Interactions between proteins encoded within the human cytomegalovirus UL133-UL138 locus

We previously described a novel genetic locus within the ULb' region of the human cytomegalovirus (HCMV) genome that, while dispensable for replication in fibroblasts, suppresses replication in hematopoietic progenitors and augments replication in endothelial cells. This locus, referred to as the UL133-UL138 locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. In this work, we have mapped the interactions among these proteins. An analysis of all pairwise interactions during transient expression revealed a robust interaction between pUL133 and pUL138. Potential interactions between pUL136 and both pUL133 and pUL138 were also revealed. In addition, each of the UL133-UL138 locus proteins self-associated, suggesting a potential to form higher-order homomeric complexes. As both pUL133 and pUL138 function in promoting viral latency in CD34(+) hematopoietic progenitor cells (HPCs) infected in vitro, we further focused on this interaction. pUL133 and pUL138 are the predominant complex detected when all proteins are expressed together and require no other proteins in the locus for their association. During infection, the interaction between pUL133 and pUL138 or pUL136 can be detected. A recombinant virus that fails to express both pUL133 and pUL138 exhibited a latency phenotype similar to that of viruses that fail to express either pUL133 or pUL138, indicating that these proteins function cooperatively in latency and do not have independent functions that additively contribute to HCMV latency. These studies identify protein interactions among proteins encoded by the UL133-UL138 locus and demonstrate an important interaction impacting the outcome of HCMV infection.     

An Endothelial Cell-Specific Requirement for the UL133-UL138 Locus of Human Cytomegalovirus for Efficient Virus Maturation

Human cytomegalovirus (HCMV) infects a variety of cell types in humans, resulting in a varied pathogenesis in the immunocompromised host. Endothelial cells (ECs) are considered an important target of HCMV infection that may contribute to viral pathogenesis. Although the viral determinants important for entry into ECs are well defined, the molecular determinants regulating postentry tropism in ECs are not known. We previously identified the UL133-UL138 locus encoded within the clinical strain-specific ULb′ region of the HCMV genome as important for the latent infection in CD34⁺ hematopoietic progenitor cells (HPCs). Interestingly, this locus, while dispensable for replication in fibroblasts, was required for efficient replication in ECs infected with the TB40E or fusion-inducing factor X (FIX) HCMV strains. ECs infected with a virus lacking the entire locus (UL133-UL138_NULL virus) complete the immediate-early and early phases of infection but are defective for infectious progeny virus production. ECs infected with UL133-UL138_NULL virus exhibited striking differences in the organization of intracellular membranes and in the assembly of mature virions relative to ECs infected with wild-type (WT) virus. In UL133-UL138_NULL virus-infected ECs, Golgi stacks were disrupted, and the viral assembly compartment characteristic of HCMV infection failed to form. Further, progeny virions in UL133-UL138_NULL virus-infected ECs inefficiently acquired the virion tegument and secondary envelope. These defects were specific to infection in ECs and not observed in fibroblasts infected with UL133-UL138_NULL virus, suggesting an EC-specific requirement for the UL133-UL138 locus for late stages of replication. To our knowledge, the UL133-UL138 locus represents the first cell-type-dependent, postentry tropism determinant required for viral maturation.     

The cytomegaloviral protein pUL138 acts as potentiator of tumor necrosis factor (TNF) receptor 1 surface density to enhance ULb'-encoded modulation of TNF-α signaling

Human cytomegalovirus is a ubiquitous herpesvirus that establishes lifelong latent infection. Changes in immune homeostasis induce the reactivation of lytic infection, which is mostly inapparent in healthy individuals but often causes overt disease in immunocompromised hosts. Based on discrepant tumor necrosis factor receptor 1 surface disposition between human cytomegalovirus AD169 variants differing in the ULb' region, we identified the latency-associated gene product pUL138, which also is expressed during productive infection, as a selective potentiator of tumor necrosis factor receptor 1, one of the key receptors of innate immunity. Ectopically expressed pUL138 coprecipitated with tumor necrosis factor receptor 1, extended the protein half-life, and enhanced its signaling responses, thus leading to tumor necrosis factor receptor 1 hyperresponsiveness. Conversely, the targeted deletion of UL138 from the human cytomegaloviral genome strongly reduced tumor necrosis factor receptor 1 surface densities of infected cells. Remarkably, the comparison of UL138 deficiency to ULb' deficiency revealed the presence of further positive modulators of tumor necrosis factor alpha signal transduction encoded within the human cytomegalovirus ULb' region, identifying this region as a hub for multilayered tumor necrosis factor alpha signaling regulation.     

