Showing papers in "Current Topics in Microbiology and Immunology in 1990"

Analysis of the Protein-Coding Content of the Sequence of Human Cytomegalovirus Strain AD169

Abstract: Large-scale sequence analysis of the AD169 strain of human cytomegalovirus (HCMV) began in this laboratory in 1984 when very little was known about the sequence or location of genetic information in the viral genome. At that time sequence analysis was confined to the major immediate-early gene (Stenberg et al. 1984), a region of the Colburn strain that contained CA tracts (Jeang and Hayward 1983), the L-S junction region (Tamashiro et al. 1984), and what has been termed the transforming region (Kouzarides et al. 1983). This chapter is being written in March 1989 when the sequence is complete except for some remaining polishing of certain areas which is still going on (manuscript in preparation). As far as we know there are no major discrepancies in the data which might lead to the sequence changing although of course this cannot be ruled out. We present a preliminary analysis of the HCMV genome and limit ourselves mainly to the potential protein-coding content of over 200 reading frames.

Retrovirus envelope glycoproteins

Abstract: The envelope glycoprotein complex of replication competent retroviruses is comprised of two polypeptides, an external, glycosylated, hydrophilic polypeptide (SU) and a membrane-spanning protein (TM), that form a knob or knobbed spike on the surface of the virion Both polypeptides are encoded in the env gene and are synthesized in the form of a polyprotein precursor that is proteolytically cleaved during its transport to the surface of the cell While these proteins are not required for the assembly of enveloped virus particles, they do play a critical role in the virus replication cycle by recognizing and binding to specific receptors (SU) and by mediating the fusion of viral and cell membranes (TM): virus particles lacking envelope glycoproteins are thus noninfectious

The role of bacterial polysaccharide capsules as virulence factors.

Abstract: Carbohydrates are universally present on the surface of living cells. On eukaryotic cells, many different carbohydrates are attached as glycoproteins and glycolipids; the oligosaccharide moieties are known to act as receptors and it seems likely that they play an important role in cell-to-cell recognition processes. Polysaccharide capsules, in prokaryotes characteristically composed of repeating oligosaccharides, are found on the surface of many bacteria. These capsules are typically composed of only one polysaccharide and lie outside the outer membrane of gram-negative cells and the peptidoglycan layer of gram-positive cells. In general, individual bacteria do not exhibit variation of these antigens as has been described for the variant glycoproteins of trypanosomes (Cross1978). Comprising 99% water, these highly hydrated, polyanionic polysaccharide capsules serve many functions. These include determining access of molecules and ions to the bacterial cell envelope and the cytoplasmic membrane, the promotion of adherence to the surfaces of inanimate objects or living cells and the formation of biofilms and microcolonies (Costerton and Irwin1981). Among certain gram-positive and gram-negative bacteria, capsules have evolved distinctive structural and functional characteristics which are of cardinal importance in the pathogenesis of infections of animals, plants and insects (Sutherland1977).

Structure and Biosynthesis of the Capsular Antigens of Escherichia coli

Abstract: A characteristic feature of gram-negative bacteria, to which Escherichia coli belong, is the presence of an outer membrane on the external side of the murein sacculus. This layer consists of proteins, lipoproteins, phospholipids, and lipopolysaccharides (LPSs). LPSs of smooth wild-type bacteria, consisting of lipid A, core, and O-specific polysaccharide, are O antigens and those of rough mutants consisting only of lipid A and core are R antigens (Jannand Westphal1975; Jannand Jann1984).

The Evolution of B-Cell Clones

Abstract: Large numbers of B cells are produced throughout life as the result of both primary B lymphopoiesis (Opstelten and Osmond 1983) and antigen-driven B- cell proliferation (Fliedner et al. 1964). It is a feature of the system that a high proportion of the cells produced have a brief lifespan (Kumararatne et al. 1985; Freitas et al. 1986; MacLennan and Gray 1986). Some, however, survive for much longer following positive selection. This can be antigen-dependent (MacLennan and Gray 1986; Gray et al. 1986). but may also be antigen- independent (MacLennan 1987). The B cells which migrate between the follicles of secondary lymphoid tissues and those which are located in the marginal zones of the spleen have been positively selected in this way. These cells, which are not in cell cycle, have an average lifespan of around 1 month (Gray 1988a; Gray and Skarvall 1988). If they are not activated by antigen within this period they die.

