Ilija Brizić

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

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.

The viral chemokine MCK-2 of murine cytomegalovirus promotes infection as part of a gH/gL/MCK-2 complex

Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination.

Inflammatory monocytes and NK cells play a crucial role in DNAM-1–dependent control of cytomegalovirus infection

Jonjic et al. show that inflammatory macrophages play an essential role in the control of murine CMV (MCMV) infection through a DNAM-1–PVR pathway.

MCMV avoidance of recognition and control by NK cells.

Natural killer (NK) cells play an important role in virus control during infection. Many viruses have developed mechanisms for subversion of NK cell responses. Murine cytomegalovirus (MCMV) is exceptionally successful in avoiding NK cell control. Here, we summarize the major MCMV evasion mechanisms targeting NK cell functions and their role in viral pathogenesis. The mechanisms by which NK cells regulate CD8+ T cell response, particularly with respect to the role of NK cell receptors recognizing viral antigens, are discussed. In addition, we discuss the role of NK cell receptors in generation and maintenance of memory NK cells. Final part of this review illustrates how the NK cell response and its viral regulation can be exploited in designing recombinant viral vectors able to induce robust and protective CD8+ T cell response.

Human cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α 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-α (PDGFR-α) on the surface of fibroblasts and that gH/gL/gO either directly or indirectly recruits gB to this complex. PDGFR-α functions as an entry receptor for HCMV expressing gH/gL/gO, but not for HCMV mutants lacking the gH/gL/gO complex. PDGFR-α-dependent entry is not dependent on activation of PDGFR-α. We could also show that the gH/gL/gO—PDGFR-α 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-α or by the gH/gL/UL128,130,131A complex. PDGFR-α-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.

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 viruses, we wondered whether deletion of pUL74 would also affect virus growth in this background. To address this question, we took advantage of the bacterial artificial chromosome (BAC)-cloned virus Merlin-RL13tetO, which grows cell associated due to the inducible expression of the viral RL13 gene, thereby resembling clinical isolates. Stop codons were introduced by seamless mutagenesis into UL74 and/or the UL128 locus to prevent expression of the trimeric or pentameric complex, respectively. Virus mutants were reconstituted by transfection of the respective genomes into cultured cells and analysed with respect to 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 with 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.

Systemic Virus Infections Differentially Modulate Cell Cycle State and Functionality of Long-Term Hematopoietic Stem Cells In Vivo

Quiescent long-term hematopoietic stem cells (LT-HSCs) are efficiently activated by type I interferon (IFN-I). However, this effect remains poorly investigated in the context of IFN-I-inducing virus infections. Here we report that both vesicular stomatitis virus (VSV) and murine cytomegalovirus (MCMV) infection induce LT-HSC activation that substantially differs from the effects triggered upon injection of synthetic IFN-I-inducing agents. In both infections, inflammatory responses had to exceed local thresholds within the bone marrow to confer LT-HSC cell cycle entry, and IFN-I receptor triggering was not critical for this activation. After resolution of acute MCMV infection, LT-HSCs returned to phenotypic quiescence. However, non-acute MCMV infection induced a sustained inflammatory milieu within the bone marrow that was associated with long-lasting impairment of LT-HSC function. In conclusion, our results show that systemic virus infections fundamentally affect LT-HSCs and that also non-acute inflammatory stimuli in bone marrow donors can affect the reconstitution potential of bone marrow transplants.

Virus-Induced Interferon-γ Causes Insulin Resistance in Skeletal Muscle and Derails Glycemic Control in Obesity

Summary Pro‐inflammatory cytokines of a T helper‐1‐signature are known to promote insulin resistance (IR) in obesity, but the physiological role of this mechanism is unclear. It is also unknown whether and how viral infection induces loss of glycemic control in subjects at risk for developing diabetes mellitus type 2 (DM2). We have found in mice and humans that viral infection caused short‐term systemic IR. Virally‐induced interferon‐&ggr; (IFN‐&ggr;) directly targeted skeletal muscle to downregulate the insulin receptor but did not cause loss of glycemic control because of a compensatory increase of insulin production. Hyperinsulinemia enhanced antiviral immunity through direct stimulation of CD8+ effector T cell function. In pre‐diabetic mice with hepatic IR caused by diet‐induced obesity, infection resulted in loss of glycemic control. Thus, upon pathogen encounter, the immune system transiently reduces insulin sensitivity of skeletal muscle to induce hyperinsulinemia and promote antiviral immunity, which derails to glucose intolerance in pre‐diabetic obese subjects. Graphical Abstract Figure. No caption available. HighlightsVirus‐induced IFN‐&ggr; downregulates insulin receptor expression of skeletal muscleMuscle insulin resistance results in compensatory hyperinsulinemia to keep euglycemiaInsulin directly boosts anti‐viral effector CD8+ T cell responsesIn obese mice with hepatic IR, viral infection causes rapid progression to diabetes &NA; It is unknown how viral infections contribute to the progression of type 2 diabetes. Šestan and colleagues demonstrate that virus‐induced interferon‐&ggr; increases muscle insulin resistance, which drives hyperinsulinemia to keep euglycemia and to boost anti‐viral CD8+ T cell responses. This mechanism in obese subjects with hepatic IR derails glycemic control.

Brain-resident memory CD8+ T cells induced by congenital CMV infection prevent brain pathology and virus reactivation

Congenital HCMV infection is a leading infectious cause of long‐term neurodevelopmental sequelae. Infection of newborn mice with mouse cytomegalovirus (MCMV) intraperitoneally is a well‐established model of congenital human cytomegalovirus infection, which best recapitulates the hematogenous route of virus spread to brain and subsequent pathology. Here, we used this model to investigate the role, dynamics, and phenotype of CD8+ T cells in the brain following infection of newborn mice. We show that CD8+ T cells infiltrate the brain and form a pool of tissue‐resident memory T cells (TRM cells) that persist for lifetime. Adoptively transferred virus‐specific CD8+ T cells provide protection against primary MCMV infection in newborn mice, reduce brain pathology, and remain in the brain as TRM cells. Brain CD8+ TRM cells were long‐lived, slowly proliferating cells able to respond to local challenge infection. Importantly, brain CD8+ TRM cells controlled latent MCMV and their depletion resulted in virus reactivation and enhanced inflammation in brain.

Immune responses to congenital cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the most common cause of viral infection acquired in utero. Even though the infection has been studied for several decades, immune determinants important for virus control and mechanisms of long-term sequelae caused by infection are still insufficiently characterized. Animal models of congenital HCMV infection provide unique opportunity to study various aspects of human disease. In this review, we summarize current knowledge on the role of immune system in congenital CMV infection, with emphasis on lessons learned from mouse model of congenital CMV infection.