplicities of infection (MOI). Here, we demonstrate that SPOC1-mediated restriction of IE expression is neutralized by increasing viral titers. Interestingly, our study reveals that SPOC1 exerts an additional antiviral function beyond the IE phase of HCMV replication. Expression of SPOC1 under conditions of high MOI resulted in severely impaired viral DNA replication and viral particle release, which may be attributed to inefficient viral transcription. With the use of click chemistry, the localization of viral DNA was investigated at late time points after infection. Intriguingly, we detected a co-localization of SPOC1, RNA polymerase II S5P and polycomb repressor complex 2 (PRC2) components in close proximity to viral DNA in areas that are hypothesized to harbor viral transcription sites. We further identified the N-terminal domain of SPOC1 to be responsible for interaction with EZH2, a subunit of the PRC2 complex. With this study, we report a novel and potent antiviral function of SPOC1 against HCMV that is efficient even with unrestricted IE gene expression.

'Getting Better'-Is It a Feasible Strategy of Broad Pan-Antiherpesviral Drug Targeting by Using the Nuclear Egress-Directed Mechanism?

The herpesviral nuclear egress represents an essential step of viral replication efficiency in host cells, as it defines the nucleocytoplasmic release of viral capsids. Due to the size limitation of the nuclear pores, viral nuclear capsids are unable to traverse the nuclear envelope without a destabilization of this natural host-specific barrier. To this end, herpesviruses evolved the regulatory nuclear egress complex (NEC), composed of a heterodimer unit of two conserved viral NEC proteins (core NEC) and a large-size extension of this complex including various viral and cellular NEC-associated proteins (multicomponent NEC). Notably, the NEC harbors the pronounced ability to oligomerize (core NEC hexamers and lattices), to multimerize into higher-order complexes, and, ultimately, to closely interact with the migrating nuclear capsids. Moreover, most, if not all, of these NEC proteins comprise regulatory modifications by phosphorylation, so that the responsible kinases, and additional enzymatic activities, are part of the multicomponent NEC. This sophisticated basis of NEC-specific structural and functional interactions offers a variety of different modes of antiviral interference by pharmacological or nonconventional inhibitors. Since the multifaceted combination of NEC activities represents a highly conserved key regulatory stage of herpesviral replication, it may provide a unique opportunity towards a broad, pan-antiherpesviral mechanism of drug targeting. This review presents an update on chances, challenges, and current achievements in the development of NEC-directed antiherpesviral strategies.

Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection.

Herpesviruses modulate immune control to secure lifelong infection. The mechanisms Human Cytomegalovirus (HCMV) employs in this regard can reveal unanticipated aspects of cellular signaling involved in antiviral immunity. Here, we describe a novel relationship between the TGF-ß family cytokine BMP9 and HCMV infection. We identify a cross-talk between BMP9-induced and IFN receptor-mediated signaling, showing that BMP9 boosts the transcriptional response to and antiviral activity of IFNß, thereby enhancing viral restriction. We also show that BMP9 is secreted by human fibroblasts upon HCMV infection. However, HCMV infection impairs BMP9-induced enhancement of the IFNß response, indicating that this signaling role of BMP9 is actively targeted by HCMV. Indeed, transmembrane proteins US18 and US20, which downregulate type I BMP receptors, are necessary and sufficient to cause inhibition of BMP9-mediated boosting of the antiviral response to IFNß. HCMV lacking US18 and US20 is more sensitive to IFNß. Thus, HCMV has a mutually antagonistic relationship with BMP9, which extends the growing body of evidence that BMP signaling is an underappreciated modulator of innate immunity in response to viral infection.