UL146 variability among clinical isolates of human cytomegalovirus from Japan

Human cytomegalovirus (HCMV) is a herpesvirus associated with serious diseases in immunocompromised subjects. The region between ORF UL133 and UL151 from HCMV, named ULb' is frequently deleted in attenuated AD169 and in highly passaged laboratory strains. However, this region is conserved in low-passaged and more virulent HCMV, like the Toledo strain. The UL146 gene, which is located in the ULb' region, encodes a CXC-chemokine analogue. The diversity of UL146 gene was evaluated among fifty-six clinical isolates of HCMV from Japan. Results show that UL146 gene was successfully amplified by the polymerase chain reaction (PCR) in only 17/56 strains (30%), while the success rate for UL145/UL147 gene was 18/56 strains (32%). After DNA sequencing, the 35 amplified strains were classified into 8 groups. When compared, variability of UL146 ranged from 25.1% to 52.9% at the DNA level and from 34.5% to 67% at the amino acid level. Seven groups had the interleukin-8 (IL-8) motif ERL (Glu-Leu-Arg) CXC and one group had only the CXC motif, suggesting the absence of the IL-8 function of UL146. In conclusion, we found that UL146 gene of HCMV is hyper-variable in clinical strains from Japan suggesting the possibility of a different function in each sequence group.     

The Human Cytomegalovirus UL133-138 Gene Locus Attenuates the Lytic Viral Cycle in Fibroblasts

The genomes of HCMV clinical strains (e.g. FIX, TR, PH, etc) contain a 15 kb region that encodes 20 putative ORFs. The region, termed ULb’, is lost after serial passage of virus in human foreskin fibroblast (HFF) cell culture. Compared to clinical strains, laboratory strains replicate faster and to higher titers of infectious virus. We made recombinant viruses with 22, 14, or 7 ORFs deleted from the ULb’ region using FIX and TR as model clinical strains. We also introduced a stop codon into single ORFs between UL133 and UL138 to prevent protein expression. All deletions within ULb’ and all stop codon mutants within the UL133 to UL138 region increased to varying degrees, viral major immediate early RNA and protein, DNA, and cell-free infectious virus compared to the wild type viruses. The wild type viral proteins slowed down the viral replication process along with cell-free infectious virus release from human fibroblast cells.     

Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis

Clinical and low passage strains of human CMV (HCMV) encode an additional MHC class I-related molecule UL142, in addition to the previously described UL18. The UL142 open reading frame is encoded within the ULb' region which is missing from a number of common high passage laboratory strains. Cells expressing UL142 following transfection, and fibroblasts infected with a recombinant adenovirus-expressing UL142, were used to screen both polyclonal NK cells and NK cell clones, in a completely autologous system. Analysis of 100 NK cell clones derived from five donors, revealed 23 clones that were inhibited by fibroblasts expressing UL142 alone. Small-interfering RNA-mediated knockdown of UL142 mRNA expression in HCMV-infected cells resulted in increased sensitivity to lysis. From these data we conclude that UL142 is a novel HCMV-encoded MHC class I-related molecule which inhibits NK cell killing in a clonally dependent manner.     

Human cytomegalovirus regulates surface expression of the viral protein UL18 by means of two motifs present in the cytoplasmic tail

UL18 is a trans-membrane viral protein expressed on human cytomegalovirus (HCMV)-infected cells, and its surface expression determines the interaction of infected cells with lymphocytes expressing the CD85j (LIR-1/ILT2) receptor. We previously showed that the UL18-CD85j interaction elicits activation of T lymphocytes. However, in in vitro cell models UL18 displays mostly undetectable surface expression. Thus, we asked how surface expression of UL18 is regulated. Domain-swapping experiments and construction of specific mutants demonstrated that two motifs on its cytoplasmic tail, homologous to YXXPhi and KKXX consensus sequences, respectively, are responsible for impairing UL18 surface expression. However, the presence of the whole HCMV genome, granted by HCMV infection of human fibroblasts, restored surface expression of either UL18 or chimeric proteins carrying the UL18 cytoplasmic tail, starting from the third day after infection. It is of note that the two motifs responsible for cytoplasmic retention are identical in all 17 HCMV strains examined. We disclosed a control mechanism used by the HCMV to regulate the availability of UL18 on the infected-cell surface to allow interaction with its ligand on T and NK cells.     

Human Cytomegalovirus Latency-Associated Proteins Elicit Immune-Suppressive IL-10 Producing CD4+ T Cells

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.     

