1

MOLECULAR BASIS OF MICROBIAL PATHOGENESISSpring 2001

LECTURE 1INTRODUCTION TO MICROBIAL PATHOGENESIS:

GENETIC APPROACHES TO STUDIES OF VIRAL PATHOGENESIS

Terence S. Dermody

OBJECTIVES

1) To understand the various stages in which pathogenic microorganisms interact with their host cells.

2) To understand the various stages in which pathogenic microorganisms interact with their host organisms.

3) To appreciate the importance of cell and tissue tropism as a critical determinant of microbial pathogenesis.

4) To understand that pathogen-receptor interactions determine the tropism of many pathogens.

RECOMMENDED READING

Mechanisms of Microbial Disease, third edition, Schaechter, Engleberg, Eisenstein, and Medoff, editors, Chapter 1, Establishment of Infectious Diseases.

Mechanisms of Microbial Disease, third edition, Schaechter, Engleberg, Eisenstein, and Medoff, editors, Chapter 31, Biology of Viruses.

I. FUNDAMENTAL CONCEPTS IN PATHOGEN-HOST INTERACTIONS

A. Pathogenesis.

Mechanism by which a microorganism injures specific cells or tissues leading to production of disease

B. Virulence.

The capacity of a particular microorganism to produce disease

C. Tropism.

2

The capacity of a microorganism to infect discrete cells or tissues

II. KEY QUESTIONS IN PATHOGENESIS RESEARCH

A. How is it known that a particular microbe causes a given disease? (Koch's postulates)

1) The organism can be demonstrated in association with the disease

2) The organism can be cultured from an individual with the disease

3) Inoculation with the organism produces the disease in experimental animals

4) The organism can be re-isolated from experimentally infected animals

B. How does a microbe interact with its host to produce disease? (stages in pathogen-host interaction)

C. How does the immune system combat microbial pathogens? (innate and acquired immunity)

III. DIVERSITY OF PATHOGENIC MICROBES

A. Viruses.

B. Bacteria.

C. Fungi.

D. Parasites.

IV. PATHOGEN-CELL INTERACTIONS

A. Pathogen structure.

1. Delivery systema. environmental stabilityb. receptor binding

2. Payloada. protein synthesisb. genome replication

3

B. Pathogen replication programs.

1. Stages in viral replication

a. attachmentb. penetrationc. uncoatingd. transcriptione. translationf. replicationg. assemblyh. release

These steps can occur in the host-cell cytoplasm or nucleus. Transcription and replication of viral genomes can be mediated by viral or cellular polymerases.

A prototypical viral replication program.

Nucleus

Release of genome

Viral proteinsynthesis

Release

Mature progeny

Attachment

Replication of genome

Entry

4

2. Stages in bacterial replication

a. attachmentb. ± penetrationc. ± intracellular replicationd. ± toxin production

• endotoxins• exotoxins

V. PATHOGEN-HOST INTERACTIONS: VIRAL PATHOGENESIS

The mechanism by which viruses produce disease in infected host organisms is termed viral pathogenesis. In order to produce disease, a virus must interact with its host in a discrete series of stages. After entry into the host, the virus must replicate in host cells, spread within the host, and overcome host defenses. At each stage of the infectious process, virus pathogenicity is determined by the concerted action of a number of viral and host factors. Illness is commonly produced by direct cellular injury at a site of viral replication. However, disease resulting from viral infection also can be produced by the host immune response elicited by the virus. Occasionally, viral infections are associated with an immune response directed against self antigens. There are many diseases, such as diabetes mellitus and multiple sclerosis, in which autoimmunity in association with viral infection is hypothesized to play a role in disease pathogenesis. Finally, some viruses produce disease by producing life-long persistent infections. These viruses occasionally are associated with immunosuppression or neoplasia in their host organisms.

A. Stages in pathogen-host interaction.

1. Entry into the host

2. Primary replication

3. Spread

4. Cell and tissue tropism

5. Secondary replication

6. Cell injury or persistence

5

7. Host immune response

Three types of viral infection.

B. Immune elements involved in defense against viral infections.

1. humoral immunityinhibition of viral infectivity

2. cell-mediated immunitylysis of virus-infected cells

3. soluble mediators, such as interferoninhibition of cellular macromolecular synthesis

VI. TROPISM

The capacity of a virus to infect a distinct group of cells or tissues is termed tropism. Viral tropism is thus an important determinant of viral pathogenesis.