Crystal structure of the tegument protein UL82 (pp71) from human cytomegalovirus.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that infects a majority of the world population. It may cause severe disease in immunocompromised people and lead to pregnancy loss or grave disabilities of the fetus upon congenital infection. For effective replication and lifelong persistence in its host, HCMV relies on diverse functions of its tegument protein UL82, also known as pp71. Up to now, little is known about the molecular mechanisms underlying the multiple functions of this crucial viral protein. Here, we describe the X-ray structure of full-length UL82 to a resolution of 2.7 Å. A single polypeptide chain of 559 amino acids mainly folds into three ß-barrels. We show that UL82 forms a dimer in the crystal as well as in solution. We identify point mutations that disturb the dimerization interface and show that the mutant protein is monomeric in solution and upon expression in human cells. On the basis of the three-dimensional structure, we identify structural homologs of UL82 from other herpesviruses and analyze whether their functions are preserved in UL82. We demonstrate that UL82, despite its structural homology to viral deoxyuridinetriphosphatases (dUTPases), does not possess dUTPase activity. Prompted by the structural homology of UL82 to the ORF10 protein of murine herpesvirus 68 (MHV68), which is known to interact with the RNA export factor ribonucleic acid export 1 (Rae1), we performed coimmunoprecipitations and demonstrated that UL82 indeed interacts with Rae1. This suggests that HCMV UL82 may play a role in mRNA export from the nucleus similar to ORF10 encoded by the gammaherpesviruses MHV68.

The incredible bulk: Human cytomegalovirus tegument architectures uncovered by AI-empowered cryo-EM.

The compartmentalization of eukaryotic cells presents considerable challenges to the herpesvirus life cycle. The herpesvirus tegument, a bulky proteinaceous aggregate sandwiched between herpesviruses' capsid and envelope, is uniquely evolved to address these challenges, yet tegument structure and organization remain poorly characterized. We use deep-learning-enhanced cryogenic electron microscopy to investigate the tegument of human cytomegalovirus virions and noninfectious enveloped particles (NIEPs; a genome packaging-aborted state), revealing a portal-biased tegumentation scheme. We resolve atomic structures of portal vertex-associated tegument (PVAT) and identify multiple configurations of PVAT arising from layered reorganization of pUL77, pUL48 (large tegument protein), and pUL47 (inner tegument protein) assemblies. Analyses show that pUL77 seals the last-packaged viral genome end through electrostatic interactions, pUL77 and pUL48 harbor a head-linker-capsid-binding motif conducive to PVAT reconfiguration, and pUL47/48 dimers form 45-nm-long filaments extending from the portal vertex. These results provide a structural framework for understanding how herpesvirus tegument facilitates and evolves during processes spanning viral genome packaging to delivery.

A cytomegalovirus inflammasome inhibitor reduces proinflammatory cytokine release and pyroptosis.

In response to viral infection, cells can initiate programmed cell death (PCD), leading to a reduction in the release of viral progeny. Viruses have therefore evolved specific mechanisms to curb PCD. Cytomegaloviruses (CMVs) are sophisticated manipulators of cellular defenses and encode potent inhibitors of apoptosis and necroptosis. However, a CMV inhibitor of pyroptosis has not been clearly identified and characterized. Here we identify the mouse cytomegalovirus M84 protein as an inhibitor of pyroptosis and proinflammatory cytokine release. M84 interacts with the pyrin domain of AIM2 and ASC to inhibit inflammasome assembly. It thereby prevents Caspase-1-mediated activation of interleukin 1ß (IL-1ß), IL-18, and Gasdermin D. Growth attenuation of an M84-deficient MCMV in macrophages is rescued by knockout of either Aim2 or Asc or by treatment with a Caspase-1 inhibitor, and its attenuation in infected mice is partially rescued in Asc knockout mice. Thus, viral inhibition of the inflammasome-pyroptosis pathway is important to promote viral replication in vivo.

Structural determinants of phosphorylation-dependent nuclear transport of HCMV DNA polymerase processivity factor UL44.

