Human Cytomegalovirus Replication In Fibroblasts Research Articles

The Pentamer glycoprotein complex inhibits viral Immediate Early transcription during Human Cytomegalovirus infections

The Pentamer complex of Human Cytomegalovirus (HCMV) consists of the viral glycoproteins gH, gL, UL128, UL130, and UL131 and is incorporated into infectious virions. HCMV strains propagated extensively in vitro in fibroblasts carry UL128, UL130, or UL131 alleles that do not make a functional complex and thus lack Pentamer function. Adding functional Pentamer to such strains decreases virus growth in fibroblasts. Here, we show that the Pentamer inhibits productive HCMV replication in fibroblasts by repressing viral Immediate Early (IE) transcription. We show that ectopic expression of the viral IE1 protein, a target of Pentamer-mediated transcriptional repression, complements the growth defect of a Pentamer-positive virus. Furthermore, we show that the Pentamer also represses viral IE transcription in cell types where HCMV in vitro latency is studied. Finally, we identify UL130 as a functional subunit of the Pentamer for IE transcriptional repression and demonstrate that cyclic AMP Response Element (CRE) and NFkB sites within the Major Immediate Early Promoter that drives IE1 transcription contribute to this repression. We conclude that the HCMV Pentamer represses viral IE transcription.

Turning the tables on cytomegalovirus: targeting viral Fc receptors by CARs containing mutated CH2\u2013CH3 IgG spacer domains

BackgroundDuring infection with human cytomegalovirus (HCMV) several viral proteins occur on cell surfaces in high quantity. We thus pursue an HLA-independent approach for immunotherapy of HCMV using chimeric antigen receptors (CARs) and bispecific BiTE® antibody constructs. In this context, HCMV-encoded proteins that mediate viral immune evasion and bind human IgG might represent particularly attractive target antigens. Unlike in observations of similar approaches for HIV and hepatitis B and C viruses, however, HCMV-infected cells develop a striking resistance to cytotoxic effector functions at later stages of the replication cycle. In our study we therefore wanted to test two hypotheses: (1) CAR T cells can efficiently inhibit HCMV replication independently from cytotoxic effector functions, and (2) HCMV can be targeted by CH2–CH3 IgG spacer domains that contain mutations previously reported to prevent exhaustion and to rescue CAR T cell function in vivo.MethodsReplication of GFP-encoding recombinant HCMV in fibroblasts in the presence and absence of supernatants from T cell co-cultures plus/minus cytokine neutralizing antibodies was analyzed by flow cytometry. CARs with wild type and mutated CH2–CH3 domains were expressed in human T cells by mRNA electroporation, and the function of the CARs was assessed by quantifying T cell cytokine secretion.ResultsWe confirm and extend previous evidence of antiviral cytokine effects and demonstrate that CAR T cells strongly block HCMV replication in fibroblasts mainly by combined secretion of IFN-γ and TNF. Furthermore, we show that fibroblasts infected with HCMV strains AD169 and Towne starting from day 3 have a high capacity for binding of human IgG1 and also strongly activate T cells expressing a CAR with CH2–CH3 domain. Importantly, we further show that mutations in the CH2–CH3 domain of IgG1 and IgG4, which were previously reported to rescue CAR T cell function by abrogating interaction with endogenous Fc receptors (FcRs), still enable recognition of FcRs encoded by HCMV.ConclusionsOur findings identify HCMV-encoded FcRs as an attractive additional target for HCMV immunotherapy by CARs and possibly bispecific antibodies. The use of specifically mutated IgG domains that bind to HCMV-FcRs without recognizing endogenous FcRs may supersede screening for novel binders directed against individual HCMV-FcRs.

A human cytomegalovirus deleted of internal repeats replicates with near wild type efficiency but fails to undergo genome isomerization.

The class E genome of human cytomegalovirus (HCMV) contains long and short segments that invert due to recombination between flanking inverted repeats, causing the genome to isomerize into four distinct isomers. To determine if isomerization is important for HCMV replication, one copy of each repeat was deleted. The resulting virus replicated in cultured human fibroblasts with only a slight growth impairment. Restriction and Southern analyses confirmed that its genome is locked in the prototypic arrangement and unable to isomerize. We conclude that efficient replication of HCMV in fibroblasts does not require (i) the ability to undergo genome isomerization, (ii) genes that lie partially within the deleted repeats, or (iii) diploidy of genes that lie wholly within repeats. The simple genomic structure of this virus should facilitate studies of genome circularization, latency or persistence, and concatemer packaging as such studies are hindered by the complexities imposed by isomerization.

