Chapter 8: Viruses and Virology

Retrovirus

A virus whose RNA genome is replicated via a DNA intermediate

Temperate virus

A virus whose genome can replicate along with that of its host without causing cell death, in a state called lysogeny (bacterial viruses) or latency (animal viruses)

Plaque

A zone of lysis or growth inhibition caused by virus infection of a lawn of sensitive host cells

8.7.1 Transcription and Translation

After T4 DNA enters the host cytoplasm, the host DNA and RNA synthesis ceases and transcription and translation of specific phage genes begins. T4 genome encodes 3 majors sets of proteins called early proteins, middle proteins and late proteins in order of their appearance in cell. T4 genome does not encode its own RNA polymerase; T4-specific proteins modify the specificity of the host RNA polymerase (using its early proteins: phage-encoded anti-sigma factor that binds to host RNA and shut down host transcription) so that it recognizes only phage promoters. Then later modify host RNA polymerase to recognize middle gene promoters, then late genes.

8.7 Replication of Bacteriophage T4

After a T4 virion penetrates a host cell, viral genes are expressed and regulated so as to redirect the host synthetic machinery to make viral nucleic acid and protein. Early viral genes encode viral genomes replication events; middle and late viral genes encode structural proteins and capsid assemble. Once T4 components have been synthesized, new virions are made, primarily by self-assembly, and the virions released after lysis of the host cell.

RNA replicase

An enzyme that can produce RNA form an RNA template

8.10.1 Classification of Animal Viruses

Animal viruses are classified by the structure of their genomes. Majority of human viral diseases are caused by RNA viruses (most are single-stranded, except reoviruses whose genomes consist of double stranded RNA). Other events beside lysis or lysogeny (in bacteriophage) are possible in animal virus infection: persistent infections (budding), and transformation (convert normal cells into tumor cells)

8.6.2 Restriction and Modification

Bacteria lack immune systems of animals but have other mechanisms: - CRISPR, an antiviral system, - restriction: destroy double-stranded viral DNA using restriction endonucleases (bacterial enzymes that cleave foreign DNA at specific sites. - modification of its DNA: protect its own DNA from restriction enzyme by methylation of nucleotides at sites where the enzyme cut. Some double-stranded DNA virus can overcome this mechanism by also modifying their own DNA so it's no longer subject to restriction enzymes.

8.6 The T4 Genome

Bacteriophage T4 contains a double-stranded DNA genome that is both circularly permuted and terminally redundant. T4 encodes its own DNA polymerase and several other replication proteins. Cell employs restriction enzymes in attempts to destroy viral and other foreign DNA, but T4 has chemically modified its DNA to make it resistant to such attack. Cells also modify their own DNA to protect it from their own restriction enzymes.

8.9.1 Bacteriophages with Single-Stranded Genomes

Bacteriophage contains plus sense RNA are converted into a double-stranded replication form before replication occurs; then single-stranded genome copies are derived & transcription of viral genes takes place. RNA replicase is needed for viral RNA genome replication: bacterial & animal cells do not possess enzymes that synthesize RNA from an RNA template. Overlapping genes: feature of many small DNA/RNA phage where genome contain overlap of open reading frames to allow more production of of single polypeptide from a given gene.

Penetration

Bacteriophages abandon the capsid outside the cell & only viral genome (and specific viral proteins, eg. virus RNA polymerase) reaches the cytoplasm. Bacteriophage T4 attaches using tail fibers. Retracts tail fibers and contact cell wall by tail pin. then T4 lyzosome forms small pore (tail lube) penetrating through the outer membrane & peptidoglycan layer and tail sheath contracts. T4 DNA enters the the cytoplasm.

8.4.1 Detecting and Counting Viruses: The Plaque Assay

Can quantify viral suspension to determine infectious virions number present per volume of fluid (a quantity called titer). Done by using plaque assay: bacteria grow & form turbid layer (lawn) during incubation, then with successful viral infection, cells are lysed & form plaque. Plaques are counted to calculate titer. Plating efficiency: in any given preparation, the # of plaque-forming units is always lower than actual counts of virial particles made microscopically.