Human cytomegalovirus DNA polymerase processivity factor UL44 is transported into the nucleus by importin (IMP) α/ß through a classical nuclear localization signal (NLS), and this region is susceptible to cdc2-mediated phosphorylation at position T427. Whilst phosphorylation within and close to the UL44 NLS regulates nuclear transport, the details remain elusive, due to the paucity of structural information regarding the role of negatively charged cargo phosphate groups. We addressed this issue by studying the effect of UL44 T427 phosphorylation on interaction with several IMPα isoforms by biochemical and structural approaches. Phosphorylation decreased UL44/IMPα affinity 10-fold, and a comparative structural analysis of UL44 NLS phosphorylated and non-phosphorylated peptides complexed with mouse IMPα2 revealed the structural rearrangements responsible for phosphorylation-dependent inhibition of UL44 nuclear import.

Dissecting the cytomegalovirus CC chemokine: Chemokine activity and gHgLchemokine-dependent cell tropism are independent players in CMV infection.

Like all herpesviruses, cytomegaloviruses (CMVs) code for many immunomodulatory proteins including chemokines. The human cytomegalovirus (HCMV) CC chemokine pUL128 has a dual role in the infection cycle. On one hand, it forms the pentameric receptor-binding complex gHgLpUL(128,130,131A), which is crucial for the broad cell tropism of HCMV. On the other hand, it is an active chemokine that attracts leukocytes and shapes their activation. All animal CMVs studied so far have functionally homologous CC chemokines. In murine cytomegalovirus (MCMV), the CC chemokine is encoded by the m131/m129 reading frames. The MCMV CC chemokine is called MCK2 and forms a trimeric gHgLMCK2 entry complex. Here, we have generated MCK2 mutant viruses either unable to form gHgLMCK2 complexes, lacking the chemokine function or lacking both functions. By using these viruses, we could demonstrate that gHgLMCK2-dependent entry and MCK2 chemokine activity are independent functions of MCK2 in vitro and in vivo. The gHgLMCK2 complex promotes the tropism for leukocytes like macrophages and dendritic cells and secures high titers in salivary glands in MCMV-infected mice independent of the chemokine activity of MCK2. In contrast, reduced early antiviral T cell responses in MCMV-infected mice are dependent on MCK2 being an active chemokine and do not require the formation of gHgLMCK2 complexes. High levels of CCL2 and IFN-γ in spleens of infected mice and MCMV virulence depend on both, the formation of gHgLMCK2 complexes and the MCK2 chemokine activity. Thus, independent and concerted functions of MCK2 serving as chemokine and part of a gHgL entry complex shape antiviral immunity and virus dissemination.

Mechanism of Human Cytomegalovirus-Induced Epithelial-Mesenchymal Transition in Glioma Cells via the Upregulation of RIP2 Expression.

Human cytomegalovirus (HCMV) is associated with epithelial-mesenchymal transition (EMT) in glioma cells; however, its underlying action mechanism remain ambiguous. In this study, we investigated the effects of receptor-interacting protein 2 (RIP2) and nuclear factor (NF)-κB on EMT in HCMV-infected glioma LN-18 cells. Wound healing and invasion assays were used to evaluate the migration and invasion of cells. Western blotting and immunofluorescence microscopy were used to determine the protein expression levels. We found that HCMV induced enhanced migration and invasion of LN-18 cells, activation of the RIP2/NF-κB signaling pathway, downregulation of epithelial cell marker (E-cadherin) expression, and upregulation of mesenchymal cell marker (N-cadherin and vimentin) expression. Moreover, inhibition of RIP2 or NF-κB inhibited the induction of HCMV in LN-18 cells. Therefore, HCMV induces EMT in glioma cells by promoting the activation of NF-κB signaling pathway via the upregulation of RIP2 expression.

Current Knowledge on the Interaction of Human Cytomegalovirus Infection, Encoded miRNAs, and Acute Aortic Syndrome.