The polymorphic HCMV glycoprotein UL20 is targeted for lysosomal degradation by multiple cytoplasmic dileucine motifs

Human cytomegalovirus (HCMV) is a widespread and persistent beta-herpesvirus. The large DNA genome of HCMV encodes many proteins that are non-essential for viral replication including numerous proteins subverting host immunosurveillance. One of them is the barely characterized UL20, which is encoded adjacent to the well-defined immunoevasins UL16 and UL18. UL20 is a type I transmembrane glycoprotein with an immunoglobulin-like ectodomain that is highly polymorphic among HCMV strains. Here, we show that the homodimeric UL20, by virtue of its cytoplasmic domain, does not reach the cell surface but is targeted to endosomes and lysosomes. Accordingly, UL20 exhibits a short half-life because of rapid lysosomal degradation. Trafficking of UL20 to lysosomes is determined by several, independently functioning dileucine-based sorting motifs in the cytoplasmic domain of UL20 and involves the adaptor protein (AP) complex AP-1. Combined substitution of three dileucine motifs allowed strong cell surface expression of UL20 comparable to UL20 mutants lacking the cytoplasmic tail. Finally, we show that the intracellularly located UL20 also is subject to lysosomal degradation in the context of viral infection. Altogether, from these data, we hypothesize that UL20 is destined to efficiently sequester yet-to-be defined cellular proteins for degradation in lysosomes.     

Effects of human cytomegalovirus infection on ligands for the activating NKG2D receptor of NK cells: up-regulation of UL16-binding protein (ULBP)1 and ULBP2 is counteracted by the viral UL16 protein

Human CMV (HCMV) interferes with NK cell functions at various levels. The HCMV glycoprotein UL16 binds some of the ligands recognized by the NK-activating receptor NKG2D, namely UL16-binding proteins (ULBP) 1 and 2 and MHC class I-related chain B, possibly representing another mechanism of viral immune escape. This study addressed the expression and function of these proteins in infected cells. HCMV induced the expression of all three ULBPs, which were predominantly localized in the endoplasmic reticulum of infected fibroblasts together with UL16. However, while at a lower viral dose ULBP1 and 2 surface expression was completely inhibited compared to ULBP3, at a higher viral dose cell surface expression of ULBP1 and ULBP2 was delayed. The induction of ULBPs correlated with an increased dependency on NKG2D for recognition; however, the overall NK sensitivity did not change (suggesting that additional viral mechanisms interfere with NKG2D-independent pathways for recognition). Infection with a UL16 deletion mutant virus resulted in a different pattern compared to the wild type: all three ULBP molecules were induced with similar kinetics at the cell surface, accompanied by a pronounced, entirely NKG2D-dependent increase in NK sensitivity. Together our findings show that upon infection with HCMV, the host cell responds by expression of ULBPs and increased susceptibility to the NKG2D-mediated component of NK cell recognition, but UL16 limits these effects by interfering with the surface expression of ULBP1 and ULBP2.     

Genetic deletion of TNF receptor suppresses excitatory synaptic transmission via reducing AMPA receptor synaptic localization in cortical neurons

The distribution of postsynaptic glutamate receptors has been shown to be regulated by proimmunocytokine tumor necrosis factor α (TNF-α) signaling. The role of TNF-α receptor subtypes in mediating glutamate receptor expression, trafficking, and function still remains unclear. Here, we report that TNF receptor subtypes (TNFR1 and TNFR2) differentially modulate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) clustering and function in cultured cortical neurons. We find that genetic deletion of TNFR1 decreases surface expression and synaptic localization of the AMPAR GluA1 subunit, reduces the frequency of miniature excitatory postsynaptic current (mEPSC), and reduces AMPA-induced maximal whole-cell current. In addition, these results are not observed in TNFR2-deleted neurons. The decreased AMPAR expression and function in TNFR1-deleted cells are not significantly restored by short (2 h) or long (24 h) term exposure to TNF-α. In TNFR2-deleted cells, TNF-α promotes AMPAR trafficking to the synapse and increases mEPSC frequency. In the present study, we find no significant change in the GluN1 subunit of NMDAR clusters, location, and mEPSC. This includes applying or withholding the TNF-α treatment in both TNFR1- and TNFR2-deleted neurons. Our results indicate that TNF receptor subtype 1 but not 2 plays a critical role in modulating AMPAR clustering, suggesting that targeting TNFR1 gene might be a novel approach to preventing neuronal AMPAR-mediated excitotoxicity.     

Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser²²). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser²² is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.     

Cutting edge: a novel viral TNF receptor superfamily member in virulent strains of human cytomegalovirus.

The UL144 open reading frame found in clinical isolates of human CMV (HCMV) encodes a structural homologue of the herpesvirus entry mediator, a member of the TNFR superfamily. UL144 is a type I transmembrane glycoprotein that is expressed early after infection of fibroblasts; however, it is retained intracellularly. A YXXZ motif in the highly conserved cytoplasmic tail contributes to UL144 subcellular distribution. The finding that no known ligand of the TNF family binds UL144 suggests that its mechanism of action is distinct from other known viral immune evasion genes. Specific Abs to UL144 can be detected in the serum of a subset of HCMV seropositive individuals infected with HIV. This work establishes a novel molecular link between the TNF superfamily and herpesvirus that may contribute to the ability of HCMV to escape immune clearance.     

