Herpesviridae
The Herpesviridae family of viruses are enveloped, dsDNA viruses which generally establish a lytic infection that kills the cell upon release, as well as a latent infection for life-long persistence in the host. Historically, they were divided into alpha, beta and gamma herpesviruses based on biological characteristics, and more recently the classification was confirmed based on sequencing data. Broadly, Alphaherpesvirinae have a variable host range, relatively short replication cycle, and establish latent infections predominantly in neuronal cells. These includes HSV, and varicella-zoster virus (VZV), the causative agent of chickenpox. Betaherpesvirinae, such as HCMV, tend to have a more restricted host range and longer replication cycles, and establish latency in secretory glands, leukocytes, kidneys and other tissues. Gammaherpesvirinae include Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV). These viruses are restricted to the family of their natural host, and usually to T or B lymphocytes, establishing latency in lymphoid tissues (Pellett & Roizman, 2013).
Impact of Human Cytomegalovirus
HCMV is a member of the betaherpesvirinae family. It is an ubiquitous pathogen, with a global seroprevalence of 83 %, with the highest seroprevalence in the WHO Eastern Mediterranean region (~90 %) and lowest in the WHO European region (~66 %) (Zuhair
et al, 2019). For most individuals, transmission occurs during childhood, with the virus
being acquired from contact with infected bodily secretions, and the infection is asymptomatic. However, reactivation during pregnancy may lead to transplacental transmission. Congenital HCMV infection can result in life-long sensorineural hearing loss, with ~10-15 % of babies infected showing symptoms, and others developing symptoms later on. Of the symptomatic infants, fatality is estimated to be between 4-30 % (Dollard et al, 2007). Initial infection with the virus during pregnancy increases the chance of transplacental transmission from ~1 % to around ~33 %, and is linked with severe disease (Kenneson & Cannon, 2007). Infection or reactivation is also damaging in immunocompromised individuals, particularly those undergoing organ transplants (Azevedo et al, 2015) or with acquired immunodeficiency disease syndrome (AIDS) (Emery, 2001).
Three drugs are currently available for treatment of symptomatic individuals, and pre- emptive therapy is sometimes employed where infection is the result of a medical intervention and can be predicted, such as organ transplants. These are all inhibitors of the viral polymerase ((val)Ganciclovir, Cidofovir and Foscarnet). There are a couple of other antivirals more commonly used for prophylaxis (Letermovir, Valacyclovir). However, these treatments have numerous side effects, such as leukopenia, renal dysfunction and ophthalmologic toxicity, as well as development of resistant viral infection. Additionally teratogenic effects have been observed in animals models so they are not recommended for use during pregnancy (Kotton, 2019; Malm & Engman, 2007). As HCMV is a leading cause of congenital disease, and due to the potential severity of infection, a vaccine, or improved antivirals, would therefore be highly desirable.
The Virus and its Life Cycle
HCMV has a the largest genome of the herpesviruses, at 236 kbp (Dolan et al, 2004), and coding for ~170 canonical proteins. Expression of these genes is temporally regulated,
and can be broadly divided into immediate early (IE), delayed early (DE) and late (L) viral genes (Wathen & Stinski, 1982), or into five classes (temporal profile (Tp) 1-5) by proteomic data (Weekes et al, 2014), and the replication cycle takes 48-72 h. The structure is characteristic of herpesviruses, with the dsDNA genome being encased in an icosahedral capsid, within an envelope acquired from the host cell membrane. HCMV tropism includes a wide range of cells such as fibroblasts, epithelial cells, endothelial cells, smooth muscle cells and macrophages (Sinzger et al, 1995).
HCMV can enter cells either by fusion of the viral envelope with the cell membrane (as during infection of fibroblasts) or following endocytosis (as in endothelial and epithelial cells). Microtubules then deliver the nucleocapsid to nuclear pore complexes where the viral genome enters the nucleus. The virus may enter a lytic life cycle. In this instance, IE genes regulate the transcription of host and viral genes. Around 14 - 24 hpi in fibroblasts, DE genes control viral DNA replication, followed by expression of L viral genes. The viral capsid assembles in the nucleus and then translocates to the cytoplasm. Throughout this process, it acquires a temporary envelope at the inner nuclear membrane, and then undergoes de-envelopment at the outer nuclear membrane. The nucleocapsid acquires tegument proteins at the cytoplasmic viral assembly compartment (AC) and undergoes secondary envelopment at the endoplasmic reticulum – golgi intermediate compartment. Finally, the virion is released from the cell by exocytosis (Fields et al, 2013; Beltran & Cristea, 2015) (Figure 1.4).
Alternatively, HCMV can establish a life-long latent infection. As with other herpesviruses, in latent infection, no new virions are produced, and it was thought that a smaller subset of viral genes are expressed (Poole & Sinclair, 2015). However, recent research found that gene expression during latency is similar to that of late lytic infection, but at lower levels, suggesting the differences between lytic and latent infection may be quantitative rather than qualitative (Shnayder et al, 2018). Latency occurs in myeloid cells, however on differentiation to macrophages or DCs this can result in reactivation of the virus. It is important to understand latency as it may provide new therapeutic avenues for clearing latent infection before reactivation occurs during transplant (Poole et al, 2014).
Figure 1.4 HCMV life cycle
CMV virions initiate infection by attaching to receptors at the cell surface and then fusing with the PM, or by endocytosis. The tegument proteins and capsid are released to the cytosol. Infectious HCMV particles enter the cell through receptor interactions, and the capsid and tegument proteins are delivered to the cytosol. The capsid travels to the nucleus along microtubules, where the viral genome is released and the gene expression cascade occurs (expression of IE, DE and then L viral genes). L gene expression triggers capsid assembly in the nucleus. Nuclear egress occurs, and the virus acquires tegument proteins in the cytosol. The virus is trafficked to AC where it acquires more tegument proteins and a viral envelope. Infectious particles are released by exocytosis. Figure reprinted from (Beltran & Cristea, 2015).