Aortic dissection is a clinicopathological entity caused by rupture of the intima, leading to a high mortality if not treated. Over time, diagnostic and investigative methods, antihypertensive therapy, and early referrals have resulted in improved outcomes according to registry data. Some data have also emerged from recent studies suggesting a link between Human Cytomegalovirus (HCMV) infection and aortic dissection. Furthermore, the use of microRNAs has also become increasingly widespread in the literature. These have been noted to play a role in aortic dissections with elevated levels noted in studies as early as 2017. This review aims to provide a broad and holistic overview of the role of miRNAs, while studying the role of HCMV infection in the context of aortic dissections. The roles of long non-coding RNAs, circular RNAs, and microRNAs are explored to identify changes in expression during aortic dissections. The use of such biomarkers may one day be translated into clinical practice to allow early detection and prognostication of outcomes and drive preventative and therapeutic options in the future.

Expression of viral CD45 ligand E3/49K on porcine cells reduces human anti-pig immune responses.

Transgenic expression of protective molecules in porcine cells and tissues is a promising approach to prevent xenograft rejection. Viruses have developed various strategies to escape the host's immune system. We generated porcine B cells (B cell line L23) expressing the human adenovirus protein E3/49K or the human cytomegalovirus protein pUL11 and investigated how human T, NK and B cell responses are affected by the expression of the viral proteins. Binding studies revealed that E3/49K and pUL11 interact with CD45 on human but not porcine peripheral blood mononuclear cells. T cell proliferation in response to L23-E3/49K cells was significantly reduced and accompanied by development of an anti-inflammatory cytokine milieu (low: TNF-alpha, IFN-gamma, IL-6; high: IL-4, IL-10). Human peripheral blood mononuclear cells which had been primed for four weeks by L23-E3/49K cells included an extended population of regulatory T cells. Cytotoxicity of effector T and natural killer cells against L23 cells was significantly reduced (40 to 50%) by E3/49K expression. B cell activation and antibody production to E3/49K expressing cells was also diminished. Surprisingly, pUL11 expression showed no effects. Reduction of human anti-pig immune responses by transgenic expression of selected viral genes may be a novel approach for protection of porcine xenografts.

Non-classical HLA-E restricted CMV 15-mer peptides are recognized by adaptive NK cells and induce memory responses.

Introduction: Human cytomegalovirus (HCMV) reactivation causes complications in immunocompromised patients after hematopoietic stem cell transplantation (HSCT), significantly increasing morbidity and mortality. Adaptive Natural Killer (aNK) cells undergo a persistent reconfiguration in response to HCMV reactivation; however, the exact role of aNK cell memory in HCMV surveillance remains elusive. Methods: We employed mass spectrometry and computational prediction approaches to identify HLA-E-restricted HCMV peptides that can elucidate aNK cell responses. We also used the K562 cell line transfected with HLA-E0*0103 for specific peptide binding and blocking assays. Subsequently, NK cells were cocultured with dendritic cells (DCs) loaded with each of the identified peptides to examine aNK and conventional (c)NK cell responses. Results: Here, we discovered three unconventional HLA-E-restricted 15-mer peptides (SEVENVSVNVHNPTG, TSGSDSDEELVTTER, and DSDEELVTTERKTPR) derived from the HCMV pp65-protein that elicit aNK cell memory responses restricted to HCMV. aNK cells displayed memory responses towards HMCV-infected cells and HCMV-seropositive individuals when primed by DCs loaded with each of these peptides and predicted 9-mer versions. Blocking the interaction between HLA-E and the activation NKG2C receptor but not the inhibitory NKG2A receptor abolished these specific recall responses. Interestingly, compared to the HLA-E complex with the leader peptide VMAPRTLIL, HLA-E complexes formed with each of the three identified peptides significantly changed the surface electrostatic potential to highly negative. Furthermore, these peptides do not comprise the classical HLA-E-restriction motifs. Discussion: These findings suggest a differential binding to NKG2C compared to HLA-E complexes with classical leader peptides that may result in the specific activation of aNK cells. We then designed six nonameric peptides based on the three discovered peptides that could elicit aNK cell memory responses to HCMV necessary for therapeutic inventions. The results provide novel insights into HLA-E-mediated signaling networks that mediate aNK cell recall responses and maximize their reactivity.

