Introduction

Human adenoviruses (Ads) possess two well-studied mechanisms for modulation of MHC class I expression. The mechanism that is predominant in infected cells involves down-regulation of surface class I antigens caused by a block in the transport of class I heavy chains to the cell surface. This block is effected by a protein product of a viral early gene (termed E3) and seems to operate in cells infected with most virus serotypes. In adenovirus-transformed cells, the level of surface class I antigens can be either elevated or decreased depending on the serotype of the transforming adenovirus and is controlled mainly at the step of transcription of class I genes. Modulation of the rate of initiation of class I gene transcription in these cells is mediated by the product(s) of a different viral early gene (E1A). Interestingly, it is the expression of the E1A gene of highly oncogenic adenoviruses (such as Adl2) that results in down-regulation of class I transcription, providing a possible mechanism whereby oncogenic Adl2-transformed cells can escape host immune surveillance and form tumours. Adenovirus-infected and adenovirus-transformed cells are, therefore, interesting experimental systems for the study of class I modulation.

Adenovirus transformation and oncogenicity have been extensively reviewed (Knippers & Levine, 1989; Boulanger & Blair, 1991; Chinnadurai, 1992; Moran, 1993) and comprehensive reviews on virus structure, biology and pathogenicity are also available (Ginsberg, 1984; Doerfler, 1986; Horwitz, 1990a,b). The wider aspects of the immunobiology of adenoviruses have also been described (Wold & Gooding, 1991; Braithwaite et al, 1993).

Introduction

Human cytomegalovirus (CMV) is a ubiquitous agent that rarely causes disease in healthy individuals but is an important pathogen in the immunocompromised or the immunologically immature foetus. Individuals at high risk are those with deficiencies in cell-mediated immunity and it is, therefore, believed that in the immunocompetent host the cell-mediated immune response is the most important form of defence against CMV (Grundy, 1991). In the immunocompromised, CMV infection can be associated with a wide range of symptoms. The most serious complication in allogeneic transplant recipients is CMV interstitial pneumonitis, which is associated with a high mortality (Meyers, Flournoy & Thomas, 1982), and the virus remains the most important infectious cause of death following bone marrow transplantation. In AIDS patients, CMV can cause sight-threatening retinitis (Collaborative DHPG Treatment Study Group, 1986) and serious disease throughout the gastrointestinal tract. Congenital CMV infection can be associated with disseminated disease, including in the CNS, and the virus is an important cause of deafness and mental retardation (Alford et al., 1990). Thus, CMV causes disease in many organ systems in various patient populations. However, even in the immunocompetent host, the virus is not eradicated from the body following primary infection and can persist in a latent form throughout life. Hence, whilst the normal host immune response is usually able to prevent disease associated with primary CMV infection, the virus ultimately evades the host response and becomes latent.

Introduction

This chapter deals with transcriptional regulation of MHC class I genes in normal physiology and development. The main focus is transcription factors involved in regulating class I gene expression and the mechanism of action of these factors.

Class I genes are widely expressed in many adult tissues. However, levels of class I expression vary among different tissues; some tissues do not express class I genes at a significant level (Klein, 1975). While lymphoid tissues express class I genes at high levels, there is virtually no expression in the CNS. The lack of class I gene expression in the CNS constitutes a unique immunological environment, which accounts for persistent infection by some viruses (Joly, Mucke & Oldstone, 1991). Other tissues, such as pancreas and muscle, appear to be low in class I gene expression (David-Watine, Israel & Kourilsky, 1990a). Class I gene expression is developmentally controlled: class I mRNAs are not found until mouse embryos reach mid-somite stage (Ozato, Wan & Orrison, 1985). Levels of MHC class I expression in embryonic tissues vary but are higher in haemato/lymphopoietic tissues than in other tissues (Hedley et al, 1989). Class I mRNA levels rise sharply during the first week of neonatal development in the mouse (Kasik, Wan & Ozato, 1987). So far, the molecule responsible for the primary onset of class I expression during embryogenesis, or that which triggers the second, post-natal upsurge of class I expression, has not been identified.

Introduction

The MHC class II (also known as immune response-associated antigens, or Ia) genes are a multigene family encoded on chromosome 6 in humans (Fig. 5.1) and chromosome 17 in mice (see Chapter 2). The functional role of the MHC class II antigens in the immune response is described extensively in Chapters 1 and 3. This includes their function in antigen presentation, thymic education and T cell recognition/activation, their contribution to transplant acceptance or rejection, their genetic association with diseases, their aberrant up-regulation in a number of autoimmune and inflammatory diseases as well as their absence in specific immunodeficiencies. The involvement of MHC class II gene products in a large number of clinical conditions confers practical importance to the study of their regulation. Furthermore, the tissue-, cellspecific, differentiation-dependent and cytokine-regulated expression of MHC class II genes represent features that have made these genes a model system to study gene regulation. In this chapter, the expression and regulation of MHC class II genes in normal and disease states will be discussed, as well as the molecular basis for the constitutive and inducible modes of class II gene regulation.

