Chapter 13 Key Concepts

Discuss the ways that plant viruses can be transmitted to their hosts.

Can be transmitted through soil where infected plants have grown, and by the growers themselves. A small percentage are transmitted through contaminated seeds/tubers/pollen. Viruses can also spread through grafting of a diseased plant onto a healthy plant.

Define a bacteriophage, or 'phage'

Viruses that infect bacteria.

Describe the process by which prions accumulate in tissues.

Proteins are misfolded and become infectious. These infectious proteins are referred to as PrPSC. Their 'normal' counterparts are referred to as PrPC. Three main reasons they accumulate: Their shape makes them resistant to degradation by host cell proteases that normally destroy older proteins. Their shape causes the molecules to become insoluble so that they aggregate in cells. Interaction between PrPC and PrPSC appears to convert the former to the latter and also contributes to the prion's accumulation in tissues.

Describe the roles of proto-oncogenes and tumor suppressor genes in controlling cell growth, and how some viruses can circumvent this control, using HPV as an example

Proto-oncogenes stimulate cell growth and tumor suppressor genes inhibit cell growth. Mutations that increase proto-oncogenes or inhibit tumor suppressor genes are the most common cause of tumors. This results from multiple mutations at different sites, not just one mutation. Oncogenes are proto-oncogenes that have been changed to promote uncontrolled growth, and viruses can create them when they insert their genome into a host cell's chromosome. Inflammation from chronic, long term viral infection can also cause this conversion. Majority of virus induced tumors are not caused by oncogenes, but by mutations in host genes that regulate cell growth. Example: HPV-encoded proteins appear to interfere with the function of a tumor suppressor gene product.

Compare and contrast the mechanisms by which plant and animal viruses enter host cells.

When plant viruses infect a cell, they do not attach to specific receptors. Instead, they enter through wound sites in the cell wall.

List the two possible outcomes of phage replication.

Productive infection: new viral particles are produced Latent state: viral genome remains silent but is replicated along with the host cell genome.

Describe the chemical structure and hosts of prions and viroids.

Viroids and prions have an even simpler structure than viruses. Viroids: single-stranded RNA that forms a ring. Less than 1/10 size of virus. Infect only plants, where they cause serious diseases. Prions: composed only of protein and have no nucleic acid. Infect humans and have been linked to a number of slow, fatal human diseases including Creutzfeldt-Jakob disease and kuru, and well as animal diseases in sheep and goats, mad cow disease, etc. Always accumulate in neural tissue--referred to as transmissible spongiform encephalopathies.

Describe the general features of viral architecture, including the terms virion, capsid, capsomers, nucleocapsid, enveloped viruses, matrix protein, naked viruses, RNA, DNA, tail fibers, and spikes.

Viruses are small: smallest are 10nm in diameter and with very little nucleic acid--sometimes as few as 10 genes. A virion (viral particle) consists of nucleic acid surrounded by a protein coat, called a capsid, which protects the nucleic acid from enzymes and toxic chemicals in the environment. Capsids are made up of capsomers. The capsule + the nucleic acid = nucleocapsid. Enveloped viruses have a lipid bilayer outside of the capsid. Between the capsid and the envelope is the matrix protein. More susceptible to disinfectants. Non-enveloped viruses have no lipid bilayer. Most phages are non-enveloped. Viruses contain RNA or DNA, but never both. Genome may be linear, circular and single or double stranded. Phages have tail fibers that allow them to latch onto the host cell. Many animal viruses have protein structures called spikes that stick out from lipid bilayer or capsid that serve the same purpose.

Compare and contrast acute infections and the two types of persistent infections caused by animal viruses.

Acute infections: result in a burst of virions being released from infected host cells. Cells usually die, but the host may survive. Examples: influenza, mumps, and poliomyelitis. Symptoms result from tissue damage following cell death as well as damage caused by the animal's immune response. Persistent infections: Remain for years, sometimes without any symptoms. Two kinds, chronic and latent. Chronic: continuous low-level production of viral particles. Disease can be transmitted even in absence of symptoms. Example: hepatitis b virus. Latent: viral genome remains silent within a host cell, yet can reactivate to cause a productive infection. Not always integrated into host cell DNA--sometimes can replicate independently, like a plasmid. The silent viral genome is called a provirus, and it cannot be eliminated from the body. Examples include shingles, herpes simplex.

Define a cytopathic effect and inclusion body

Cytopathic effect: distinct morphological alterations in cells infected with a virus. Usually visible in cell cultures and can help researchers detect viruses. Host cells may change shape, detach from surface, lyse, etc. Inclusion body: caused by certain viruses that form a distinct region at the site of viral replication. The position of the inclusion body depends on the type of virus and are often characteristic to a specific virus.

Describe filamentous phage infections and name the model virus.

