Chapter 2: The Infectious Cycle

T Introduction Viruses are unique: often made up of nothing more than a nucleic acid molecule wrapped in protein, they parasitize the cellular machinery to produce thousands of progeny Despite such variety, viruses are amenable to study because all viral propagation can be described in the context of three fundamental properties, as noted in chapter 1: viral genomes are packaged inside particles that mediate their transmission from cell to cell; the viral genome contains the information for initiating and completing an infectious cycle; viruses establish themselves in a host population to ensure virus survival Cells can be infected In such a way as to ensure that a single reproduction cycle occurs synchronously in every infected cell, called one-step growth The infectious cycle The infectious cycle comprises attachment and entry of the particle, production of viral mRNA and its translation by host ribosomes, genome replication, and assembly and release of progeny particles containing the genome The term virus reproduction is another name for the sum total of all events that occur during the infectious cycle While virus particles readily attach to cells in culture, in nature they must encounter a host, no mean feat for nano particles without any means of locomotion The cell Viral reproduction requires many different functions of the host cell Ex. Machinery for translation of viral mRNAs, sources of energy, and enzymes for genome replication The cellular transport apparatus brings viral genomes to the correct cellular compartment and ensures that viral subunits reach locations where they may be assembled into virus particles Entering cells A virion may not infect every cell it encounters: it must first come in contact with the tissues that contain cells to which it can bind Such cells [the ones virions can bind to] are normally recognized by means of the specific interaction of a virus particle with a cell surface receptor Virus-receptor interactions can be either promiscuous or highly selective, depending on the virus and the distribution of the cell receptor The presence of such receptors determines whether the cell will be susceptible to the virus. However, whether a cell is permissive for the reproduction of a particular virus depends on other, intracellular components found only in certain cell types Cells must be both susceptible and permissive if an infection is to be successful SusceptibleThe state of being predisposed to, sensitive to, or of lacking the ability to resist something PermissiveAllowing; tolerant Viral RNA synthesis Although the genomes of viruses come in a number of configurations, they share a common requirement: they must be efficiently copied into mRNAs for the synthesis of viral proteins and progeny genomes for assembly With the exception of retroviruses, all RNA viruses encode an RNA-dependent RNA polymerase to catalyze the synthesis of both mRNAs and genomes For the majority of DNA viruses and retroviruses, synthesis of viral mRNA is accomplished by RNA polymerase II, the enzyme that produces cellular mRNA Viral protein synthesis All viruses are parasites of translation: their mRNAs must be translated by the host's cytoplasmic protein-synthesizing machinery However, viral infection often results in modification of the host's translational apparatus so that viral mRNAs are translated selectively Analysis of viral translation has also led to the discovery of new mechanisms, such as internal ribosome binding and leaking scanning, that have been subsequently found to occur in uninfected cells Viral genome replication Replication of viral genomes requires the cell's synthetic machinery in addition to viral proteins. The cell provides nucleotide substrates, energy, enzymes, and other proteins Transport systems are required because the cell is compartmentalized: essential components might be found only in the nucleus, the cytoplasm, or within subcellular organelles Assembly of progeny virus particles The various components of a virus particle, the nuclei acid genome, capsid protein(s), and in some cases envelope proteins, are often synthesized in different cellular compartments The primary sequences of viral structural proteins contain sufficient information to specify assembly; this property is exemplified by the remarkable in vitro assembly of tobacco mosaic virus from coat protein and RNA Successful virus reproduction depends on redirection of the host cell's metabolic and biosynthetic capabilities, signal transduction pathways, and trafficking systems Viral pathogenesis Viral pathogenesis is the process by which viruses cause disease. The study of viral pathogenesis requires investigating not only the relationships of viruses with the specific cells that they infect but also the consequences of infection for the host organism The nature of viral disease depends on the effects of viral reproduction on host cells, the responses of the host's defense systems, and the ability of the virus to spread in and among hosts Overcoming host defenses Organisms have many physical barriers to protect themselves from dangers in their environment, such as invading parasites Studies of the interactions between viruses and the immune system are particularly instructive, because of the many viral countermeasures that can frustrate this system Cultivation of viruses Cell culture Types of cell culture To prepare a cell culture, tissues are disassociated into a single-cell suspension by