The replication of bluetongue virus in Culicoides vectors.

Abstract: Bluetongue virus (BTV) has long been known to be transmitted biologically by certain species of biting midge belonging to the genus Culicoides (Latreille). Du Toit (1944) in South Africa was the first to implicate a Culicoides species in the transmission of this virus when he showed that C. imicola (pallidipennis) was able to transmit bluetongue from infected to susceptible sheep. Since that time numerous authors have confirmed his observations (Walker and Davies 1971; Braverman and Galun 1973; Braverman et al. 1981, 1985; Mellor et al. 1984a). However, although there are well over 1000 species of Culicoides in the world (Boorman 1988), only 17 have been connected with BTV and to date only six, C. variipennis, C. imicola, C. fulvus, C. actoni, C. wadai and C. nubeculosus, have been proven to transmit the virus (Table 1). This number may soon be increased to eight since on the basis of epidemiological evidence and virus isolations it is Table 1 Field and laboratory BTV infections of Culicoides Subgenus Species Virus isolation Laboratory infection Transmission Avaritia C. actoni − + + C. brevipalpis − + − C. brevitarsis + + − C. fulvus + + + C. imicola + + + C. obsoletus + − − C. tororoensis + − − C. wadai − + + Culicoides C. peregrinus − ± − Diphaeomia C. debilipalpis − + − Hoffmania C. insignis + − − C. milnei + - - C. venustus − + − Monoculicoides C. variipennis + + + C. nubeculosus − + + Oecacta C. oxytoma − + − Pulicaris C. impunctatus − ± − likely that C. insignis and C. brevitarsis will also prove to be competent BTV vectors (Greiner et al. 1985; Standfast et al. 1985). Although some of the remaining species of Culicoides may eventually be shown to be fully competent BTV vectors most species will be refractory to infection. Why this should be is not yet entirely clear, but it is known that the mechanism or mechanisms controlling the oral infection of Culicoides with BTV operate chiefly at the level of the mid-gut wall (Jennings and Mellor 1988), a single layer of cells of epithelial origin supported by a basement lamina (Hardy et al. 1983; Megahed 1956).

Retroviral RNA packaging: sequence requirements and implications.

Abstract: Unlike many animal viruses, infection by retroviruses generally does not lead to cessation of host RNA synthesis. Despite the high levels of host RNA in infected cells, the vast majority of retroviral particles contain a precise genomic complex consisting of two molecules of genomic RNA, rather than cellular or subgenomic viral mRNAs. Thus, the retroviral genome must be selected for encapsidation against a high background of cellular RNAs. It is therefore surprising that the retroviral genome is structurally similar to that of cellular mRNA. For instance, both molecules contain a 5′ m7 G cap and several hundred A residues at the 3′ terminus (reviewed in Coffin 1984a, 1985). Viral subgenomic mRNAs are even more similar to genomic RNA. The ability of the retroviral particle to choose correctly genomic RNA from the vast excess of heterologous molecules implies that specific sequences are present within the genome which direct the efficient encapsidation of the correct RNAs. Analysis of spontaneous and engineered mutants of both avian and murine retroviruses has in fact revealed that cis-acting sequences are involved and are present in the retroviral genome.

Antigenic Structure of Picornaviruses

Abstract: The detailed antigenic structure of the picornaviruses has been intensively studied over the past few years using a variety of different methods which have frequently given conflicting results. The resolution of the atomic structures of at least one member of each of the four genera of picornavirus has contributed greatly to the accepted views.