Role of the MHC class II antigens in the immune response

The specificity of the immune system is conferred by the binding of foreign antigens through receptors on the surfaces of B and T lymphocytes, the immunoglobulin (Ig) molecule and the T cell receptor (TCR), respectively.

Introduction

Cervical cancer and pre-cancer form a disease continuum ranging from cervical intraepithelial neoplasia (CIN) through microinvasion to invasive carcinoma; about 70% of the tumours are squamous and 30% are adeno- and adenosquamous carcinomas (Buckley & Fox, 1989). Most tumours are thought to develop from an area of intraepithelial neoplasia within the transformation zone (Coppelston & Reid, 1967). Cervical cancer is estimated to be the second most common female cancer with approximately 500 000 new cases per annum worldwide (Parkin, Laara & Muir, 1980). Sexually transmitted infections are recognized as one of the major risk factors and the active agents are thought to be specific types of human papillomavirus (HPV) (Munoz et al., 1992).

Papillomaviruses are small DNA viruses associated with benign and malignant proliferative lesions of cutaneous epithelium. Over 60 different types of papillomavirus have been described and they can be segregated into groups distinguished by DNA sequence homology and the specific lesions with which they are associated (de Villiers, 1989). HPV 6 and 11 are found most commonly in cervical condyloma, benign lesions that tend to regress spontaneously, and low-grade CIN. HPV 16 and 18 are the types most commonly associated with high-grade CIN lesions and invasive carcinoma of the cervix. The viruses will replicate only in specific differentiation stages of epithelia, which limits the use of in vitro culture methods for producing HPV. To circumvent this, molecular biological techniques have been utilized extensively to characterize HPV.

Introduction

The expression of MHC products by pancreatic islet cells has been extensively studied because these cells are the targets of the destructive autoimmune response that leads to insulin-dependent diabetes mellitus (IDDM) and also because they constitute a set of distinct cell populations ideal for the study of peripheral tolerance by gene targeting in transgenic (tg) mice. Many of the studies described in this chapter were first prompted by concepts previously proposed in the aberrant class II expression hypothesis of endocrine autoimmunity (Bottazzo et al., 1983).

The aberrant class II expression hypothesis of endocrine autoimmunity

Aberrant (actually ectopic) HLA class II expression, i.e. the expression of HLA class II by cells of lineages that are normally class II negative, was first detected in the thyroid follicular cells of glands resected from patients suffering from autoimmune thyrotoxicosis, or Graves' disease (Hanafusa et al., 1983). This finding, together with the demonstration that thyroid follicular cells can be induced to express HLA class II antigens in vitro (Pujol-Borrell et al., 1983), led to the formulation of the aberrant class II expression hypothesis. This is based on the assumption that the lack of active immune response to endocrine cells and other scarce and differentiated cells present in peripheral tissues is the result of their low expression of HLA proteins, which makes them ‘invisible’ to T cells (immunological silence).

Introduction

This paper aims to give an overview of the dynamics and genetics of interactions between populations of viruses and their hosts. I emphasize that ‘successful’ parasites are those individuals who leave the most offspring, and that this does not necessarily mean evolving toward being harmless to the host (even though this may be the best for the viral population as a whole); many evolutionary trajectories are possible, depending on the constraints and trade-offs among viral virulence, transmissibility, and the costs of host resistance. These general ideas are illustrated by the particular cases of myxoma virus and Australian rabbits, and of HIV-1 and human hosts (where the role of ‘escape mutants’ of the virus, and of a possible ‘diversity threshold’ beyond which the immune system can no longer control the virus, are emphasized).

Viruses as ‘microparasites’

Viruses and other infectious agents that afflict humans and other animals are conventionally classified along taxonomic lines. In discussing the ecology or evolution of host/parasite associations, however, it is often more useful to make distinctions on the basis of the population biology of the interaction.

Microparasites (sensu Anderson & May, 1979) are those which have direct reproduction, usually at very high rates, within the host. They characteristically have small sizes and short generation times; the duration of infection is typically very short relative to the expected lifespan of the host, and hosts that recover from infection usually acquire immunity against re-infection for some time, often for life. The result is that most microparasitic infections are of a transient nature in individual hosts.

Introduction

When the key biological molecules, proteins and nucleic acids, were first studied, they were characterized by their sizes, compositions and, for proteins, their antigenicity. These simple characters were often used to infer the relationships of the organisms from which the molecules were obtained, and the results seemed to be sensible. However, it was not until the sequences of the amino acids in the proteins were determined, and more recently, the nucleotides in the nucleic acids, that the molecular basis of the relationships were shown to reside in the sequences and their three-dimensional structures.

