The contribution of pUL74 to growth of human cytomegalovirus is masked in the presence of RL13 and UL128 expression

The glycoproteins gH and gL of human cytomegalovirus (HCMV) form a complex either with pUL74 (trimeric complex) or with proteins of the UL128 locus (pentameric complex). While the pentameric complex is dispensable for viral growth in fibroblasts, deletion of pUL74 causes a small plaque phenotype in HCMV lab strains, accompanied by greatly reduced cell-free infectivity. As HCMV isolates shortly after cultivation from clinical specimens do not release cell-free infectious virus, we wondered whether deletion of pUL74 would also affect virus growth in this background. To address this question, we took advantage of the BAC-cloned virus Merlin-RL13tetO that resembles such clinical isolates by growing cell-associated due to inducible expression of the viral RL13 gene. Stop codons were introduced by seamless mutagenesis into UL74 and/or the UL128 locus to abolish expression of the trimeric or pentameric complex, respectively. Virus mutants were reconstituted by transfection of the respective genomes into cultured cells and analyzed regarding focal growth. When the UL128 locus was intact, deletion of pUL74 did not notably affect focal growth of Merlin, irrespective of RL13 expression. In the absence of UL128 expression, foci were increased compared to wild type, and infectious cell-free virus was produced. Under these conditions, disruption of UL74 completely prevented virus spread from initially transfected cells to surrounding cells. In conclusion the contribution of pUL74 is masked when the UL128 locus is expressed at high levels, and its role in cell-free virus spread is only revealed when expression of the pentameric complex is inhibited

Human cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-alpha as a key for entry

Herpesvirus gH/gL envelope glycoprotein complexes are key players in virus entry as ligands for host cell receptors and by promoting fusion of viral envelopes with cellular membranes. Human cytomegalovirus ( HCMV) has two alternative gH/gL complexes, gH/gL/gO and gH/gL/UL128,130,131A which both shape the HCMV tropism. By studying binding of HCMV particles to fibroblasts, we could for the first time show that virion gH/gL/gO binds to platelet-derived growth factor-alpha (PDGFR-alpha) on the surface of fibroblasts and that gH/gL/gO either directly or indirectly recruits gB to this complex. PDGFR-alpha functions as an entry receptor for HCMV expressing gH/gL/gO, but not for HCMV mutants lacking the gH/gL/gO complex. PDGFR-alpha-dependent entry is not dependent on activation of PDGFR-alpha. We could also show that the gH/gL/gO-PDGFR-alpha interaction starts the predominant entry pathway for infection of fibroblasts with free virus. Cell-associated virus spread is either driven by gH/gL/gO interacting with PDGFR-alpha or by the gH/gL/UL128,130,131A complex. PDGFR-alpha-positive cells may thus be preferred first target cells for infections with free virus which might have implications for the design of future HCMV vaccines or anti-HCMV drugs

Cytomegalovirus protein m154 perturbs the adaptor protein-1 compartment mediating broad-spectrum immune evasion

Cytomegaloviruses (CMVs) are ubiquitous pathogens known to employ numerous immunoevasive strategies that significantly impair the ability of the immune system to eliminate the infected cells. Here, we report that the single mouse CMV (MCMV) protein, m154, downregulates multiple surface molecules involved in the activation and costimulation of the immune cells. We demonstrate that m154 uses its cytoplasmic tail motif, DD, to interfere with the adaptor protein-1 (AP-1) complex, implicated in intracellular protein sorting and packaging. As a consequence of the perturbed AP-1 sorting, m154 promotes lysosomal degradation of several proteins involved in T cell costimulation, thus impairing virus-specific CD8+ T cell response and virus control in vivo. Additionally, we show that HCMV infection similarly interferes with the AP-1 complex. Altogether, we identify the robust mechanism employed by single viral immunomodulatory protein targeting a broad spectrum of cell surface molecules involved in the antiviral immune response

Murine Models of Central Nervous System Disease following Congenital Human Cytomegalovirus Infections

Human cytomegalovirus infection of the developing fetus is a leading cause of neurodevelopmental disorders in infants and children, leading to long-term neurological sequela in a significant number of infected children. Current understanding of the neuropathogenesis of this intrauterine infection is limited because of the complexity of this infection, which includes maternal immunological responses that are overlaid on virus replication in the CNS during neurodevelopment. Furthermore, available data from human cases are observational, and tissues from autopsy studies have been derived from only the most severe infections. Animal models of this human infection are also limited by the strict species specificity of cytomegaloviruses. However, informative models including non-human primates and small animal models have been developed. These include several different murine models of congenital HCMV infection for the study of CMV neuropathogenesis. Although individual murine models do not completely recapitulate all aspects of the human infection, each model has provided significant information that has extended current understanding of the neuropathogenesis of this human infection. This review will compare and contrast different murine models in the context of available information from human studies of CNS disease following congenital HCMV infections