Filamentous phages (M13 as model) are single-stranded DNA phages that look like long fibers. They cause productive infections but the process does not kill the host cells--just makes them grow slower. Infections are initiated by attaching to host cell and inserting its single-stranded genome into the bacterial cell, where DNA polymerase synthesizes the complementary strand. This double stranded DNA is called the replicatory form (RF). One strand of the RF is used to make mRNA as well as multiple copies of the phage cell's single stranded genome. Phage particles are assembled in extrusion, which the virus uses to exit the cell. Phage coat protein molecules are inserted into the host cell's cytoplasmic membrane. Simultaneously, other phage proteins form pores that span the cytoplasmic and outer membrane of the host cell. Phage DNA is secreted through the pores, coat protein molecules cover the single stranded DNA to form the nucleocapsids.

Describe the three general shapes of viruses.

Icosahedral: appear spherical when viewed with an electron microscope, but their surface is actually 20 flat triangles arranged like a soccer ball. Helical:appear cylindrical when viewed with a microscope, but they are arranged in a helix. Complex: includes phages. These viruses have more complicated structures, many with a head and a tail.

Explain the importance of lysogeny in the disease causing ability of certain phages.

Lysogeny is advantageous to a phage in nutrient deficient environments, as lytic infections will be hindered by slow bacterial growth/metabolism. Can hide and wait until conditions are right. Describe the concept of immunity to superinfection in temperate phage infections. | Lysogens are protected against infection by the same phage because the repressor that maintains the prophage in the integrated state will also bind to the operator in incoming phage DNA. The operator is a regulatory protein that controls the expression of the genes that direct a lytic infection.

Describe four methods used to quantitate animal viruses.

Plaque assay -- clear zones surrounded by uninfected cells are counted to determine the viral titer, similar to a bacteriophage plaque assay but tissue cells serve as the host instead of bacteria. If there's a high enough concentration of viruses to be seen with an electron microscope, direct cell counts can be used. Quantal assay -- Several dilutions of the virus preparation are administered to a number of animals, cells, or chick embryos. The titer is the dilution at which 50% of the inoculated hosts are infected or killed. This can be reported as the ID50/ infective dose or the LD50/lethal dose. Red blood cells -- certain viruses cause red blood cells to hemagglutinate/clump. Visible at high concentrations of virus and is useful in determining the relative concentration of viral particles. The highest dilution showing maximum agglutination is the titer of the virus.

Describe antigenic drift in the context of viral RNA replication

Replicases (RNA-dependent RNA polymerases used to replicate viral RNA in host cell) lack proofreading ability and make more mistakes than DNA polymerase, generating mutations during replication. These mutations lead to antigenic variation and allow RNA viruses to adapt to selective pressures. One example of this variation is antigenic drift: occurs when mutations accumulate in genes encoding proteins that are recognized by the immune system. This leads to a person whose immune response protected him or her against influenza virus one year to possibly not be protected the next.

Define burst size

The number of phage particles released upon the lysing of a host cell by a virus.

Describe how animal cells are cultivated in the laboratory.

Cell culture/tissue cultures are used to cultivate most animal viruses. Animal cells are grown in a liquid medium in special screw-capped flasks and provided with the proper nutrients to divide repeatedly. They grow much more slowly than bacteria. Different types of animal cells can be grown (white blood cells, solid tissue cells, etc). Tumor cells are often used for their increased growth rate.

Describe how animal proteins synthesize viral proteins and replicate the genome for DNA viruses, RNA viruses, and reverse transcribing viruses.

DNA viruses: replicate in the nucleus of the host cell and use host cell components for DNA synthesis and gene expression. Often encode their own DNA polymerase. Double stranded DNA is transcribed to produce mRNA, which is then translated to make viral proteins. Double stranded DNA also serves as a template for DNA replication. Single stranded viruses will need to have a complementary strand sequenced first to create double-stranded DNA. RNA viruses: most are single stranded and replicate in the cytoplasm. Their replication ALWAYS requires a RNA-dependant RNA polymerase (replicase). (+) single stranded RNA functions as mRNA and can be simply translated to produce viral proteins, including proteins that synthesize more copies of the viral genome from the RNA strand. (-) single stranded RNA cannot be translated directly into proteins. First, it must be copied into a (+) RNA. Then it can be translated to make proteins or used as a template for synthesizing new (-) RNA strands. Double stranded RNA must carry its own replicase to create single stranded (+) RNA because cells are unable to translate double-stranded RNA. Reverse transcribing viruses: In reverse transcription, DNA is synthesized from an RNA template. Reverse transcribing viruses encode the enzyme reverse-transcriptase, an RNA-dependant DNA polymerase that synthesizes DNA from RNA. Retroviruses have (+) RNA and carry reverse transcriptase within the virion.After entering cell, the reverse transcriptase uses the RNA genome to make a strand of DNA. That strand is then synthesized to make double stranded DNA which integrates into host cell chromosome. Once integrated, the DNA may remain in a latent state (like temperate phage) or it may be transcribed into RNA that is used to produce new virions. Note: Hepatitis B virus uses reverse transcriptase, but in a very different way.

Compare and contrast generalized and specialized transduction.

