Department of Virology, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK
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Abstract |
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Introduction |
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Cytomegalovirus (CMV) strain AD169 was initially isolated in fibroblast cell cultures in 1956. It was sequenced in its entirety in 1990 and consists of four equimolar isomers produced by inversion of either the unique long (UL) or unique short (US) regions. These UL and US regions are bounded by terminal repeat (TR) and inverted repeat (IR) regions.1 Predicted open reading frames are numbered sequentially within each region and annotated using the abbreviations p for protein, gp for glycoprotein, pp for phosphoprotein, followed by any common non-systematic name; e.g. gpUL55 (gB) is the 55th open reading frame in the UL region and encodes a glycoprotein known as glycoprotein B. The original report describes 208 open reading frames in strain AD169, which, after allowance for known splicing events, were predicted to produce 203 proteins, 189 of which were unique while the remaining were present in two copies in the repeat regions.2
This impressive sequencing effort and gene analysis has stood the test of time, with only minor revisions required subsequently. However, in 1996 it was reported that clinical strains of CMV contained a series of genes not found in the AD169 or Towne strains.3 The details are complex (reviewed in Prichard et al.4), but essentially mean that 22 additional genes are present in wild-type strains (19 additional to Towne). None of these genes has a homologue in other herpesviruses and most are predicted to be type 1 glycoproteins. At least one has been shown to have interesting biological activity; gpUL146 is an alpha chemokine, the first such molecule described in a viral genome.5 Thus, overall, CMV clinical strains have 225 genes.
An annotated overview of the CMV genome, incorporating these changes, is shown in Figure 1. Genes marked in pale green (capsid), red (tegument) or pale blue (glycoproteins) form the structural proteins of the virion. They are all contained in approximately half of the genome spanning 2 o'clock to 8 o'clock when Figure 1
is visualized as a clock face. What then is the function of the remaining genes in clinical strains? Presumably, they facilitate survival of the virus in its natural host, and the emerging functions of one set (coloured dark green) are particularly intriguing for they interact with the human immune system.
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Figure 2 shows a simplified scheme of how the cellmediated immune system detects cells infected with a virus such as CMV. Proteins encoded by the CMV genome are digested in the proteasome and the resulting peptides translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen presentation (TAP). There, the peptides associate with the heavy chains of HLA class I molecules and are transported through the trans-Golgi network to the plasma membrane. This combination of host HLA class I containing its cognate viral peptide is normally recognized by a specific CD8+ cytotoxic T-lymphocyte (CTL), leading to destruction of the cell via fas- or perforin-dependent mechanisms. CMV has evolved to interfere with this pathway at a series of steps, which can be characterized into those which act at immediate-early times (before any viral protein translation takes place), at early times (before viral DNA replication takes place) and at late times (during formation of the physical virion).68 At immediate-early times, CMV must take transcriptional control of the cell, and the major immediate-early region (UL123/122) is key.
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In addition, gene UL16 interferes with a positive signal provided by two distant members of the major histocompatability complex (MHC) family of proteins present on lymphocytes.9
In combination, these effects enable the CMV-infected cell to escape the surveillance of several types of cellmediated response. In addition, gene US2 also blocks HLA class II expression,10 CMV expresses an Fc receptor to evade humoral immunity and usurps human complement control proteins to degrade complement bound to the virion. Thus, in these ways, and others yet to be defined, CMV enters into a chronic replicative state within its human host, leading to the persistence of virus and an increased opportunity for horizontal and vertical transmission of infectivity.
Replication
In fibroblast cell cultures CMV replicates to produce cytopathic effect (CPE) after 24 weeks incubation. This slow evolution of CPE is typical of the Betaherpesvirinae and represents a major impediment to studying CMV replication in the laboratory.
In retrospect, we recognize that this slow appearance of CPE is misleading about the true nature of CMV replication. Serial clinical investigations using quantitative competitive polymerase chain reaction (QC-PCR) show that, in vivo, in its natural human host, CMV replicates rapidly.11 This contrast between findings in vivo and in vitro is only one of the disconnections seen between cultures and the real world' (see Table 1). Taken together, it is the behaviour of strain AD169 in fibroblast cell cultures that is aberrant. Thus, CMV should be seen as a rapidly replicating virus, with all the implications that has for control of CMV infection and disease via antiviral chemotherapy.
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Diseases caused by CMV |
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CMV has long been recognized to cause a series of end-organ diseases (see Table 2) collectively called direct effects or CMV disease. These are defined according to criteria agreed at the International CMV Workshop in 1996 and updated regularly thereafter.12 Essentially, these require the patient to have characteristic symptoms, to have clinical signs in the affected organ and to have CMV detected in biopsies from the same organ. The definition is stringent and useful as an end-point for early clinical trials of anti-CMV compounds.
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Allograft recipients: indirect effects
In addition, CMV is statistically associated with several clinical conditions collectively termed indirect effects.16 These include graft rejection, immunosuppression manifest as secondary fungal or bacterial infections, opportunistic neoplasms, accelerated atherosclerosis after heart transplant and death. Clearly, each of these conditions is multifactorial, but the results of double-blind, randomized, placebo-controlled trials (discussed later) show that CMV makes an active contribution to their genesis.