Examples of viruses that spread systemically and have a distinct tropism for the nervous system:

A. Poliovirus.ingestion hematogenous (?neural) spread CNS

B. Eastern equine encephalitis virus.inoculation hematogenous spread CNS

C. Rabies virus.inoculation neural spread CNS

D. Reovirus.

6

ingestion neural spread CNSVII. GENETIC APPROACHES TO STUDIES OF VIRAL PATHOGENESIS:

REOVIRUSES AND MECHANISMS OF VIRAL NEUROPATHOGENESIS

Reoviruses belong to the family Reoviridae, which includes the human pathogens, rotavirus and Colorado tick fever virus. Virtually all mammals, including humans, serve as hosts for mammalian reoviruses. However, infections of humans are either asymptomatic or associated with mild upper respiratory disease or gastroenteritis, hence the name respiratory enteric orphan (reo) viruses. Rotavirus is the most important cause of diarrheal disease in the world; Colorado tick fever virus is a rare cause of viral encephalitis.

VIII. REOVIRUS STRUCTURE

A. Coat components.

Mammalian reoviruses are non-enveloped, icosahedral viruses. The 1, 3, and 1 proteins form the outer capsid, and the 2, 2, 1, 2, 3 proteins form the core. There are three reovirus serotypes, and each serotype is represented by a prototype strain isolated from a human host; these strains are type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D).

B. Genome.

The reovirus genome consists of 10 double-stranded RNA gene segments. Each segment is monocistronic with the exception of the S1 gene, which encodes virion structural protein 1 and non-structural protein 1s, in overlapping reading frames.

The reovirus virion. The reovirus virion consists of an icosahedral outer capsid and an inner core containing 10 double-stranded RNA gene segments. Viral gene segments are classified as L, M, or S (large, medium, or small) based on relative

L1L2L3

M1M2M3

S1S2S3S4

2

3

21

1

1

OuterCapsid

Core

7

electrophoretic mobilities. S1, the largest member of the S-class gene segments, encodes the 1 protein.

IX. OVERVIEW OF THE REOVIRUS REPLICATION CYCLE

A. Attachment.Virions bind receptors via the 1 protein.

B. Internalization.Virions are internalized by receptor-mediated endocytosis.

C. Disassembly and penetration.Proteolytic processing of outer-capsid proteins 3 and 1 is accomplished by cellular proteases. Proteolysis of the viral outer capsid is inhibited by treatment of cells with ammonium chloride, which indicates that reovirus entry is acid dependent. The processed form of the virion, termed the infectious subvirion particle (ISVP), penetrates the endocytic compartment liberating the core particle into the cytoplasm.

D. Transcription and translation.The viral transcriptase is contained within the viral core. It facilitates synthesis and transport of viral mRNAs into the cytoplasm.

E. Replication.Virus-encoded RNA-dependent RNA polymerase synthesizes double-stranded genome segments from positive-sense RNA templates.

F. Assembly and release.

The reovirus growth cycle. Reoviruses are lytic viruses with an entirely cytoplasmic life cycle.

Nucleus

Uncoating

Release of core

Latetranscription

ReplicationEarly

complexes

Viral proteinsynthesis

Release

Mature progeny

Attachment

Endocytosis

Earlytranscription

10

10

8

X. REOVIRUS PATHOGENESIS

A. Reovirus serotypes cause distinctly different CNS diseases.

Mammalian reoviruses provide a powerful experimental system to study viral neuropathogenesis. Reovirus serotypes 1 and 3 display markedly different patterns of pathogenesis in newborn mice, an established model of reovirus infection. Following oral or intramuscular inoculation, strains of both serotypes invade the CNS, yet by different routes and with distinct pathologic consequences. T1 reovirus spreads to the CNS hematogenously and infects ependymal cells, resulting in hydrocephalus. In contrast, T3 reovirus spreads to the CNS neurally and infects neurons, causing lethal encephalitis.

STAGES OF REOVIRUS INFECTION

9

SEROTYPE-DEPENDENT DIFFERENCES IN REOVIRUS PATHOGENESIS

DISEASE PARAMETER SEROTYPE 1 SEROTYPE 3

PATTERN OF SPREAD hematogenous neural

CNS TROPISM ependymal cells neurons

CNS INJURY hydrocephalus encephalitis

B. Determination of the genetic basis for serotype-dependent differences in reovirus pathogenesis through the use of reassortant viruses.