Integration of Retroviral DNA

Abstract: Integration of a DNA copy of the viral genome into host cellular DNA is an essential step in the life cycle of most, if not all, retroviruses (Donehower and Varmus 1984; Schwartzberg et al. 1984a; Panganiban and Temin 1984b; Colicelli and Goff 1985; Harris et al. 1984). Since retroviral DNA molecules are not ordinarily able to replicate autonomously as episomes, they depend upon integration for stable maintenance in dividing cells. Once integrated, the viral genome, or provirus, is transmitted as an integral element of the host genome. Integration appears moreover to be important, though perhaps not essential, for the transcription of viral DNA into new copies of the viral genome and messenger RNAs that encode viral proteins. Thus, integration of a provirus defines the critical switch in the viral life cycle from mere subsistence to multiplication.

Streptozotocin interactions with pancreatic beta cells and the induction of insulin-dependent diabetes.

Abstract: The single most consistent finding in insulin-dependent diabetes mellitus (IDDM) is a substantial reduction in insulin secreting β cells (GEPTS 1965). The pathogenic factors responsible for this cellular destruction are complex and most likely differ among different subgroups in this category. Although these factors have not yet been definitively elucidated, it has become apparent that genetic influences and both humoral and cell mediated immunological phenomena are involved (EISENBARTH 1986; LEFEBVRE 1988). Also, a role for environmental factors in the etiology of IDDM has recently been indicated by epidemiological studies which have demonstrated that there is a marked increase in newly diagnosed cases of IDDM, which can only be explained by changes in environmental influences such as chemicals and viruses (KROWLEWSKI et al. 1987). Direct evidence that an ingested chemical can cause IDDM in humans comes from case reports of individuals who ate the rat poison Vacor in suicide attempts. Many of these individuals developed ketosis prone diabetes mellitus (KARAM et al. 1980; PROSSER and KARAM 1978). Studies in laboratory animals have provided additional evidence that xenobiotics can cause a critical reduction in insulin secreting cells. It is well established that nitrosamides like streptozotocin (SZ) and chlorozotocin and other complex amines like alloxan cause severe diabetes in laboratory animals (DULIN and SORET 1977; COOPERSTEIN and WATKINS 1981; MOSSMAN et al. 1985).

Structure and function of C3a anaphylatoxin.

Abstract: The C3a molecule is one of three activation fragments from the complement cascade, a family of factors that include C3a, C4a, and C5a. Early recognition that more than one bio active fragment was generated during complement activation cascade must be credited to COCHRANE and MULLER-EBERHARD (1968) and to DIAS DA SILVA et al. (1967). These investigators were the first to use purified components of human complement to demonstrate, and then to characterize, the activation fragment from C3. In previous studies complement was activated in serum, and the bioassays that were used detected only the factor C5a (actually C5adesArg). DIAS DA SILVA et al. realized, even using purified components of the complement C1 esterase, C4, C2, and C3, that the anaphylatoxin released from C3 was not stablized unless the digest was acidified to pH 2-5, according to earlier observations of STEGEMANN et al. (1964). Later work by BOKISCH and MuLLER-EBERHARD (1970) would explain why the acid treatment was successful. Acidification presumably prevented residual carboxypeptidase (serum carboxypeptide N) in the isolates from removing an essential C-terminal arginine and inactivating the newly formed C3a anaphylatoxin. It was this lability of C3a bioactivity in serum that had prevented its discovery prior to isolation and activation of the purified components. These investigators went on to separate an active principal from the larger protein components in the reconstituted C3—C3 convertase system by gel filtration. They demonstrated that the lower molecular weight factor: (a) induced smooth-muscle contraction and was tachyphylactic to itself but not cross-tachyphylactic to the C5-derived anaphylatoxin; (b) enhanced vascular permeability when injected into skin; (c) degranulated guinea pig ileal mast cells; and (d) promoted histamine release from rat peritoneal mast cells. DIAS DA SILVA’S group originally termed the active fragment F(a)C3, i.e., activation (a) fragment from the third component of complement. COCHRANE and MuLLER-EBERHARD identified the fragment as the C3 anaphylatoxin, assuming it to be an analog to the “classical” C5a anaphylatoxin. We will refer to the fragment here primarily as C3a because the biologic action of the factor is not anaphylactoid in nature, and anaphylatoxin is functionally an inaccurate designation. However, the recognized nomenclature from common usage still refers to fragments C3a, C4a, and C5a collectively as anaphylatoxins.

