Cytomegalovirus Infection, Defense Strategies and Development of Latency

CMV is a double-stranded DNA virus belonging to the р-herpesvirus family and is an omnipresent pathogen worldwide. In developing countries, the percentage of the population infected with CMV early in life approximates 100 %, but in industrialized societies this is far less and depends on subject age and socioeconomic factors [8]. Thus, it may be considered that not being infected with CMV is an artefact of civilization and hygiene. However, the fraction of the infected population in developed countries is commonly higher in the aged than the young, either because of new infections and seroconversion throughout the lifespan [8] and/or because younger and older people experienced different exposures in childhood and later life. This quandary again illustrates a basic problem with studies of this type in humans: they are cross-sectional and therefore it is formally impossible to distinguish between increases over time within individuals, and differences in the circumstances of the populations studied, as in this case. Hence, we must be careful to state “differences” when the data are from cross-sectional studies and limit “changes” to those that we know for certain do occur over time.

The genome of wild-type CMV strains is of about 235 kb with ca. 200 genes; it is the largest known human virus. Its double-stranded DNA is enclosed in an icosahedral viral capsid, which itself is surrounded by tegument or matrix proteins (Fig. 4.1). CMV has a class E genome: the two unique regions, unique long (UL) and unique short (US), are flanked by pairwise inverted repeats (terminal or internal repeat long, TRL/IRL) and internal/terminal repeat short (IRS/TRS): 5' TRL—IRL-IRS—TRS. The “a sequence” is shared by both repeats. Additional regions include the “b and c sequence”. The genome exists in four genomic isomers by inversion of the UL/US regions. In the laboratory, one must discriminate between high (Ad169, Towne) and low passage (Toledo, Merlin) CMV strains: Ad169 and Towne are highly passaged laboratory mutants, which lack either 15 or 13 kb, respectively, at the 3'end of the UL region (UL/b'region). Nearly all strains primarily isolated and propagated in fibroblast culture show mutations in the UL128 locus (UL128L) and in genes of the RL11 family, which has an impact on cell

Basic components of cytomegalovirus

Fig.4.1 Basic components of cytomegalovirus

tropism. UL128L includes the UL128, UL130, and UL131A regions, which form a pentameric complex with viral glycoprotein genes gH and gL. This pentameric complex is not essential for viral entry into fibroblasts but it is essential for endothelial and epithelial entry. Additionally, 14 genes located at the 5' end of the genome (RL5A, RL6, RL11—UL1, UL4-UL11) are not essential for viral growth in fibroblasts [9].

The central part of the UL region contains clusters of genes which have homologs in other herpesviruses, such as DNA polymerase, glycoprotein B and glycoprotein H, while the rest of the genome contains genes that appear primarily only in P-herpes viruses or exclusively in human CMV [10, 11]. Thus, there is great potential complexity in the interactions of CMV and its products with infected cells and the immune system. These are still far from clarified.

CMV is usually naturally acquired as an asymptomatic primary infection in infancy or during the first months of life via breastfeeding, after which the virus establishes lifelong persistence. Transmission of the virus occurs through exposure to infectious body fluids, including saliva, urine, breast milk, semen and blood [12]. Both T cells and antibodies specific for CMV remain present throughout life, although the possibility that some individuals may harbor CMV and specific T cells, but no antibody, has not been completely excluded [10, 13].

The initial infection with CMV activates a whole series of complex reactions of the host in order to limit the replication of the virus (Fig. 4.2). The diverse defense systems, in which the innate and adaptive immune system are involved, are able to detect the foreign nature of the virus very early after contact. Specialized pathogen- associated receptors recognize viral glycoproteins and even the entire viral genome as such. In the early phase of the immune response interferons and other cytokines play an important role in keeping the viral infection in check [11]. Additionally, NK cells as well as T cells and B cells with their specific cytokines and cytotoxic activity and antibody production, all contribute to CMV surveillance [10, 11]. Neutralizing antibodies produced by B cells are thought to play an important role in controlling CMV infection and re-infection. The impaired production of effector B cells secreting antibody specific for envelope glycoprotein and the induction of atypical memory B cells may reduce the production of neutralizing antibodies during primary CMV infection and support virus dissemination and establishment of CMV latency

Immune responses to CMV. DC

Fig. 4.2 Immune responses to CMV. DC: dendritic cells; Th cell: T helper cell; NK cell: natural killer cell; IFN: interferon; TLR: toll-like receptor; CMV: cytomegalovirus

[14]. As the proportion of the older population infected with CMV is greater than the young, the effects of CMV may be confused with those of chronological age.