HCMV and the Immune Response
The ability of HCMV infection to remain asymptomatic in healthy individuals, whilst it can cause devastating disease in the immunocompromised, highlights the effectiveness of the immune response in controlling the infection. HCMV has a multitude of mechanisms for subverting the immune response.
Diagram of HCMV life cycle removed for copyright reasons. Copyright holder is Taylor & Francis.
HCMV infection is initially controlled in a number of ways by the intrinsic and innate immune response, including detection of the envelope glycoproteins B and H by TLR2, subsequent signalling through NF-B and production of IFN (Boehme et al, 2006). Additionally, NK cells play a crucial role in control of HCMV infection (Biron et al, 1989; Gazit et al, 2004; Vivier et al, 2008). Several restriction factors are involved in the immune response (see section 1.2.2), including the subnuclear structure ND10. This is composed of promyelocytic leukaemia protein (PML), death domain-associated protein 6 (Daxx) and nuclear autoantigen Sp-100 (Sp100). Viral genomes are found in close proximity to the ND10 bodies, and knockdown of any of these proteins leads to enhanced replication of HCMV (Tavalai et al, 2008; Adler et al, 2011). Daxx limits transcription of IE genes by generating a repressive chromatin structure at the viral major immediate early promoter (Woodhall et al, 2006). Sp100 is an IFN-stimulated protein, and also limits viral transcription. HCMV evades this restriction by delivering the viral protein pp71 in the virion and this targets Daxx for proteasomal degradation, stimulating expression of IE genes (Saffert & Kalejta, 2006); HCMV IE1 downregulates Sp100 (Tavalai et al, 2011).
IE proteins also disrupt the IFN mediated immune response, with IE1 preventing STAT1, STAT2 and IRF9 associating to form ISGF3 (Paulus et al, 2006), and IE2 blocking the production of IFNβ (Taylor & Bresnahan, 2005). Other examples of HCMV proteins involved in immune evasion include US2, one of several HCMV proteins able to modulate MHC class I expression. It also downregulates integrins and thrombomodulin, and can act synergistically with UL141 to downregulate the NK cell ligand CD112 (Hsu
et al, 2015). Alongside downregulating MHC class I, HCMV encodes UL18, an MHC
class I homologue which binds the inhibitory leukocyte immunoglobulin-like receptor 1 (Lir-1) (Yang & Bjorkman, 2008).
Adaptive immunity is also crucial in controlling infection, as evidenced by HCMV reactivation in immunosuppressed transplant recipients (Azevedo et al, 2015). Presentation of HCMV antigens on APCs leads to a large HCMV-specific T cell response, similar in size to that seen with HIV and larger than for most other viruses, composed of virus-specific CD4+ and CD8+ T cells. The scale of the T cells response varies between individuals, but examination of memory T cells in seropositive individuals found ~10% of CD4+ and CD8+ cells were reactive to HCMV (Rosa & Diamond, 2012; Sylwester et
al, 2005). Adoptive transfer of HCMV specific CD8+ T cells reduced viremia in patients,
emphasising the importance of the T cell response in controlling infection (Cobbold et
al, 2005). B cells are also important in the adaptive immune response, with antibodies
generated against tegument, envelope and non-structural proteins (Landini et al, 1988; Urban et al, 1996).
Proteomic studies of HCMV used in this thesis
A previous study used quantitative temporal viromics to analyse the proteomic changes observed in the host and virus during HCMV infection (Weekes et al, 2014). The data from Weekes et al. has been used here for comparison with VACV infection, and for the identification of ARFs. Fibroblasts were infected with the Merlin strain of HCMV, and either whole cell lysate (WCL) or PM enriched samples harvested at various time points throughout a single viral replication cycle. Each sample was digested and labelled with a different tandem mass tag (TMT), prior to analysis by triple-stage mass spectrometry (MS3). This gave the relative quantitation of each protein across the samples (proteomic techniques are discussed further in section 1.5. For each of the WCL and PM experiments, samples were harvested either at the time points described in Figure 1.5 (WCL2 and PM2) or in duplicate at mock, 24, 48 and 72 hpi (WCL1 and PM1). More than 8000 human proteins were quantified over the time-course, including ~1200 proteins with PM related gene ontology (GO) annotations, as well as 139 of the 171 canonical HCMV proteins.
Figure 1.5 Schematic of HCMV time-course proteomics experiment
Fibroblasts were infected with the Merlin strain of HCMV, and samples harvested at various time points throughout infection for either WCL or PM analysis (Weekes et al, 2014). The proteins were then digested, labelled with TMT and subjected to mass spectrometry analysis. The time points displayed are those relating to the WCL2 and PM2 experiments.
Proteins downregulated by HCMV infection were identified in the data and used to predict those that might be important in infection. Examination of the downregulated proteins identified multiple protocadherins, some of which are putative NK cell ligands, as well as modulation of proteins in the IFN induction and signalling pathways, and ISGs. Additionally, analysis of the kinetics of expression of viral proteins defined five temporal classes of protein expression (Tp1-5).
This data demonstrates the power of multiplexed proteomics in large scale studies of infection. It also provides a comprehensive resource which is used in this thesis to aid identification of candidate ARFs. Several additional proteomic studies on HCMV have been conducted during my PhD in the Weekes lab. As they are not central to this project, they are not discussed further, however they focus on protein degradation during HCMV infection, leading to the identification of HLTF as a novel ARF (Nightingale et al, 2018), and the generation of a global interactome of HCMV proteins (Nobre et al, 2019).