AIDS patients: direct effects
The same principles of CMV viraemic dissemination and high viral load indicating high risk of CMV disease apply equally to AIDS patients.17,18 Nevertheless, it is remarkable that 85% of viraemic spread localizes to the retina in contrast to c. 1% in transplant patients. Other CMV diseases in AIDS patients include enteritis, polyradiculopathy and encephalopathy.
AIDS patients: indirect effects
A high CMV viral load is associated statistically with an increased death rate,19 and this effect is independent of HIV viral load.20 Multiple mechanisms have been shown whereby CMV (or other herpesviruses) could facilitate the pathogenicity of HIV.21 It is thus interesting that the increased mortality associated with CMV can be reversed through administration of drugs acting against CMV.22 The full significance of these observations remains to be defined in the era of highly active antiretroviral therapy (HAART).
Intra-uterine infection
Neonates born with cytomegalic inclusion disease may have many or all of the conditions listed in Table 3. More frequently they are born without these symptoms or signs but develop progressive hearing loss and/or mental retardation on follow up.23 Again, the neonates most at risk are those with a high viral load.24
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Characteristics of a drug ideal for CMV |
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The drug discovery process |
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As an example, the compound with the best safety profile in vivo (aciclovir) was not identified by such screening as having useful activity against CMV. Fortunately, aciclovir was developed because of its activity against herpes simplex virus (HSV) and varicella-zoster virus (VZV) and so was available for clinical studies of CMV. Even when its effect on CMV was noted this was dismissed for more than a decade, despite encouraging results from controlled clinical trials2527 and support from basic biochemistry.28 Thus, if precedence was given to an in vitro assay of unproven validity for this compound, is it possible that other useful molecules were dismissed during other drug discovery programmes?
In the modern era, pharmaceutical companies are hopefully screening against defined molecular targets known to provide essential functions for viral replication (see Table 5).
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Strategies for deploying anti-CMV compounds: results of controlled clinical trials |
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In clinical practice, decisions about the use of anti-CMV drugs are based on the balance of efficacy, toxicity and risk of disease for an individual patient. Four distinct strategies have been evaluated in controlled clinical trials (Table 6).
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The effects of compounds on CMV disease, listed in Table 7c, depend on patient sample size as well as the potency of the compound. Thus, in bone marrow transplant patients, ganciclovir reduced CMV disease when used for prophylaxis in one study,29 with a strong trend in a second.30 It also reduced CMV disease when given as pre-emptive therapy,31 but not when given for treatment of established disease.32 Since there is no evidence-based medicine from double-blind, randomized, placebocontrolled trials to show that ganciclovir (or any other drug) can speed the resolution of CMV disease, this drug should be deployed to prevent disease becoming established. Whether this is best achieved by antiviral prophylaxis or by pre-emptive therapy is hotly debated.33,34
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AIDS patients
In reviewing these treatment trials, the reader may be surprised by the omission of a column in Table 7ad for AIDS patients. These patients cannot be included because no trials have been designed based on virological criteria. Instead, AIDS physicians randomized patients with early CMV retinitis to receive immediate or delayed treatment with ganciclovir, foscarnet or cidofovir (reviewed in Jacobson40). They thereby provided evidence of clinical efficacy for all three drugs and rediscovered the maxim that infectious diseases should be treated soon after diagnosis, rather than waiting for extensive end-organ damage to occur. Nevertheless, controlled trials of clinical prophylaxis, i.e. administration of a drug when disease is not apparent clinically, can be interpreted to provide evidence in favour of virological prophylaxis and pre-emptive therapy.41,42 Inevitably, some patients recruited into such studies have PCR evidence of CMV viraemia and so can be examined for a pre-emptive therapeutic effect.
Such patients who received valaciclovir as part of a randomized clinical trial showed that this drug has efficacy as pre-emptive therapy.43 The same was not true for oral ganciclovir, although the dose chosen was only 1 g tds.42 In contrast, patients without evidence of CMV infection at trial entry, i.e. true prophylaxis, benefited from either valaciclovir or oral ganciclovir.4143 These findings support the results from natural history studies showing that the viraemic component of CMV pathogenesis is similar in transplant and AIDS patients.17,18,44,45
Neonates
The results of an important controlled clinical trial conducted by the Collaborative Antiviral Study Group have recently been reported in abstract form.46 Neonates born with CNS symptoms due to CMV infection were randomized to receive iv ganciclovir for 6 weeks at a dose previously evaluated in neonates,47 or to receive no therapy. Those who received ganciclovir had significantly less hearing loss than the controls. This important observation extends to neonates the concept of pre-emptive therapy and demonstrates that some of the damage caused to these neonates occurs after birth.
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Desired potency of antiviral compounds |
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Resistance |
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A common feature of these two papers is the use of long-term oral ganciclovir. It is to be hoped that the recent licensure of valganciclovir will replace this sub-optimal use of ganciclovir.
It is interesting to note that the UL97 mutations which confer resistance to ganciclovir all give impaired fitness in vivo when compared with the wild type.11,48 A series of individual point mutations can confer resistance to ganciclovir, as can deletions or double mutants.51 Nevertheless, the fitness deficits for each of these ranges from 3% to 12%.
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The future |
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Acknowledgements |
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Notes |
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References |
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