The role of viruses and environmental factors in the induction of diabetes.

Abstract: The development of IDDM results from the destruction of pancreatic beta cells. Genetic factors, various immune system alterations, and environmental factors have been studied as the possible causes of IDDM. The concordance rate for developing IDDM between monozygotic twins approaches 50%, suggesting that genetic factors are necessary, but nongenetic factors such as various immune system alterations and environmental factors also influence the clinical expression of genetic susceptibility. Environmental factors (e.g., viruses, chemicals, and diet) affecting the induction of diabetes may act as primary injurious agents which damage pancreatic beta cells or as triggering agents of autoimmunity. Certain viruses including EMC-D and Mengo virus 2T can directly infect pancreatic beta cells and replicate in the cells. The replication of viruses in the beta cells results in the destruction of the cells within 3 days, and the infected mice develop a diabeteslike syndrome in 3-4 days without the involvement of autoimmunity. In contrast, rubella virus appears to be somewhat weakly associated with autoimmune IDDM in hamsters. In addition, endogenous retrovirus expressed in pancreatic beta cells is clearly associated with the development of insulitis and diabetes in NOD mice. In man, there appears to be no correlation between the detection of islet cell autoantibodies and anti-Coxsackie B viral antibodies in newly diagnosed IDDM. In contrast, persistent infection of CMV and rubella virus appears to be associated with the presence of autoantibodies in newly diagnosed IDDM patients. It is particularly noteworthy that human CMV can induce islet cell autoantibodies that react specifically with a 38 kDa islet cell protein which may represent islet cell-specific antigens in a proportion of CMV-associated IDDM cases. These observations suggest that the association of diabetes with Coxsackie B viruses might be due to cytolytic infection of the beta cells with no link to autoimmunity, while both rubella virus and CMV are probably associated with autoimmune IDDM. A number of structurally diverse chemicals including alloxan, streptozotocin, chlorozotocin, Vacor, and cyproheptadine are diabetogenic mainly in rodents and sometimes in man. Possible mechanisms for beta cell destruction by these chemicals include (a) generation of oxygen free radicals and alteration of endogenous scavengers of these reactive species; (b) breakage of DNA and a consequent increase in the activity of poly-ADP-ribose synthetase, an enzyme depleting nicotinamide adenine dinucleotide in beta cells; and (c) inhibition of active calcium transport and calmodulin-activated protein kinase activity. (ABSTRACT TRUNCATED AT 400 WORDS)

Abnormalities of the immune system induced by dysregulated bcl-2 expression in transgenic mice.

Abstract: Dysregulated expression of the putative cellular oncogene bcl-2 by chromosomal translocation has been strongly implicated in follicular center B cell lymphoma, one of the most common hematologic malignancies in humans. These tumors usually contain a 14;18 chromosomal translocation (Fukuhara et al 1979) that juxtaposes bcl-2 with the immunoglobulin heavy chain (Igh) locus (Tsujimoto et al 1984; Cleary and Sklar 1985; Bakshi et al 1985). The recombination presumably subjects bcl-2 to the control of Igh regulatory sequences which enforce its constitutive expression in B lymphoid cells. The bcl-2 gene encodes a 24 kD non-glycosylated protein located on the cytoplasmic face of the plasma membrane (Tsujimoto et al 1987; Chen-Levy et al 1989). It is normally expressed in pre-B cells, quiescent in resting B cells, expressed again in activated B cells and then downregulated upon their terminal differentiation (Reed et al 1987; Gurfinkel et al 1987; Chen-Levy et al 1989). The first indication of the function of this putative oncogene was revealed by our previous studies on interleukin-3-dependent cell lines infected with a bcl-2 retrovirus. The cells survived withdrawal of growth factor, but entered a Go state, suggesting that constitutive bcl-2 expression promotes cell survival rather than proliferation (Vaux et al 1988).

Translational suppression in gene expression in retroviruses and retrotransposons.