In this chapter we outline some of the methods used for inferring the phylogenetic or other relationships of sequences. We distinguish the various components of analysis; the ways that nucleotide and amino acid sequences are recorded for analysis, the means by which that data can be transformed to improve the chances of discovering the true relationships, and the ways by which the relationships are inferred and displayed. We have omitted to discuss the more traditional ways for indirectly comparing sequences using, for example, their composition, RFLP similarity or nearest neighbour frequency. However, it is worth emphasizing that, if such methods indicate relationships that correlate with those inferred from sequence data, then a much larger body of older, simpler data may become available and permit broader comparisons and extrapolations.

Sequence analysis

Aims of sequence analysis

The most appropriate way to analyse a sequence depends on the goal of the analysis.

Introduction

The influenza viruses of the Orthomyxoviridae family totally depend on higher vertebrates as hosts, and are transmitted by a respiratory or a faecal-oral route. In contrast, two new members of the family, Thogoto (THO) and Dhori (DHO) viruses, here called the orthoacarivirus group, replicate in both vertebrate and tick cells, and are transmitted by tick bite. This chapter compares orthoacariviruses with orthomyxoviruses (influenza viruses), and speculates on their evolutionary origins.

Natural history of Thogoto and Dhori viruses

The first reported isolation of THO virus was from a pool of ticks removed from cattle in Thogoto forest near Nairobi, Kenya, in 1960 (Haig, Woodall & Danskin, 1965). To date, the virus has been isolated from ixodid tick species collected in several countries extending across central Africa, and in Egypt, Iran, Sicily, and Portugal (Davies, Jones & Nuttall, 1986). Dhori virus was first isolated in 1971 from ticks removed from camels in north-west India (Anderson & Casals, 1973). Subsequent isolations have been made from ticks in the former USSR, Portugal, and Egypt (Jones et al., 1989). Experimental studies confirmed that both THO and DHO viruses are true arboviruses (Davies et ai, 1986; Jones et al., 1989) in that uninfected ticks become infected as they feed on THO or DHO virus-infected vertebrate hosts; the viruses replicate in the ticks, and are then transmitted by tick bite when the infected ticks take their next bloodmeal.

Both THO and DHO viruses have a wide vertebrate host range (Table 28.1).

Introduction

The vertebrate immune system contains both broadly reactive and highly specific mechanisms to detect and destroy foreign proteins and nucleic acids. The specific arms of the immune system, antibodies and T cells, change extremely rapidly in response to the appearance of a new antigen, while the non-specific mechanisms of antiviral defence, principally interferons (IFN) and natural killer (NK) cells, are relatively invariant.

To escape destruction by the immune system, viruses use many tactics and strategies that allow them to survive in the individual, at least long enough to be transmitted to another host. The immune system therefore exerts a powerful selection pressure on a virus in one individual, a pressure that can sometimes be demonstrated experimentally. However, the extent to which the resulting variation in the virus is reflected in evolutionary changes in the virus population is largely a matter of inference. Recent advances in our knowledge of how the immune system, particularly the T cell, recognizes its target antigen, and the ability to sequence quickly many variants of viral genomes, should soon lead to a better understanding of the importance of immunity in affecting viral evolution.

I shall consider here the mechanisms used by viruses of vertebrates to avoid the immune system. There are three main categories of mechanism: i) changes in antigenic structure, allowing immediate escape from immune recognition; ii) changes in gene composition of the virus, usually allowing escape from non-specific viral destruction, and iii) changes in viral behaviour, such as its tissue tropism or the time-course of infection.

Introduction

Poxviruses are a family of viruses with large DNA genomes that include pathogens of vertebrates (subfamily Chordopoxvirinae) and of invertebrates (subfamily Entomopoxvirinae) (Table 18.1). The prototype poxvirus is vaccinia virus, a member of the genus Orthopoxvirus (Dales & Pogo, 1981; Buller & Palumbo, 1991; Moss, 1990, 1991). Vaccinia virus was successfully used for almost 200 years for smallpox vaccination. The origin and passage history of vaccinia virus are obscure; however, analysis of vaccine strains indicate that the virus is most closely related to, but is distinct from, variola virus, which causes smallpox, and also cowpox and monkeypox viruses.