An important feature of the reovirus system is the ability to employ reassortant genetics to identify specific viral genes involved in various aspects of virus-host interaction. Parental virus strains that differ with respect to a given phenotype (e.g., tissue tropism) are used to generate progeny reassortant viruses containing mixtures of gene segments from each of the parents. The progeny can be used to determine which gene is associated with the phenotype.

Generation of reassortant viruses. Biological polymorphisms exhibited by different reovirus strains can be mapped to specific gene segments through the use of reassortant viruses. Reovirus strains are distinguishable by signature electrophoretic profiles of their double-stranded RNA gene segments. Coinfection of cells with different strains produces a collection of progeny reassortant viruses containing various combinations of gene segments from each parent. A phenotypic difference between two parental strains can be genetically mapped by screening the reassortant viruses in appropriate assays and correlating expression of the phenotype with a specific parental gene segment. The electropherotypes of wt strains type 1 Lang (T1L) and type 3 Dearing (T3D) are illustrated in this figure.

Using viruses containing reassorted genomes derived from crosses of T1L and T3D (T1L x

T1L T1L x T3D ProgenyT3D

10

T3D reassortants), serotype-specific patterns of viral spread in the host and tropism for particular cells and tissues have been mapped to the S1 gene segment, which encodes viral attachment protein 1. This approach also was used to show that serotype-specific differences in binding primary cultures of ependymal cells and neurons are determined by the 1-encoding S1 gene. These findings indicate that the 1 protein dictates the types of cells in the CNS that serve as targets for reovirus infection.

Reovirus tropism is determined by receptor binding. In reovirus-infected mice, tropism for particular cells and tissues is determined by the capacity of different viral serotypes to bind distinct classes of receptors. Receptor recognition is a property of the viral attachment protein, 1. T1 1 binds receptors expressed on ependymal cells, whereas T3 1 binds receptors on neurons.

XI. REOVIRUS ATTACHMENT PROTEIN 1

Reovirus attachment protein 1 forms an elongated fiber topped with a globular head. The 1 protein binds two types of cellular receptors using independent receptor-binding domains. A domain in the fibrous tail of T3 1 binds -linked sialic acid. T1 1 also binds a cell-surface carbohydrate, but this molecule has not been defined. A second RBD is located in the globular head of both T1 and T3 1. This domain binds junction adhesion molecule (JAM). JAM is a type I transmembrane protein with two extracellular Ig domains and a short cytoplasmic tail. JAM is an important component of TJs between endothelial and epithelial cells, and it may function to organize TJ formation by interaction with other TJ proteins and the cytoskeleton. JAM is highly conserved among mammals, with human, murine, bovine, and rat JAM displaying approximately 70% amino acid identity. Given the broad host range of mammalian reovirus, it is not surprising that a reovirus receptor would display a high degree of sequence conservation.

Ependymal Cell Neuron

T1-R T3-R

T1 -1 T3 -1

11

Reovirus attachment protein, 1. Computer-processed electron micrograph of 1. Image adapted from Fraser et al. (B) Predicted secondary structures of 1 morphologic regions: T(i), T(ii), T(iii), T(iv), and H. Morphologic regions T(i) and T(ii) are predicted to be formed by -helical coiled coil. Region T(iii) is predicted to be -sheet and T(iv) to be two shorter -helical coiled coils that flank -sheet. Morphologic region H is predicted to assume a more complex arrangement of secondary structures corresponding to the globular 1 head. Sequences of T3 1 required for binding SA and influencing protease sensitivity are contained in the 1 tail, whereas sequences required for attachment of virions to JAM reside in the 1 head.

XII. KEY POINTS

A. Virus particles are simple structures that contain two components: a delivery system and a payload.

B. All viruses must disassemble to replicate.

C. Viral pathogenesis is dependent on an ordered series of interactions between a virus and its host.

D. Tropism of many viruses is determined by the capacity of the virus to bind particular types of receptors.

Unidentifiedcarbohydrate

JAM

Type 1 1

Sialic acid JAM

Type 3 1

12

XII. GLOSSARY OF TERMS

ANTIBODY

A molecule produced by animals in response to a foreign substance, known as an antigen. An antibody has the property of combining specifically with the antigen that elicited its formation. Monoclonal antibodies are produced by a single clone of lymphocytes and recognize a single antigenic epitope. Polyclonal antibodies are produced by different lymphocytes and recognize many antigenic epitopes.

ANTIBODY-MEDIATED NEUTRALIZATION

The capacity of an antibody to block viral infectivity in cell culture. Neutralization-resistant viral variants can grow in the presence of neutralizing antibodies.