Abstract: The past 5 years have brought an exciting and very unexpected solution to a longstanding question in retrovirology: the mechanism of expression of the pol gene Since the earliest studies of retroviral gene expression, the mechanism by which pol, the gene that encodes the critical enzymes reverse transcriptase, integrase, and sometimes protease, acts had remained an enigma Experiments carried out recently seem to have finally settled this issue, as the pol genes of several retroviruses and one retrotransposon have been shown to be expressed by one or another form of translational suppression This solution to the problem of pol gene expression is as unexpected as it is unusual Even 5 year ago there was general agreement in this field that mRNA splicing would ultimately be found to be responsible for expression of the pol functions

Cellular and molecular basis of the protective immune response to cytomegalovirus infection.

Abstract: Cytomegalovirus (CMV) infection can occur throughout life. Similar to other herpesviruses, after primary infection CMV remains in the host in a latent state. Infection of the immunocompetent host does not cause clinical symptoms, whereas infection of the immunocompromised host can cause severe and even fatal disease. Morbidity and mortality associated with primary CMV infection or with reactivation from latency is common in immunosuppressed transplant recipients and in patients with immunodeficiency caused by HIV infection. CMV pneumonia is considered the immediate cause of death in these patients.

Capsular polysaccharides as vaccine candidates.

Abstract: The discovery of a “specific soluble substance” secreted by pneumococci during growth (Dochez and Avery 1917) and the identification of this substance as a carbohydrate (Heidelberger and Avery 1923) were new and important developments in vaccine technology. This became evident when following the initial finding that the isolated capsular polysaccharides of pneumococci were immunogenic in mice (Schiemannand Casper 1927) it was demonstrated that they were also immunogenic in man (Francis and Tillet 1930). The potential of capsular polysaccharides as vaccines was fully confirmed when it was demonstrated unequivocally that multivalent pneumococcal polysaccharide vaccines were able to provide type-specific protection in humans against the acquisition of pneumococcal infection (MacLeod et al. 1945). However, at this time the phenomenal success of antibiotic therapy in the treatment of bacterial infections caused a lengthy hiatus in the further development of polysaccharide vaccines.

The biologic significance of bacterial encapsulation.

Abstract: Capsules are important determinants of the behavior of bacteria within the animal host. To survive within the host, bacteria must be able to evade a diverse array of defense mechanisms that include complement-mediated bacteriolysis, uptake and killing by phagocytes as well as cell-mediated immune mechanisms. In this chapter the manner in which bacterial capsules, particularly those of Escherichia coli, enable the organism to survive in this hostile environment will be detailed.

Structure of the bluetongue virus genome and its encoded proteins.

Abstract: Bluetongue virus (BTV) particles were initially suggested to be morphologically similar to reovirus particles (Owen and Muntz 1966; Studdert et al. 1966; Els and Verwoerd 1969) and were subsequently confirmed to contain a. double-stranded RNA (dsRNA) genome as in reovirus (Els 1973; Verwoerd 1969; Verwoerd et al. 1970; Bowne and Ritchie 1970). BTV, along with other morphologically related viruses, is classified as an orbivirus within the family Reoviridae (Borden et al. 1971; Murphy et al. 1971). To date 24 different BTV serotypes have been identified from different parts of the world including North and South America, Australia, Africa, and Southeast Asia (Howell 1960, 1970; Howell and Verwoerd 1971; Gorman et al. 1983; Knudson and Shope 1985). In the early 1970s Verwoerd and coworkers carried out the first biochemical studies of the BTV particle and since then many further studies on the genome of BTV have been done. In this chapter we intend to define the current state of our understanding of the genetics and the structure of the bluetongue genes and gene products.

Genetics of capsular polysaccharide production in bacteria

Abstract: A feature of many bacteria of diverse genera is the production of extracellular acidic polysaccharides. These polysaccharides may be organised into distinct structures termed capsules, or may be excreted as an extracellular slime. However, this distinction is arbitrary and in practice may be of no functional significance.

Recombinant vaccinia viruses as vectors for studying T lymphocyte specificity and function.