Poxvirus virions are large (300-400 nm) oval particles that contain at least two lipoprotein envelopes. The virion is composed of a DNA molecule and various amounts of more than 100 protein species, a number of which possess enzymatic activity. The poxviruses replicate in the cytoplasm of the cell, where their enzymes involved in nucleotide metabolism, DNA replication and transcription are expressed. The coding of these enzymes makes the virus independent of the corresponding host cell enzymes and, consequently, the virus is able to carry out gene expression and DNA replication in enucleated cells. African swine fever virus (ASFV) occupies an anomalous taxonomic position and has not been assigned to any major virus family (Vinuela, 1985, 1987; Wilkinson, 1989). Although it was originally classified as a member of the family Iridoviridae, its unique characteristics justified its separation from this group. ASFV naturally infects warthogs and bush pigs in southern and eastern Africa. It also infects the domestic pig, causing severe economic losses in the affected countries.

‘Classical’ has connotations of ‘ancient’, but for this book the word relates to studies on virus evolution dating from the time before the sequencing of viral genomes became a way of life for evolutionary virologists. Classical ideas on the origins of viruses were pure speculation and are not worth pursuing further; it was suggested that viruses had arisen either from host cell components in some unknown way, or that some of the more complex viruses may have evolved by progressive degeneration along the sequence bacterium, mycoplasma, rickettsia, chlamydia, poxvirus. Since the focus of this book is the role of molecular biology in understanding viral evolution, I have selected as examples the only two viruses that were discussed in an evolutionary context in books on animal virology published before the emergence of recombinant DNA technology in the early 1970s, namely influenza A virus and myxoma virus (Burnet, 1955, 1960; Andrewes, 1967; Fenner et al., 1974).

Early studies on the evolution of influenza A virus

Here I describe our knowledge of the evolution of influenza viruses as it appeared in 1975, when the only methods of study were serology and peptide mapping of selected proteins. Chapter 34 takes over from that time and tells how molecular biological methods have expanded our understanding of the evolution of these very interesting viruses.

Structure of the virion

First, a little background is needed. There are three species of influenza virus, A, B, and C, each of which can produce respiratory disease in humans.

Introduction

Plant systematics has experienced a methodological revolution in the last three decades. Parsimony analysis of character-state transformations and recognition of strictly monophyletic groups have given systematists the ability to explore the evolutionary relationships of plants with more rigour than in the past. Additionally, recent technological advances and developments in molecular biology have provided systematists with valuable new sources of data for phylogeny reconstruction. ‘Molecular systematics’ is a rapidly expanding field that includes the use of restriction endonuclease sites and nucleotide sequences, among other sources of information (Hillis & Moritz, 1990). Within the embryophytes and in some algal groups (e.g. Rhodophyta, Chlorophyta) chloroplast DNA is the molecule of choice for most plant systematists (Palmer et al., 1988). The chloroplast genome is small, highly conserved, present in a large number of copies and usually inherited only from the female parent. All green plants possess a chloroplast genome that is structurally conserved across most major groups. Thus the chloroplast genome provides a source of data potentially comparable at high taxonomic levels.

Other sources of molecular data are the mitochondrial genome (mtDNA; Palmer, 1992) and ribosomal RNA and DNA (rRNA and rDNA; Hamby & Zimmer, 1992). The mitochondrial genome in plants is large, and its nucleotide sequence evolves slowly. However, it is subject to frequent structural rearrangements and insertions and deletions. These attributes render plant mtDNA of little systematic use in studies involving either restriction sites, which are dependent on a relatively stable genome, or sequences (Palmer, 1992).

The genes for rDNA code for ribosomal rRNAs, and are very conserved owing to structural features.

Introduction

One of the notable features of viruses is the exceptional diversity of their genome organization and of the modes of their genome expression. Viruses whose genome is RNA rather than DNA, as in all cellular organisms (Fig. 4.1), contribute greatly to virus diversity. The origin and evolution of RNA viruses has intrigued researchers for decades. Three classes of RNA viruses can be distinguished, depending on the type of genomic molecule (Baltimore, 1971; Fig. 4.1). These are RNA viruses that employ positive messenger-sense molecules as genomes (+RNA), those that have genome molecules with complementary polarity (negative) to that of their mRNAs (–RNA), and those that have genomes of double-stranded (ds) RNA molecules. A unique virusencoded enzyme RNA-dependent RNA polymerase (RdRp) directs the synthesis of all of these RNA species. Sometimes, viruses with RNA genomes which are replicated through a DNA stage, the retro viruses, are also regarded as RNA viruses (Chapter 27).

In order to sort out the diversity of viruses, a classification has been introduced which uses about a dozen different phenotypic characteristics (Francki et al., 1991). Thus, more than 60 RNA viral families and separate groups have been delineated, but this classification does not attempt to reflect the evolutionary relationships between the taxa.

A lack of virus fossils, and the apparent high rate of turnover of genome sequences, seemed to preclude our ability to understand RNA virus evolution. However, through the last decade, thanks primarily to the enormous increase in our knowledge of viral genome sequences, it became evident that some progress in evolutionary inference might be possible.