Non-redundant and Redundant Roles of Cytomegalovirus gH/gL Complexes in Host Organ Entry and Intra-tissue Spread

Abstract Herpesviruses form different gH/gL virion envelope glycoprotein complexes that serve as entry complexes for mediating viral cell-type tropism in vitro; their roles in vivo, however, remained speculative and can be addressed experimentally only in animal models. For murine cytomegalovirus two alternative gH/gL complexes, gH/gL/gO and gH/gL/MCK-2, have been identified. A limitation of studies on viral tropism in vivo has been the difficulty in distinguishing between infection initiation by viral entry into first-hit target cells and subsequent cell-to-cell spread within tissues. As a new strategy to dissect these two events, we used a gO-transcomplemented ΔgO mutant for providing the gH/gL/gO complex selectively for the initial entry step, while progeny virions lack gO in subsequent rounds of infection. Whereas gH/gL/gO proved to be critical for establishing infection by efficient entry into diverse cell types, including liver macrophages, endothelial cells, and hepatocytes, it was dispensable for intra-tissue spread. Notably, the salivary glands, the source of virus for host-to-host transmission, represent an exception in that entry into virus-producing cells did not strictly depend on either the gH/gL/gO or the gH/gL/MCK-2 complex. Only if both complexes were absent in gO and MCK-2 double-knockout virus, in vivo infection was abolished at all sites. Author Summary The role of viral glycoprotein entry complexes in viral tropism in vivo is a question central to understanding virus pathogenesis and transmission for any virus. Studies were limited by the difficulty in distinguishing between viral entry into first-hit target cells and subsequent cell-to-cell spread within tissues. Employing the murine cytomegalovirus entry complex gH/gL/gO as a paradigm for a generally applicable strategy to dissect these two events experimentally, we used a gO-transcomplemented ΔgO mutant for providing the complex exclusively for the initial cell entry step. In immunocompromised mice as a model for recipients of hematopoietic cell transplantation, our studies revealed an irreplaceable role for gH/gL/gO in initiating infection in host organs relevant to pathogenesis, whereas subsequent spread within tissues and infection of the salivary glands, the site relevant to virus host-to-host transmission, are double-secured by the entry complexes gH/gL/gO and gH/gL/MCK-2. As an important consequence, interventional strategies targeting only gO might be efficient in preventing organ manifestations after a primary viremia, whereas both gH/gL complexes need to be targeted for preventing intra-tissue spread of virus reactivated from latency within tissues as well as for preventing the salivary gland route of host-to-host transmission

HCMV infection of fibroblasts is not dependent on phosphorylation of PDGFR-α.

<p>(a) Serum-starved HFF or MRC-5 were co-incubated with wt TB40 (m.o.i of 10 in 0.05% DMEM), PDGF-AA (100 ng ml^-1), PDGF-BB (100 ng ml^-1) or 0.05% DMEM (mock) for 1 hour at 4°C and then shifted to 37°C for 20 min. p-PDGFR-α or PDGFR-α and p-Akt or Akt were detected by Western blot analysis of total cell lysates. (b) HFF were pre-incubated with different concentrations of imatinib mesylate for 1 hour at 37°C and then infected with wt TB40-luc (m.o.i. of 1) for 90 min. Free virus and cell surface-bound virus were inactivated by washing the cells three times with PBS, pH 3.0. After 24 hours in medium containing the respective amounts of imatinib mesylate, a luciferase assay was performed to determine infection of cells. One representative experiment done in triplicates is shown. To control for the activity of imatinib mesylate, HFF were in parallel pre-treated with the indicated concentrations of imatinib mesylate for 1 hour at 37°C and then stimulated with PDGF-BB (100 ng ml^-1) or mock-treated for 20 min. p₇₄₂- and p₇₆₂-PDGFR-α or PDGFR-α were detected by Western blot analysis of total cell lysates. (c,d) 293 cells were transfected with pCMV-PDGFR-α, pCMV-PDGFR-α(1–558), or a control vector. (c) Total cell extracts of pCMV-PDGFR-α- or pCMV-PDGFR-α(1–558)-transfected cells were analyzed for PDGFR-α expression by Western blot 24 hours after transfection using an antibody recognizing the N-terminus of human PDGFR-α (2D2-1A11). (d) 48 hours after transfection 293 cells were infected with wt TB40-luc. 24 hours after infection, cells were analyzed by a luciferase assay. Shown are means +/- SD of three independent experiments done in triplicates.</p

Silencing of PDGFR-α reduces cell-associated spread of gH/gL/gO-positive HCMV.