Given the numerous host defense mechanisms (Fig. 4.2), successful viral infection is highly dependent on multiple evasive strategies (Fig. 4.3). These include some proteins that are delivered together with infectious virions (such as tegument protein pp65) and are activated during the early phase of infection (such as viral IE proteins) in order to block intrinsic cellular defense mechanisms [15]. CMV is also able to interfere with components of the cellular immune response. For example, products of at least seven CMV genes have been identified that inhibit NK-cell functions [16, 17]. Several other genes modulate the presentation of CMV peptides to T cells. They inhibit the loading of peptides onto MHC complexes or cause a dislocation of class I molecules from endoplasmic reticulum into the cytosol, where they are then degraded [18]. CMV also encodes a number of molecules similar to chemokines, cytokines, and their receptors, which are probably involved in immune evasion [19]. The virus also inhibits the proliferation of peripheral blood mononuclear cells and production of cytokines from these cells [10]. In addition, CMV inhibits the maturation of dendritic cells and is able also to initiate the process of apoptosis [10, 11, 20].

In this way, CMV infection initiates a complex interaction between the virus and host. Despite the sophisticated mechanisms with which CMV is equipped to subvert the innate and adaptive immune responses of the host, the latter are generally able to clear cells producing infectious virus particles in immunocompetent individuals [21, 22 i . Also intrinsic defenses, which are cell-autonomous components of the immune system protecting individual cells from viral attack, play an important role

Examples of evasive strategies of CMV. CMV

Fig. 4.3 Examples of evasive strategies of CMV. CMV: cytomegalovirus; MHC: major histocompatibility complex; PBMC: peripheral blood mononuclear cells; NK cell: natural killer cell; DC: dendritic cells; ER: endoplasmic reticulum [23]. They are mediated by constitutively expressed proteins called restriction factors , and can rapidly act at a very early stage of CMV infection by inhibiting an essential viral process. This may be one reason why herpes viruses have evolved to establish a latent state of infection, during which infectious virions are not generated, and the virus is able to persist for the lifetime of the host, but does not cause active disease (Fig. 4.4). Although various cell types may support productive infection with CMV and carry the virus, latent CMV infection has been most convincingly documented in cells of myeloid origin [21] . In the bone marrow, CD34+ myeloid progenitor cells are a site of latency, while in peripheral blood CD 14+ monocytes may carry latent CMV. Intense research on the clarification of the molecular basis for cell type-specific CMV latency remains at an early stage. It is still not definitively established how CMV establishes latency after acute infection in the face of an ongoing robust immune response involving both innate and adaptive immunity. The results of some studies indicate that the decision between permissive and latent infection may be determined by the balance between activating and repressive factors, which control transcription of viral genes upon initial infection, and this may differ among different cell types [22, 24].

It is also becoming increasingly clear that the latent state might be mediated by epigenetic factors through the process of heterochromatinization of viral genomes to silence viral gene expression. As a result, the virus can indefinitely persist in these cells because it does not express pathogen-associated molecular patterns, rendering it invisible to the host immune system [25]. Thus, CMV is able to manipulate cellular controls including regulation of the cell cycle and gene silencing extremely

Impact of CMV infection on immunosenescence well

Fig. 4.4 Impact of CMV infection on immunosenescence well. The ability to overcome cellular defenses as well as to control of proliferation and gene expression are decisive factors, which allow future viral replications and/ or viral persistence [5, 21, 22, 25, 26].

In a cell culture model of latency, it was shown that CMV expresses only a restricted number of genes. Among them is an alternatively spliced, latency- associated form of IL-10-like molecule (LAcmvIL-10) that is known to inhibit MHC class II recognition of infected cells, thus avoiding elimination by CD4+ T cells of the adaptive immune system. This immunomodulatory cytokine, secreted from latent virus-infected cells potentially over decades could contribute generally to chronic immune system dysfunction [18]. During experimental latency in CD34+ cells, cellular miRNAs (for example hsa-miR-92a) can be downregulated, leading to upregulation of the cellular myeloid transcription factor GATA2, which may be important for proliferation and survival of hematopoietic progenitor cells. In this model, viral LAcmvIL-10 induces hsa-miR-92a, which upregulates GATA2. This may lead to transcription of latent CMV genes (LUNA, UL144) and also of the cellular cytokine gene IL-10. Finally, IL-10 may block apoptosis of latently infected cells (via BCl-2, STAT3 phosphorylation and HSP-70) [24]. The summed side-effects of lifelong coexistence with CMV are likely to have profound implications for the functioning of the host’s immune system and the phenomena of immunosenescence.

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