Human Cytomegalovirus Gene UL21a Encodes a Short-Lived Cytoplasmic Protein and Facilitates Virus Replication in Fibroblasts

The human cytomegalovirus (HCMV) gene UL21a was recently annotated by its conservation in chimpanzee cytomegalovirus. Two large-scale mutagenic analyses showed that mutations in overlapping UL21a/UL21 resulted in a severe defect of virus growth in fibroblasts. Here, we characterized UL21a and demonstrated its role in HCMV infection. We mapped a UL21a-specific transcript of approximately 600 bp that was expressed with early kinetics. UL21a encoded pUL21a, a protein of approximately 15 kDa, which was unstable and localized predominantly to the cytoplasm during HCMV infection or when expressed alone. Interestingly, pUL21a was drastically stabilized in the presence of proteasome inhibitor MG132, but its instability was independent of a functional ubiquitin-mediated pathway, suggesting that pUL21a underwent proteasome-dependent, ubiquitin-independent degradation. A UL21a deletion virus was attenuated in primary human newborn foreskin fibroblasts (HFFs) and embryonic lung fibroblasts (MRC-5), whereas a marker-rescued virus and mutant viruses lacking the neighboring or overlapping genes UL20, UL21, or UL21.5-UL23 replicated at wild-type levels. The growth defect of UL21a-deficient virus in MRC-5 cells was more pronounced than that in HFFs. At a high multiplicity of infection, the UL21a deletion virus synthesized viral proteins with wild-type kinetics but had a two- to threefold defect in viral DNA replication. More importantly, although pUL21a was not detected in the virion, progeny virions produced by the mutant virus were approximately 10 times less infectious than wild-type virus, suggesting that UL21a is required for HCMV to establish efficient productive infection. We conclude that UL21a encodes a short-lived cytoplasmic protein and facilitates HCMV replication in fibroblasts.

Onset of Human Cytomegalovirus Replication in Fibroblasts Requires the Presence of an Intact Vimentin Cytoskeleton

Like all viruses, herpesviruses extensively interact with the host cytoskeleton during entry. While microtubules and microfilaments appear to facilitate viral capsid transport toward the nucleus, evidence for a role of intermediate filaments in herpesvirus entry is lacking. Here, we examined the function of vimentin intermediate filaments in fibroblasts during the initial phase of infection of two genotypically distinct strains of human cytomegalovirus (CMV), one with narrow (AD169) and one with broad (TB40/E) cell tropism. Chemical disruption of the vimentin network with acrylamide, intermediate filament bundling in cells from a patient with giant axonal neuropathy, and absence of vimentin in fibroblasts from vimentin(-/-) mice severely reduced entry of either strain. In vimentin null cells, viral particles remained in the cytoplasm longer than in vimentin(+/+) cells. TB40/E infection was consistently slower than that of AD169 and was more negatively affected by the disruption or absence of vimentin. These findings demonstrate that an intact vimentin network is required for CMV infection onset, that intermediate filaments may function during viral entry to facilitate capsid trafficking and/or docking to the nuclear envelope, and that maintenance of a broader cell tropism is associated with a higher degree of dependence on the vimentin cytoskeleton.

NF-κB activation can mediate inhibition of human cytomegalovirus replication

The activation of NF-kappaB has long been considered a positive factor for human cytomegalovirus (HCMV) replication. The HCMV immediate-early promoter, the initial transcriptional element in the HCMV replication cycle, is activated by the transcription factor NF-kappaB, and several HCMV gene products have been demonstrated to activate this transcription factor. However, the role of NF-kappaB in the full replication cycle of the virus has not been carefully examined. A series of experiments that demonstrate an important inhibitory role of NF-kappaB for HCMV replication in fibroblasts is presented here. Using both genetic and pharmaceutical methods, it was shown that blocking NF-kappaB activation in cell culture does not inhibit HCMV replication, but rather leads to a modest increase in replication. Two cytokines inhibitory for HCMV, tumour necrosis factor-alpha and interferon-gamma, no longer inhibit HCMV when NF-kappaB activation is blocked. Furthermore, forced expression of the NF-kappaB activating IkappaB kinase beta (IKKbeta), but not a kinase inactive mutant, also inhibits HCMV replication. In addition, it was shown that NF-kappaB signalling is essential for the production of an anti-viral factor in the supernatant of HCMV-infected fibroblasts, and identified interferon-beta as this factor. Thus, the role of NF-kappaB in fibroblasts is to activate a host defence against HCMV.