AUTOIMMUNITY

A process by which an immune response is directed against self antigens. Autoimmune disease can arise in association with many types of viral infections.

CAPSID

The virus coat. Some viruses are devoid of membrane (naked); others are surrounded by membranes (enveloped). If enveloped, the membrane most often derives from the nuclear or cytoplasmic membrane of the host cell, often modified by the presence of membrane-bound viral proteins.

CELLULAR RECEPTORS

Molecules located on the surface of host cells to which viruses bind in order to initiate the process of viral infection.

ENCEPHALITIS

An acute, overwhelming infection of the brain, caused most often by viral infections.

EPITOPE

A minimum antigenic determinant. An antigenic epitope is that part of the antigen molecule that combines with antibody. A single antigen can contain many epitopes.

GLYCOPROTEINS

Proteins modified to contain carbohydrate residues. Sialylated glycoproteins contain sialic acid. Type 3 reovirus can bind to sialylated glycoproteins. The glycoprotein bound by type 1 reovirus is not known.

13

HEMAGGLUTINATION

The process by which viruses cause erythrocytes to clump together. In many cases, this property is due to viruses binding to carbohydrates on the erythrocyte surface.

HYDROCEPHALUS

A condition marked by dilatation of the cerebral ventricles. Viruses can cause hydrocephalus by infecting ventricular lining cells (ependymal cells) resulting in an obstruction of cerebrospinal fluid pathways.

REASSORTANT VIRUSES

Viruses that contain segmented genomes are capable of exchanging their gene segments when two different viruses infect the same cell. Progeny viruses isolated from such mixed infections are called reassortant viruses because they contain a mixture of gene segments derived from each of the parents. Genetic studies using reassortant viruses have allowed many properties of reovirus pathogenesis to be mapped to individual gene segments. For example, if strain A causes disease X and strain B causes disease Y, studies using reassortant viruses generated from crosses between strains A and B will indicate the gene or genes responsible for each disease.

REOVIRUS

Respiratory Enteric Orphan virus. Reoviruses do not produce significant disease in humans; however, they have been established as a powerful model system for studies of viral pathogenesis in experimental animals. Reoviruses contain a double-protein shell and a genome consisting of ten segments of double-stranded RNA.

REOVIRUS SIGMA () 1 PROTEIN

The 1 protein is the reovirus attachment protein and determinant of the serotype-specific immune response. It plays an important role in reovirus pathogenesis by determining the capacity of the virus to infect particular cells and tissues.

SEROTYPE

A scheme used to classify virus strains based on the immune response elicited by viruses in infected animals. Animals are protected against reinfection by viruses of the same serotype, but they are susceptible to infection by viruses of different serotypes. Three reovirus serotypes, or types, exist.

TROPISM

The capacity of a virus to infect a distinct group of cells. Viral tropism is an important determinant of viral pathogenesis. For example, poliovirus, eastern equine encephalitis virus, rabies virus, and reovirus all have a tropism for neuronal cells, and each can produce disease involving the central nervous system.

TYPE 1 REOVIRUS

14

These viruses spread via the blood stream and infect ependymal cells in the central nervous system, producing hydrocephalus. The Lang strain is the prototype type 1 reovirus.

TYPE 3 REOVIRUS

These viruses spread through nerves and produce neuronal cell injury, resulting in an acute encephalitis. The Dearing strain is the prototype type 3 reovirus.

VIRAL ATTACHMENT PROTEINS

Viral structural proteins that mediate the binding of viruses to cellular receptors.

VIRAL PATHOGENESIS

The mechanism by which viruses produce disease in infected host organisms. Pathogenesis is dependent upon a complex interaction of both viral and host factors.

VIRAL REPLICATION:

attachment - Physical interaction between a virion and the surface of a potentially infectable cell.

penetration - Entry of attached virions across the cytoplasmic (or endocytic) membrane to the interior of the cell.

uncoating - Disassembly of the virion to liberate genomic components for expression. This step is followed by viral transcription and translation, and synthesis of new viral genomes.

assembly - Step-wise and systematic aggregation of capsid components, progeny genomes, core proteins, possible membranous components, and other virion components to mature virions.

release - A process whereby progeny virions exit from infected cells. This may occur by means of cell lysis, budding, or cell-to-cell fusion. The process may or may not jeopardize the viability of the cell.

VIRION

A single viral particle.