Describe three mechanisms that confer bacterial resistance to phage infection.| 1. Preventing Phage Attachment: If a bacterium alters or covers a given receptor, that cell becomes resistant to any phage that requires the receptor for attachment. 2. Restriction-modification systems: protect bacteria from phage infection by quickly degrading incoming foreign DNA. 3. CRISPR system: Bacterial cells that survive some phage infections retain small segments of phage DNA, incorporating them into the bacterial genome. Describe how a plaque assay is used to detect and estimate the numbers of phage particles. | Plaque assays are used to determine the concentration of phage particles in samples such as sewage, seawater, or soil. In this type of assay, a melted, cooled soft agar medium is inoculated with a bacterial host and phage and poured over the plate. Lysis of cells leads to the formation of plaques--circular zones of clearing in the bacterial growth. Each plaque represents a single phage particle infecting a cell.

Describe how viruses are classified and named, including the key characteristics used in the current classification scheme

Key characteristics used to classify viruses are genome structure (type of nucleic acid and strandedness) and the hosts they infect (bacteria, archaea, animals, plants). Shape and disease symptoms are also considered. The names of viruses all end in the suffix viridae. Names follow no consistent pattern--can be geographical, the researcher name, or the disease it causes. Viruses are commonly referred to by only their species name or informal name, neither of which is capitalized. Ex: rhabdovirus.

Describe the characteristics and steps of lytic phage infections and name the model virus.

Lytic or virulent phages (T4 is model) exit the host at the end of the infection cycle by lysing the cell. These viruses cause productive infections, meaning they produce new viral particles in the host. Phage 'takes over'/hijacks bacterial cell. Infection cycle is a five-step process: attachment, genome entry, synthesis, assembly, and release. Entire cycle takes about 30 minutes. Attachment: Phage attach to a receptor on the host cell. Genome entry: Phage injects its genome into the cell by degrading a small part of the bacterial cell wall, using an enzyme (ex: T4 uses a lysozyme). The phage tail contracts, injecting DNA into the host cell. The capsid remains on the outside of the cell. Synthesis of phage proteins and genome: viral genome is transcribed and translated by infected. Phage encoded proteins are made in a specific time sequence to control the course of infection. Assembly/maturation: once multiple copies of the phage genome and the structural components of the phage are made, they assemble to produce new phage particles, either via self assembly or with the help of certain phage proteins. Release: late in infection, the phage encoded enzyme lysozyme is produced, digesting the host cell wall from within and causing the cell to lyse and release phage.

Describe the steps of a generalized infection cycle of animal viruses.

Similar to bacteria: a five step process. Attachment: basically the same as all virus-cell interactions. Animal viruses have spikes on their surfaces that bind to receptor proteins, usually glycoproteins on host cell plasma membrane. Often more than one spike must bind. Genome entry: method varies between enveloped and non-enveloped viruses. For all animal viruses, the entire virion enters the cell--not just the nucleic acid. Enter by 1 of 2 mechanisms--fusion with host membrane or endocytosis (non enveloped viruses can only use endocytosis). Nucleic acid ALWAYS separates from its protein coat before the start of replication. 1. FUsion: envelope fuses with cytoplasmic membrane of the host cell, releasing nucleocapsid directly into the host cell. 2. Endocytosis: viruses rely on receptor-mediated endocytosis. Viral particles bind to the receptors that normally trigger endocytosis, caused the cell to take them in. Synthesis of viral proteins and replication of the genome: Replication strategy can be divided into three general categories: DNA viruses, RNA viruses, and reverse transcribing viruses. Assembly and maturation: Involve bringing the newly formed viral nucleic acid with capsid proteins and packaging them to form the nucleocapsid. Spontaneous self-assembly that occurs when an appropriate amount of viral nucleic acid and capsid proteins have accumulated in the host cell. Site depends on the characteristics of the virus: 1. Non-enveloped: mature fully in the cytoplasm 2. Enveloped: Some maturation steps occur as the virion leaves the host cell. Some DNA viruses assemble nucleocapsids in the host cell. Release: the release of the virus also depends on its characteristics. Most enveloped viruses are released by budding, a process where the virus acquires its envelope from a cytoplasmic or organelle membrane. Doesn't necessarily destroy the cell. 1Non enveloped viruses are released when the host cell dies. Different than with bacteriophages: animal viruses use apoptosis, or programmed cell death. An animal's immune response can also kill the cell and trigger the release.

Describe temperate phage infections, and name the model virus. Also include descriptions of repressor proteins and phage induction.

Temperate phages (Lambda phage is model) have the option of either directing a lytic infection (productive) or incorporating their cell into the host cell genome in a lysogenic infection, creating a lysogen cell (latent infection). This process uses the enzyme integrase. The integrated DNA in a lysogen is called a prophage, and is passed on whenever the cell divides. Later, the prophage can begin the process that leads to a productive infection when it is excised from the host chromosome by a phage-encoded enzyme. A repressor protein prevents expression of the gene required for prophage incision, and is therefore essential for maintaining the lysogenic state in a temperate phage infection. Phage induction can occur in temperate phage infections if a lysogenic culture is treated with a DNA damaging agent like UV light. The SOS repair system is activated which activates a protein that destroys the repressor protein. This causes the prophage to be excised from the chromosome, allowing it to enter to lytic cycle and cause a productive infection.