Establishment and application of a point-of-care testing and diagnosis method for early immediate expression gene IE1 of cytomegalovirus in maternal urine based on isothermal amplification.

BACKGROUND: Human Cytomegalovirus virus (HCMV) is a worldwide virus that causes no serious symptoms in most adults. However, HCMV infection during pregnancy, it may lead to a series of serious complications, such as hearing loss, mental retardation, visual impairment, microcephaly and developmental retardation. AIM: The aim of this study was to develop a simple, low dependence on equipment and accurate method for HCMV detection based on the recombinase polymerase amplification (RPA) and lateral flow chromatography strip (LFS) reading. METHODS: In order to meet the feasibility of HCMV early screening, three pairs of RPA primers were designed based on the UL123 gene encoding IE1, which was expressed immediately in the early stage of HCMV. In order to improve the specificity of the reaction and satisfy the visual detection, a specific probe was designed to insert THF site between upstream and downstream primers, fluorescein isothiocyanate (FITC) and C3spacer were used to modify the 5' end and the 3' end respectively, and Biotin was used to modify the 5' end of the reverse primer. HCMV standard strain AD169 was enriched by ARPE-19 cells culture, and its genome was extracted. The primers and probes were screened by RPA-LFS test, and the optimal reaction temperature and time were determined The specificity was verified in different viruses, bacteria and parasites. The standard curve was drawn based on the constructed recombinant plasmid of pMD18T-HCMV-UL123 and used for HCMV genomic DNA quantification and determination of the detection sensitivity. Urine samples from artificial HCMV contamination or clinical collection were prepared to evaluate the consistency with the results of real-time quantitative PCR. RESULTS: The results showed that the primers and probes for HCMV RPA-LFS detection based on UL123 gene were successfully screened, the amplification of HCMV genomic DNA with as low as 30 copies could be completed at 37 °C within 15 min, it did not react with Human herpesvirus 1, Streptococcus pyogenes, Candida albicans, Listeria monocytogenes, Y. enterocolitica, Klebsiella Pneumoniae, Enterobacter cloacae, Citrobacter freundii, Vibrio alginnolyfificus, Vibrio parahaemolyticus, S. typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa and Trichomonas vaginalis. The positive rate of PCR was 96.67 % in 30 simulated urine samples and 100 % in 127 clinical urine samples with the same UL123 gene detection. CONCLUSIONS: To sum up, we developed a diagnostic method for HCMV based on UL123 gene combined with RPA and LFS, which is low dependent on equipment, fast, sensitive and specific, provide reference for point-of-care testing HCMV in grass-roots laboratories and remote areas.

Spatially resolved protein map of intact human cytomegalovirus virions.

Herpesviruses assemble large enveloped particles that are difficult to characterize structurally due to their size, fragility and complex multilayered proteome with partially amorphous nature. Here we used crosslinking mass spectrometry and quantitative proteomics to derive a spatially resolved interactome map of intact human cytomegalovirus virions. This enabled the de novo allocation of 32 viral proteins into four spatially resolved virion layers, each organized by a dominant viral scaffold protein. The viral protein UL32 engages with all layers in an N-to-C-terminal radial orientation, bridging nucleocapsid to viral envelope. We observed the layer-specific incorporation of 82 host proteins, of which 39 are selectively recruited. We uncovered how UL32, by recruitment of PP-1 phosphatase, antagonizes binding to 14-3-3 proteins. This mechanism assures effective viral biogenesis, suggesting a perturbing role of UL32-14-3-3 interaction. Finally, we integrated these data into a coarse-grained model to provide global insights into the native configuration of virus and host protein interactions inside herpesvirions.