mechanical disruption followed by treatment with proteolytic enzymes Primary cell cultures are prepared from animal tissues. They have a limited life span, usually no more than 5 to 20 cell divisions Commonly used primary cell cultures are derived from chicken or mouse embryos, monkey kidneys, or human tissues that are otherwise typically disposed of, such as embryonic amnion, kidney, foreskin, and respiratory epithelium Primary cell cultures are used for the propagation of viruses to be used as human vaccines to avoid contamination of the product with potently oncogenic DNA from continuous cell lines Some viral vaccines are now prepared in diploid cell strains, which consist of a homogenous population of a single cell type and can divide up to 100 times before dying Continuous cell lines consist of a single cell type that can be propagated indefinitely in culture. Propagation These immortal lines are usually derived from tumor (1) to cause an organism to tissue or by treating a primary cell culture or a reproduce or breed, especially diploid strain with a mutagenic chemical or an by natural means. (2) to oncogene transmit or spread from one Such cell lines often do not resemble the cell of generation to another origin; they are less differentiated (having lost the morphology and biochemical features that they possessed in the organ), are often abnormal in chromosome morphology and number (aneuploid), and can be tumorigenic (i.e., they produce tumors when inoculated into immunodeficient mice) In contrast to cells that grow in monolayers on plastic dishes, others can be maintained in suspension cultures, in which a spinning magnet continuously stars the cells Despite the wide utility of monolayer and suspension cell cultures in virology, they are not without limitations, including the finite life span of primary cell cultures and the abnormal phenotype of continuous cell lines, such as immortality These problems can be overcome by these of induced pluripotent stem cells (iPSCs), which are adult cells that have been reprogrammed genetically to an embryonic stemcell like state by the introduction of four genes (Oct4, Sox2, Kif4, and cMyc) Monolayer and suspension cell cultures do not reproduce the cell type diversity and architecture typical of tissues and organs One way to overcome this limitation is by the use of organotypic slice cultures, which can be produced from a variety of organs, including brain, liver, and kidney Another type of three-dimensional cell system is the multicellular, self-organizing organoid that approximates the organization, function, and genetics of specific organs Air-liquid interface cultures are used to model the respiratory tract, a major site of virus entry and infection. This organ presents a challenge because its structure differs from the pharynx to the alveoli Evidence of viral reproduction in cultured cells Some viruses kill the cells in which they reproduce, and they may eventually detach from the cell culture plate. As more cells are infected, the changes become visible and are called cytopathic effects Many types of cytopathic effect can be seen with a simple light or phase-contrast microscope at low power, without fixing or staining the cells These changes include the rounding up and detachment of cells from the culture dish, cell lysis, swelling of nuclei, and sometimes the formation of a group of fused cells called a syncytium High-power microscopy is required for the observation of other cytopathic effects, such as the development of intracellular masses of virus particles or unassembled viral components in the nucleus and/or cytoplasm (inclusion bodies), formation of crystalline arrays of viral proteins, membrane blebbing, duplication of membranes, and fragmentation of organelles The development of characteristic cytopathic effects in infected cell cultures is frequently monitored in diagnostic virology after isolation of viruses from specimens obtained from infected patients or animals Embryonated eggs Before the advent of cell culture, many viruses were propagated in embryonated chicken eggs. At 5 to 14 days after fertilization, a here is drilled in the Shell and virus is injected into the site appropriate for its replication Assay of viruses Measurement of infectious units One of the most important procedures in virology is measuring the virus titer, the concentration of infectious virus particles in a sample The response may be quantitative (as in assays for plaques, fluorescent foci, infectious centers, or abnormal growth and morphology) or all-or-none, in which the presence or absence of infection is measured Plaque assay The measurement of virus titers by plaque assay was first developed for bacteriophages by d’Herelle in 1917 and then modified for animal viruses by Renato Dulbecco in 1952 If the infected cells are damaged, the plaque can be distinguished from the surrounding monolayer. In time, the plaque becomes large enough to be seen with the naked eye For the majority of animal viruses, there is a linear relationship between the number of infectious particles and the plaque count Number of Plaques 24 One infectious particle is therefore sufficient to initiate infection, and the virus is said to infect cells with one-hit kinetics twonit For one-hit kinetics, the number of plaques is directly proportional to the first power of the concentration of the virus inoculated Two-hit kinetics is when two different types of virus particle must infect a cell to ensure replication