Increased human cytomegalovirus replication in fibroblasts after treatment with therapeutical plasma concentrations of valproic acid

Valproic acid (2-propylpentanoic acid, VPA), an effective inhibitor of histone deacetylases (HDAC) is used for the treatment of epilepsia. In this study, structure–activity relationships for the action of structurally modified VPA derivatives on human cytomegalovirus (HCMV) replication and HDAC inhibition were defined. Pretreatment of human foreskin fibroblasts with VPA (0.125–1 mM) caused a concentration-dependent increase of HCMV immediate early and antigen late antigen expression. Structure–activity relationships of VPA derivatives for HCMV stimulation were compared to those for teratogenic action and those for HDAC inhibition. Side chain elongation and introduction of a triple bond in 4-position of the other chain caused teratogenicity, stimulated HCMV replication, and increased HDAC inhibition, as demonstrated by enhanced levels of acetylated histones. Teratogenic VPA derivatives with a branched chain in 3-position as well as a non-teratogenic anticonvulsive active VPA derivative did not stimulate HCMV or accumulation of acetylated histones. This demonstrates a strict correlation between inhibition of HDAC and increased HCMV replication.

Inhibition of cytomegalovirus late antigen expression and cytomegalovirus replication in human fibroblasts and differentiated monocytic cells by liposome-encapsulated foscarnet

Foscarnet is one of the drugs of choice for the treatment of cytomegalovirus (CMV) infections, but its efficacy is limited by systemic toxicity, poor penetration into cells and a short plasma half-life. In this study, two types of sized, foscarnet-containing liposomes were evaluated with respect to their in vitro efficacy against human CMV in differentiated monocytic (THP-1) cells and embryonic lung fibroblasts. Both preparations of liposomal foscarnet inhibited CMV replication and CMV late antigen expression in either cell type. In contrast to free foscarnet, the extent of inhibition was dependent on the cell type, with superior activity towards CMV in THP-1 cells. The antiviral effect was most likely mediated by direct association of liposome-encapsulated foscarnet with THP-1 cells. Free foscarnet, however, showed an equal or greater antiviral activity than liposome-encapsulated foscarnet. At the IC 50 concentrations of free and liposome-encapsulated foscarnet, toxicity towards the permissive cells was not observed. Together with our previous findings of the prolonged circulation time of one of the liposome preparations used in this study, these results indicate that liposome-encapsulated foscarnet warrants further exploration to assess its potential for the treatment of CMV-infections.

Expression of human cytomegalovirus ppUL83 (pp65) in a stable cell line and its association with metaphase chromosomes.

Astrocytoma cells were transfected with an expression vector for the structural phosphoprotein ppUL83 (pp65) of human cytomegalovirus (HCMV). Once made, pp65 showed the same characteristics as naturally produced protein and was stable for several days in astrocytoma cells, as it is during HCMV replication in fibroblasts. In permanently transfected cells, pp65 was homogeneously dispersed in the cell nucleus and showed a preferential methanol-stable association with condensed chromatin throughout cell division. Furthermore, pp65 could be detected bound to metaphase-arrested chromosomes in pp65-expressing astrocytoma cells, and in fibroblasts and astrocytoma cells during productive virus infection.

Inhibition of CMV replication in human fibroblasts by human monocyte-derived macrophages: implication for CMV persistent infection

The effect of monocytes (M) and monocyte-derived macrophages (MdM) on cytomegalovirus (CMV) replication in human fibroblasts (HF) was studied by one step growth experiments, plaque formation and dot hybridization with a labeled CMV DNA probe. HF were infected with CMV at multiplicities of infection (mol) of 0.001 to 1. After virus adsorption, M or MdM were added at an effector target ratio of 2:1. MdM reduced both infectious viral yield and the quantity of viral DNA and inhibited viral plaque formation. M, however, affected these parameters to a lesser extent. Electron microscopic studies showed that MdM treated CMF-infected HF, 4 days pi, contained only a few capsids in their nuclei and many vacuoles in their cytoplasm as compared to CMV infected HF (control). A reduction of CMV DNA inhibition was observed upon incubation of the infected HF cells with MdM separated from the infected cells by a membrane. Addition of tumor necrosis factor (TNF) antibody to CMV-infected HF incubated with MdM either in direct contact or separated by a membrane from the infected cells reduced the inhibition of CMV-DNA production. The results of this study suggest that MdM may modulate CMV replication in vivo and may also have a role in CMV persistence or chronic infection.