Abstract: Over the past 20 years, a steadily increasing portion of immunological research has been directed towards understanding the specificity and function of T lymphocytes. Vaccinia virus (VV) has proven to be the preferred vector for determining the specificity of T lymphocytes for individual gene products derived from a variety of organisms, including viruses, protozoa, and mammalian cells (we refer to these genes as “extrinsic genes”, their products as “extrinsic antigens” and their origin as “source organisms”) and for studying the processing and presentation of antigens to T lymphocytes by antigen presenting cells (APCs). The popularity of VV recombinants has two sources. First, the technology of producing VV recombinants is relatively straightforward and VV recombinants consistently provide high levels of expression of extrinsic antigens. Second, studies of T lymphocytes require the expression of antigens in histocompatible cells. The ability of VV to infect mice and a broad variety of mouse and human cell lines of diverse lineages that can function as APCs make them ideal vectors for studies of T lymphocytes.

Regulation and tissue-specific expression of human cytomegalovirus.

Abstract: Human cytomegalovirus (HCMV) establishes a lifelong latent state after primary infection, as do the other members of the human herpesvirus family. The site of latency and the molecular mechanisms involved in the establishment and maintenance of latent virus in the cell are unknown for HCMV. In this chapter, we will discuss experimental approaches to identifying cell types naturally infected in the human host by HCMV. We will examine cell culture systems and transcription factor interactions in vitro which may identify HCMV-host cell interactions that regulate expression of the virus.

The Replication of Bluetongue Virus

Abstract: Bluetongue virus (BTV) replicates in the cytoplasm of a wide variety of cell types and infection ultimately leads to cell death. The studies of Verwoerd, Huismans and others in the late 1960s and continuing to the present (see Chap. 2, this volume) on the double-stranded, segmented genomic RNA (Verwoerd 1969; Verwoerd et al. 1970), the bishelled nature of the virus particle (Verwoerd et al. 1972), the activation of the virion-bound transcriptase, and the activity of this enzyme in vivo and in vitro (Verwoerd and Huismans 1972) indicated that BTV possesses many characteristics in common with reovirus. However, BTV and the similar African horse-sickness and epizootic hemorrhagic disease (EHD) of deer viruses differ from reovirus in several respects. They are smaller, lack a well-defined outer capsid layer, and exhibit greater pH sensitivity. In addition, they are insect transmitted. Such differences led to the grouping of these viruses (Verwoerd 1970) into a genus for which the name “orbivirus” was proposed (Borden et al. 1971). BTV is the type species of the Orbivirus genus within the Reoviridae family.

Decay-accelerating factor and membrane cofactor protein.

Abstract: Cells exposed to plasma proteins are frequently under attack from the complement system. This can arise either as a bystander process to the classical or alternative pathways of activation initiated during the immune response to foreign particles and organisms or from the constant tick-over of the alternative pathway. Thus, it is critical for the cell to regulate the complement pathway on its own surface. The plasma proteins, H and C4 binding protein (C4bp), in conjugation with the serine protease I, function to this end. Additionally, cells possess a number of membrane proteins to regulate complement deposited on their surfaces; the largest group, focused on C3 and the C3 convertases, consists of the C3b/C4b receptor (CR1), decay-accelerating factor (DAF), and membrane cofactor protein (MCP). CR1, although it has both decay-accelerating activity and serves as a cofactor for the I-mediated cleavage of C3b and C4b, acts mainly extrinsically as a receptor for C3b-bearing immune complexes. DAF exerts its decay-accelerating activity intrinsically on the cell itself (see below). Indeed, the lack of DAF in the membrane of blood cells in the disease paroxysmal nocturnal hemoglobinuria (PNH) leads to an increased complement sensitivity of these cells. Purified MCP can also regulate C3 and the C3 convertases through cofactor I activity. MCP has the same approximate size and overall structure as DAF, and hence it might also function intrinsically to control C3 convertases formed on the same cell. This chapter reviews in detail the structure, both at the protein and DNA levels, of these two complement regulatory membrane glycoproteins, DAF and MCP, and discusses their physiological roles in protecting cells from damage by autologous complement.

Poliovirus RNA replication.