For two-hit kinetics, the number of plaques is directly 0.25 0.50 o.is 1.00 Relative Virus Concentration proportional to the square of the concentration When one infectious virus particle initiates a plaque, the viral progeny within the plaque are biological clones, and virus stocks prepared from a single plaque are known as plaque purified Fluorescent-focus assay The fluorescent-focus assay, a modification of the plaque assay, can be done more rapidly and is useful in deterring the titers of viruses that do not form plaques The initial procedure is the same as in the plaque assay. However, after a period sufficient for adsorption and gene expression, galls are made permeable and incubated with an antibody raised against a viral protein Infectious-centers assay Another modification of the plaque assay, the infectious-centers assay, is used to determine the fraction of cells in a culture that are infected with A virus The number of plaques that form on the indicator cells is a measure of the number of cells infected in the original population. The fraction of infected cells can therefore be determined A typical use of the infectious-centers assay is to measure the proportion of virusproducing cells in persistently infected cultures Transformation assay The transformation assay provides A method for determining the titers of some retroviruses that do not form plaques The transformed cells form small piles, or foci, that can be distinguished easily from the rest of the monolayer. Infectivity is expressed in focus-forming units per milliliter End-point dilution assay The end-point dilution assay provided a means to determine virus titer before the development of the plaque assay In cell culture, infection may be determined by the development of cytopathic effect; in eggs or animals, infection may be gauged by virus titer, death, or disease When the end-point dilution assay is used to assess the virulence of a virus Or its capacity to cause disease, the result can be expressed in terms of 50% lethal dose per milliliter or 50% paralytic dose per milliliter, end points of death and paralysis, respectively Efficiency of plating Efficiency of plating is defined as the infectious virus titer divided by the total number of virus particles in the sample For many bacteriophages, the particle-to-PFU ratio approaches l, the lowest value that can be obtained. However, for animal viruses, this value can be much higher, ranging from 1 to 10,000 Although the linear nature of the dose-response curve indicates that a single particle is capable of initiating an infection (one-hit kinetics), the high particle-to-PFU ratio of many viruses demonstrates that not all virus particles are successful High values are sometimes caused by the presence of noninfectious particles with genomes that harbor lethal mutations or that have been damaged during growth or purification (defective particles) Measurement of virus particles Although the numbers of virus particles and infection units are often not equal, assays for particle number are frequently used to approximate the number of infectious particles present in a sample Biochemical or physical assays are usually more rapid and Easter to carry out than those for infectivity, which may be slow, cumbersome, or impossible onehit Electron microscopy With few exceptions, virus particles are too small to be observed directly by light microscopy. However, they can be seen readily in the electron microscopy A virus preparation is mixed with a known concentration of latex beads, and the numbers of virus particles and beads are then counted, allowing the concentration of the virus particles In the sample to be determined by comparison Hemagglutination Members of the Adenoviridae, Orthomyxoviridae, and Paramyxoviridae, among others, contain proteins that bind to erythrocytes (red blood cells); these viruses can link multiple cells, resulting in formation of a lattice. This property is called hemagglutination Centrifugation The use of centrifugal force to separate particles from solution according to size, shape, Or density has been a staple of virology The ultracentrifuge was invented by Theodore Svedberg in 1925, and it is the first initial of his last name that is used to describe the sedimentation coefficient of a particle as measured by centrifugation, e.g., the 16S ribosomal subunit Preformed gradients of sucrose are often used because particles that move with various velocities can be separated differentially in the increasing viscosity of the solution One application of sucrose gradients is the purification of virus particles. Another is polysome profiling, an analysis of the mRNAs associated with ribosomes Because mRNAs undergoing translation can be associated with different numbers of ribosomes, they can be separated on a sucrose gradient A more modern use of the polysome profile is to extract the RNA from each fraction and determine which mRNAs are being actively translated Another method for purifying viruses is by isopycnic centrifugation, which separates particles solely on the basis of their density Virus particles move down the tube until they reach the point which their density is the same as the gradient medium Measurement of viral enzyme activity Some animal virus particles contain nucleic acid polymerases, which can be defeated by mixing permeabilized particles with precursors and measuring their incorporation into nucleic acid If reverse transcriptase is present, a radioactive product will be