A Cysteine Residue of Human Cytomegalovirus vMIA Protein Plays a Crucial Role in Viperin Trafficking to Control Viral Infectivity.

Viperin is a multifunctional interferon-inducible protein that is directly induced in cells by human cytomegalovirus (HCMV) infection. The viral mitochondrion-localized inhibitor of apoptosis (vMIA) interacts with viperin at the early stages of infection and translocates it from the endoplasmic reticulum to the mitochondria, where viperin modulates the cellular metabolism to increase viral infectivity. Viperin finally relocalizes to the viral assembly compartment (AC) at late stages of infection. Despite the importance of vMIA interactions with viperin during viral infection, their interacting residues are unknown. In the present study, we showed that cysteine residue 44 (Cys44) of vMIA and the N-terminal domain (amino acids [aa] 1 to 42) of viperin are necessary for their interaction and for the mitochondrial localization of viperin. In addition, the N-terminal domain of mouse viperin, which is structurally similar to that of human viperin, interacted with vMIA. This indicates that the structure, rather than the sequence composition, of the N-terminal domain of viperin, is required for the interaction with vMIA. Recombinant HCMV, in which Cys44 of vMIA was replaced by an alanine residue, failed to translocate viperin to the mitochondria at the early stages of infection and inefficiently relocalized it to the AC at late stages of infection, resulting in the impairment of viperin-mediated lipid synthesis and a reduction in viral replication. These data indicate that Cys44 of vMIA is therefore essential for the intracellular trafficking and function of viperin to increase viral replication. Our findings also suggest that the interacting residues of these two proteins are potential therapeutic targets for HCMV-associated diseases. IMPORTANCE Viperin traffics to the endoplasmic reticulum (ER), mitochondria, and viral assembly compartment (AC) during human cytomegalovirus (HCMV) infection. Viperin has antiviral activity at the ER and regulates cellular metabolism at the mitochondria. Here, we show that Cys44 of HCMV vMIA protein and the N-terminal domain (aa 1 to 42) of viperin are necessary for their interaction. Cys44 of vMIA also has a critical role for viperin trafficking from the ER to the AC via the mitochondria during viral infection. Recombinant HCMV expressing a mutant vMIA Cys44 has impaired lipid synthesis and viral infectivity, which are attributed to mislocalization of viperin. Cys44 of vMIA is essential for the trafficking and function of viperin and may be a therapeutic target for HCMV-associated diseases.

Cytomegalovirus and Cardiovascular Disease: A Hypothetical Role for Viral G-Protein-Coupled Receptors in Hypertension.

Cytomegalovirus (CMV) is a member of the ß-herpesviruses and is ubiquitous, infecting 50%-99% of the human population depending on ethnic and socioeconomic conditions. CMV establishes lifelong, latent infections in their host. Spontaneous reactivation of CMV is usually asymptomatic, but reactivation events in immunocompromised or immunosuppressed individuals can lead to severe morbidity and mortality. Moreover, herpesvirus infections have been associated with several cardiovascular and post-transplant diseases (stroke, atherosclerosis, post-transplant vasculopathy, and hypertension). Herpesviruses, including CMV, encode viral G-protein-coupled receptors (vGPCRs) that alter the host cell by hijacking signaling pathways that play important roles in the viral life cycle and these cardiovascular diseases. In this brief review, we discuss the pharmacology and signaling properties of these vGPCRs, and their contribution to hypertension. Overall, these vGPCRs can be considered attractive targets moving forward in the development of novel hypertensive therapies.

Identification of Small Molecule Inhibitors of Human Cytomegalovirus pUL89 Endonuclease Using Integrated Computational Approaches.