Abstract: The biosynthesis of RNA directed by an RNA template is a reaction that is unique to RNA viruses. Although studies of polio virus RNA synthesis have been conducted in a somewhat intermittent fashion during the past 25 years in several different laboratories, no clear picture has yet emerged regarding the biochemistry of RNA replication for this or any other RNA virus. Upon entry into the cell, the positive strand, infecting RNA genome directs the synthesis of viral proteins which are required for replication of the RNA. The replication process involves first the synthesis of a negative strand RNA molecule; subsequent transcription of this negative strand produces new copies of the positive strand RNA. Historically, the experimental approach initially utilized to analyze the poliovirus RNA replication reaction was enzymological; efforts were made to isolate and purify an RNA-dependent RNA polymerase activity from virus-infected cells. Indeed, at that time, the only tools available for RNA replication studies were biochemical. The biochemistry, however, proved difficult. RNA replication was found to occur in intracellular structures that are tightly associated with or in membranes, and these proved intractable to purification and dissection. Disruption of the membrane structure in order to isolate template or enzyme components often appeared to alter their properties and/or structures. Thus, the initial approach yielded little information about the mechanism of RNA replication, and it has been only quite recently that alternative approaches have been applied.

Picornavirus protein processing--enzymes, substrates, and genetic regulation.

Abstract: The sequence of events leading to the successful completion of a Picornavirus infection of susceptible cells is ultimately controlled by proteolytic processing. As a consequence of encoding their viral-specific polypeptides within a single, open reading frame, picornaviruses must depend upon the intramolecular and intermolecular interactions of viral proteinases and their cognate substrates. This chapter will first provide an overview of how the biosynthetic activities that occur during a picornavirus life cycle are regulated by protein processing activities and signals encoded in the viral genome. An examination of the nature of picornavirus proteinases and their polyprotein substrates will be presented in order to underscore the unifying principles of proteolytic cleavage and to point out the peculiar differences in processing strategies among the different picornaviruses. The application of recombinant DNA methodologies, particularly site-specific mutagenesis, to the study of structure/function relationships of picornavirus cleavage activities will then be discussed. This discussion will also focus on the molecular genetics of viable virus mutants with engineered processing lesions and on in vitro expression of altered cleavage phenotypes. Finally, the biochemical implications of the observed picornavirus processing activities will be addressed. In particular, primary sequence versus conformational determinants of protein processing will be analyzed, as well as the importance of cis versus trans cleavage.

Bluetongue virus structural components.

Abstract: The structural components of bluetongue virus (BTV), the prototype of the orbivirus genus, has been the subject of a number of reviews (Verwoerd et al. 1979; Gorman and Taylor 1985; Spence et al. 1984). The main features can be summarized as follows: BTV is an icosahedral-shaped particle consisting of a segmented double-stranded RNA genome encapsidated in a double-layered protein coat. Removal of the outer protein layer activates a viral-associated RNA polymerase which transcribes the ten genome segments into 10 mRNAs which are in turn translated into at least seven structural and three nonstructural proteins. The characteristic features of these different structural components will be reviewed in this chapter. The key to the elucidation of the structural characteristics has been the ability to isolate and purify large amounts of BTV.

T -Cells, Stress Proteins, and Pathogenesis of Mycobacterial Infections*

Abstract: When a microbial pathogen meets a mammalian organism, different kinds of relationship may evolve. Exotoxin-producing pathogens can harm the host in a dramatic way without becoming too involved themselves. Purulent bacteria colonize extracellular niches from which they can cause acute-type diseases. In both cases, humoral immunity has a profound effect, and normally either type of pathogen is rapidly eliminated once it is taken up by professional phagocytes. So-called intracellular pathogens establish a lifestyle inside host cells, and many of them survive within macrophages at least for some time. Bacteria of this group include Mycobacterium tuberculosis, M. bovis, M. leprae, Salmonella typhi, Legionella pneumophila, and Listeria monocytogenes—the etiologic agents of tuberculosis, leprosy, typhoid fever, Legionnaire’s disease, and listeriosis, respectively. Although macrophages provide a major habitat for these microorganisms, other host cells can be affected as well, with M. leprae-infected Schwann’s cell providing a notable example.