produced by priming on the template. This product can be detected by precipitation or bound to a filter and quantified Because enzymatic activity is proportional to particle number, this assay allows rapid tracking of virus production in the course of an infection Serological methods The specificity of the antibody-antigen reaction has been used to design a variety of assays for viral proteins and anti-viral antibodies When a virus preparation is inoculated into an animal, an array of antibodies is produced. These antibodies can bind to virus particles, but not all of them can block infectivity (neutralize) Virus neutralization assays are usually conducted by mixing dilutions of antibodies with virus, incubating them, and assaying for remaining infectivity in cultured cells, eggs, or animals The end point is defined as the highest dilution of anybody that inhibits the development of cytopathic effect in cells or virus reproduction in eggs or animals Antibodies against viral proteins with hemagglutination activity can block the ability of virus to bind red blood cells In this assay, dilutions of antibodies are incubated with virus, and erythrocytes are added. After incubation, the titer is read as the highest dilution of antibody that inhibits hemagglutination Antibodies can be used to visualize viral or cellular proteins in infected cells or tissues Enzyme immunoassay. Detection of viral antigens or antiviral antibodies can be accomplished by solid-phase methods, in which an antiviral antibody or protein is adsorbed too plastic surface To detect antibodies to viruses, viral protein is first linked to the plastic support, and then the specimen is added Fluorescence microscopy Single virus particle tracking can be achieved by inserting the coding sequence for a fluorescent protein into the viral genome, often fused to the coding region of a viral protein However, when the virus particle emits a high fluorescent signal in a low background, it is possible to use a computational point tracking algorithm to locate the particle with greater precision than the diffraction limit of the light microscope The first advance was confocal microscopy, which utilizes a scanning point of light instead of full-sample illumination Super-resolution microscopy combines the advantages of fluorescent imaging (multicolor labeling and live-cell imaging) while breaking the resolution limit of light microscopy Different formats include single molecule localization microscopy, in which only a subset of fluorophores are turned on during each imaging cycle, thus allowing position determination with nanometer accuracy Structured illumination microscopy utilizes standing waves formed by interference in laser illumination to create an excitation field that allows optical sectioning at very high resolution Detection of viral nucleic acids Polymerase chain reaction. In this technique, specific oligonucleotides are used to amplify viral DNA sequences from infected cells or clinical specimens Each cycle consists of thermal denaturation, primer annealing, and extension, carried out by automated cycler machines. The result is exponential amplification of the target sequence that is located between the two DNA primers In contrast to conventional PCR, real-time PCR can be used to quantitate the amount of DNA or RNA in a sample In this procedure, also called quantitative PCR, the amplified DNA is detected as the reaction progresses, not after it is completed as in conventional PCR Viral reproduction: the burst concept A fundamental and important principle 1s that viruses are reproduced via the assembly of preformed components into particles: the parts are first made in cells and then assembled into the final product The yield from one cell is one viral generation; it was called the burst because the viruses that he studied literally burst from the infected cell Another important implication of the burst is that a cell has a finite capacity to produce virus A burst occurs for viruses that kill the cell after infection, namely, cytopathic viruses. However, some viruses do not kill their host cells, and virus particles are produced as long as the cell is alive The one-step growth cycle In their classic experiment, they added virus particles to a culture of rapidly growing E. coli. These particles adsorbed quickly to the cells. The infected culture was then diluted, preventing further adsorption of unbound particles This simple dilution step is the key to the experiment: it reduces further binding of virus to cells and effectively synchronizes the infection There is an initial lag period in which no infectious viruses can be detected. This lag period is followed by a rapid increase in the number of infectious particles, which then plateaus Particle infectivity is lost during this phase because the released genome is not infectious under the conditions of the plaque assay. Later, newly assembled infectious particles could be detected inside the cell that had not yet been released by cell lysis The results of these experiments defined two new terms in virology: the eclipse period, the phase in which infectivity is lost when virions are disassembled after penetrating cells, and the latent period, the time it takes to replicate and assemble new virus particles before lysis Synchronous infection, the key to the one-step growth cycle, is usually accomplished by infecting cells with a sufficient number of virus particles to ensure that most of the cells are infected rapidly