Replication of Human Cytomegalovirus (HCMV) requires the presence of a metal-dependent endonuclease at the C-terminus of pUL89, in order to properly pack and cleave the viral genome. Therefore, pUL89 is an attractive target to design anti-CMV intervention. Herein, we used integrated structure-based and ligand-based virtual screening approaches in combination with MD simulation for the identification of potential metal binding small molecule antagonist of pUL89. In this regard, the essential chemical features needed for the inhibition of pUL89 endonuclease domain were defined and used as a 3D query to search chemical compounds from ZINC and ChEMBL database. Thereafter, the molecular docking and ligand-based shape screening were used to narrow down the compounds based on previously identified pUL89 antagonists. The selected virtual hits were further subjected to MD simulation to determine the intrinsic and ligand-induced flexibility of pUL89. The predicted binding modes showed that the compounds reside well in the binding site of endonuclease domain by chelating with the metal ions and crucial residues. Taken in concert, the in silico investigation led to the identification of potential pUL89 antagonists. This study provided promising starting point for further in vitro and in vivo studies.

Intercellular communication within the virus microenvironment affects the susceptibility of cells to secondary viral infections.

Communication between infected cells and cells in the surrounding tissue is a determinant of viral spread. However, it remains unclear how cells in close or distant proximity to an infected cell respond to primary or secondary infections. We establish a cell-based system to characterize a virus microenvironment, distinguishing infected, neighboring, and distal cells. Cell sorting, microscopy, proteomics, and cell cycle assays allow resolving cellular features and functional consequences of proximity to infection. We show that human cytomegalovirus (HCMV) infection primes neighboring cells for both subsequent HCMV infections and secondary infections with herpes simplex virus 1 and influenza A. Neighboring cells exhibit mitotic arrest, dampened innate immunity, and altered extracellular matrix. Conversely, distal cells are poised to slow viral spread due to enhanced antiviral responses. These findings demonstrate how infection reshapes the microenvironment through intercellular signaling to facilitate spread and how spatial proximity to an infection guides cell fate.

Decoding murine cytomegalovirus.

The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.

Novel Fab-peptide-HLA-I fusion proteins for redirecting pre-existing anti-CMV T cell immunity to selective eliminate carcinoma cells.

Typically, anticancer CD8pos T cells occur at low frequencies and become increasingly impaired in the tumor micro environment. In contrast, antiviral CD8pos T cells display a much higher polyclonality, frequency, and functionality. In particular, cytomegalovirus (CMV) infection induces high numbers of 'inflationary' CD8pos T cells that remain lifelong abundantly present in CMV-seropositive subjects. Importantly, these so-called inflationary anti-CMV T cells increase with age, maintain a ready-to-go state, populate tumors, and do not become exhausted or senescent. Given these favorable attributes, we devised a novel series of recombinant Fab-peptide-HLA-I fusion proteins and coined them 'ReTARGs'. A ReTARG fusion protein consists of a high-affinity Fab antibody fragment directed to carcinoma-associated cell surface antigen EpCAM (or EGFR), fused in tandem with soluble HLA-I molecule/ß2-microglobulin, genetically equipped with an immunodominant peptide derived from CMV proteins pp65 (or IE-1). Decoration with EpCAM-ReTARGpp65 rendered EpCAM-expressing primary patient-derived carcinoma cells highly sensitive to selective elimination by cognate anti-CMV CD8pos T cells. Importantly, this treatment did not induce excessive levels of proinflammatory T cell-secreted IFNγ. In contrast, analogous treatment with equimolar amounts of EpCAM/CD3-directed bispecific T-cell engager solitomab resulted in a massive release of IFNγ, a feature commonly associated with adverse cytokine-release syndrome. Combinatorial treatment with EpCAM-ReTARGpp65 and EGFR-ReTARGIE-1 strongly potentiated selective cancer cell elimination owing to the concerted action of the corresponding cognate anti-CMV CD8pos T cell clones. In conclusion, ReTARG fusion proteins may be useful as an alternative or complementary form of targeted cancer immunotherapy for 'cold' solid cancers.