<p>(a) Confluent monolayers of HFF were infected with wt TB40 virus at a very low m.o.i. After infection, cells were either overlaid with methylcellulose or medium containing anti-gB antibodies (SM5-1, 2μg ml^-1), anti-gH antibodies (14-4B), PDGFR-α-Fc (2 μg ml^-1), or no inhibitor (mock-treated). (b) HFF infected with wt TB40 or TB40-UL131Astop virus were mixed with uninfected cells. After adherence, cells were either overlaid with methylcellulose or fresh medium was added containing 2 μg ml^-1 PDGFR-α-Fc, 2 μg ml^-1 PDGFR-β-Fc, or no inhibitor (mock-treated). (a,b) 5 days later, cells were stained for HCMV IE1 by indirect immunofluorescence and cells per focus counted. For each treatment, at least 10 (a) or 20 (b) foci were counted and depicted as means +/- SD. Shown are representative experiments. Asterisks under (a) represent P<0.001 values determined by comparing foci in mock-treated monolayers with foci in monolayers overlaid with methylcellulose, co-incubated with antibodies, or co-incubated with PDGFR-α-Fc (Mann-Whitney Rank Sum test). (c) NT siRNA or PDGFR-α siRNA-transfected HFF 48 hours after transfection were mixed with HFF infected with wt TB40, TB40-UL131Astop, or TB40-ΔgO virus. After adherence, cells were overlaid with methylcellulose or methylcellulose containing anti-UL131A rabbit antiserum (1:40) or a control rabbit antiserum (1:40). 5 days later, cells were analyzed as described under (a). The P value shown (Mann-Whitney Rank Sum test) was determined by comparing cell spread of wt TB40 virus with spread of TB40-UL131Astop virus in PDGFR-α-silenced HFF.</p

Silencing of PDGFR-α reduces infection of fibroblasts with gH/gL/gO-positive HCMV.

<p>HFF were transfected with PDGFR-α siRNAs, non-targeting (NT) siRNAs or mock-transfected. (a) 72 and 108 hours after transfection, total cell lysates were analyzed by Western blot for the expression of PDGFR-α or GAPDH. (b) 72 hours after transfection, cell surfaces of siRNA-transfected cells were stained with an anti-PDGFR-α antibody (35248) and a secondary Fluor 488-labelled anti-mouse antibody and analyzed by FACS. (c) 48 hours after transfection, cells were infected with wt TB40, TB40-UL131Astop and TB40-ΔgO viruses and 24 hours post infection stained for HCMV IE1 by indirect immunofluorescence. (d) HFF were infected as described under (c) and the percentage of IE1-positive nuclei was determined. Infection of cells transfected with NT siRNAs (set to 100%) and infection of PDGFR-α-silenced HFF is shown. Shown are means +/- SD from two independent experiments done in triplicates.</p

Anti-gH antibodies co-precipitate PDGFR-α, gO and gB from lysates of HFF co-incubated with TB40-UL131Astop virions.

<p>HFF surface proteins were biotinylated and lysates of HFF (1), lysates of HFF mixed with lysates of TB40-UL131Astop virions (2) and lysates of HFF co-incubated with TB40-UL131stop virions (3) were subjected to anti-gH immunoprecipitation. The precipitates were analyzed by Western blot using antibodies directed against PDGFR-α, gO, gH and gB.</p

Silencing of PDGFR-α reduces infection of fibroblasts with gH/gL/gO-positive HCMV.

<p>HFF were transfected with PDGFR-α siRNAs, non-targeting (NT) siRNAs or mock-transfected. (a) 72 and 108 hours after transfection, total cell lysates were analyzed by Western blot for the expression of PDGFR-α or GAPDH. (b) 72 hours after transfection, cell surfaces of siRNA-transfected cells were stained with an anti-PDGFR-α antibody (35248) and a secondary Fluor 488-labelled anti-mouse antibody and analyzed by FACS. (c) 48 hours after transfection, cells were infected with wt TB40, TB40-UL131Astop and TB40-ΔgO viruses and 24 hours post infection stained for HCMV IE1 by indirect immunofluorescence. (d) HFF were infected as described under (c) and the percentage of IE1-positive nuclei was determined. Infection of cells transfected with NT siRNAs (set to 100%) and infection of PDGFR-α-silenced HFF is shown. Shown are means +/- SD from two independent experiments done in triplicates.</p