NK cell activity during human cytomegalovirus infection is dominated by US2-11-mediated HLA class I down-regulation

A highly attractive approach to investigate the influence and hierarchical organization of viral proteins on cellular immune responses is to employ mutant viruses carrying deletions of various virus-encoded, immune-modulating genes. Here, we introduce a novel set of deletion mutants of the human CMV (HCMV) lacking the UL40 region either alone or on the background of a deletion mutant devoid of the entire US2-11 region. Deletion of UL40 had no significant effect on lysis of infected cells by NK cells, indicating that the expected enhancement of HLA-E expression by specific peptides derived from HCMV-encoded gpUL40 leader sequences was insufficient to confer target cell protection. Moreover, the kinetics of MHC class I down-regulation by US2-11 genes observed at early and late phases postinfection with wild-type virus correlated with increased susceptibility to NK lysis. Thus, the influence of HCMV genes on NK reactivity follows a hierarchy dominated by the US2-11 region, which encodes all viral genes capable of down-modulating expression of classical and non-classical MHC class I molecules. The insights gained from studies of such virus mutants may impact on future therapeutic strategies and vaccine development and incorporate NK cells in the line of defense mechanisms against HCMV infection.     

Double anti-angiogenic and anti-inflammatory protein Valpha targeting VEGF-A and TNF-alpha in retinopathy and psoriasis

Pathological angiogenesis usually involves disrupted vascular integrity, vascular leakage, and infiltration of inflammatory cells, which are governed mainly by VEGF-A and TNF-α. Although many inhibitors targeting either VEGF-A or TNF-α have been developed, there is no single inhibitor molecule that simultaneously targets both molecules. Here, we designed and generated a novel chimeric decoy receptor (Valpha) that can simultaneously bind to VEGF-A and TNF-α and block their actions. In this experimental design, we have shown that Valpha, which is an effective synchronous blocker of VEGF-A and TNF-α, can drastically increase treatment effectiveness through its dual-blocking characteristics. Valpha contains the VEGF-A-binding domain of VEGFR1, the TNF-α-binding domain of TNFR2, and the Fc domain of IgG1. Valpha exhibited strong binding characteristics for its original counterparts, VEGF-A and TNF-α, but not for the extracellular matrix, resulting in a highly favorable pharmacokinetic profile in vivo. Compared with VEGF-Trap or Enbrel, both of which block either VEGF-A or TNF-α, singularly, Valpha is a highly effective molecule for reducing abnormal vascular tufts and the number of F4/80(+) macrophages in a retinopathy model. In addition, Valpha showed superior relief effects in a psoriasis model with regard to epidermal thickness and the area of blood and lymphatic vessels. Thus, the simultaneous blocking of VEGF-A and TNF-α using Valpha is an effective therapeutic strategy and cost-efficient for treatment of retinopathy and psoriasis.     

The Human Cytomegalovirus UL76 Gene Regulates the Level of Expression of the UL77 Gene

Background

Human cytomegalovirus (HCMV) can be reactivated under immunosuppressive conditions causing several fatal pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. HCMV also causes deafness and mental retardation in neonates when primary infection has occurred during pregnancy. In the genome of HCMV at least 194 known open reading frames (ORFs) have been predicted, and approximately one-quarter, or 41 ORFs, are required for viral replication in cell culture. In contrast, the majority of the predicted ORFs are nonessential for viral replication in cell culture. However, it is also possible that these ORFs are required for the efficient viral replication in the host. The UL77 gene of HCMV is essential for viral replication and has a role in viral DNA packaging. The function of the upstream UL76 gene in the HCMV-infected cells is not understood. UL76 and UL77 are cistons on the same viral mRNA and a conventional 5′ mRNA for UL77 has not been detected. The vast majority of eukaryotic mRNAs are monocistronic, i.e., they encode only a single protein.

Methodology/Principal Findings

To determine whether the UL76 ORF affects UL77 gene expression, we mutated UL76 by ORF frame-shifts, stop codons or deletion of the viral gene. The effect on UL77 protein expression was determined by either transfection of expression plasmids or infection with recombinant viruses. Mutation of UL76 ORF significantly increased the level of UL77 protein expression. However, deletion of UL76 upstream of the UL77 ORF had only marginal effects on viral growth.

Conclusions/Significance

While UL76 is not essential for viral replication, the UL76 ORF is involved in regulation of the level of UL77 protein expression in a manner dependent on the translation re-initiation. UL76 may fine-tune the UL77 expression for the efficient viral replication in the HCMV- infected cells.