Replicative form

A double-stranded DNA molecule that is an intermediate in the replication of viruses with single-stranded DNA genomes

Virus

A genetic element containing either RNA or DNA surrounded by a protein capsid and that replicates only inside host cells. They are obligate intracellular parasites. Virology is the study of virology.

Early protein

A protein synthesized soon after virus infection and before replication of the virus genome

Middle protein

A protein with either a structural or catalytic function synthesized after the early proteins in a virus infection

Late protein

A protein, typically a structural protein, synthesized late in virus infection

Lysogeny

A state in which the viral genome is replicated in step with the genome of the host. Lysogenic conversion: confer new genetic properties on the bacterial host cell.

Enveloped

In reference to a virus, having the lipoprotein membrane surrounding the virion. Can be tightly or loosely wrapped. May or may not have spikes (fibers)

8.1 What is a Virus?

A virus is an obligate intracellular parasite that requires a suitable host cell for replication. A virion is the extracellular form of a virus and contains either an RNA or a DNA genome inside a protein shell. Once inside the cell, either the virion or its nucleic acid redirects host metabolism to support virus replication. Viruses are classified by the characteristics of their genome and hosts. Bacteriophages infect bacterial cells.

Provirus

The genome of a temperate or latent animal virus when it is replicating in step with the host chromosome

Virion

The infectious virus particle; the viral genome surrounded by a protein coat and sometimes other layers

Prophage

The lysogenic form of a bacteriophage

8.9 An Overview of Bacterial Viruses

The most common viruses on Earth are the complex bacteriophages with heads and tails, such as T4 and lambda. The double-stranded DNA genomes of these phages encode hundreds of proteins. These viruses have been used as model systems not only for virus replication but also for molecular biology and genetics.

Titer

The number of infectious virus particle; the viral genome surrounded by a protein coat and sometimes other layers

8.11 The Virosphere and Viral Ecology

The number of viruses on Earth is greater than the number of cells by 10-fold. Most of the genetic diversity on Earth resides in virus genomes, most of which are still to be investigated. Viruses affect their host cells by either culling the host population or by carrying out horizontal gene transfer from one bacterial cell to another. In the oceans, both Bacteria and Archaea are likely to be infected with viruses.

Capsid

The protein shell that surrounds the genome of a virus particle

Bacteriophage

A virus that infects bacterial cells.

Virulent virus (lytic)

A virus that lyses or kills the host cell after infection

8.10.2 Consequences of Virus Infection in Animal Cells

- Virulent infection: results in lysis of the host cell, most common - Latent infection: viral genome does not replicate, host cell unharmed - Persistent infections: release of virion by budding process (enveloped viruses), slow, host cell may not be lysed. - Transformation: convert normal cell into tumor cell viruses that infect animals often infect only certain tissues: viral receptors differ in different tissues are often the macromolecules that is specific to each type of cell tissue (eg. intercellular communication, immune systems). Must eventually lose their outer coat to expose the viral genome. The entire virion of naked animal viruses and enveloped viruses may enter the cell via endocytosis. Then picked up the part of the cell's membrane and use it as part of the viral envelope.

8.7.2 Packaging the T4 Genome and Virion Assemble and Release

3 stages: - prohead assembly with only scaffolding proteins & head structural proteins. - viral packaging motor is assembled at the opening to the prohead and pumps double-stranded T4 DNA genome--> prohead expands when pressurized by entering DNA, & scaffolding proteins are discarded. - packaging motor is discarded and the capsid head is sealed. After head is sealed, then tail, tail fibers & other components of the virion are added, primarily by self-assembly. Phage very late enzymes combine to breach host cytoplasmic membrane & peptidoglycan layer to release virion --> cell breaks open by osmotic lysis.

Rolling circle replication

A DNA replication mechanism in which one strand is nicked and unrolled for use as a template to synthesize a complementary strand.

Lysogen

A bacterium containing a prophage.

Host cell

A cell inside which a virus replicates

8.6.1 Genome Replication and Circular Permutation

Circular permutation: some population of virions of a sing virus contain genomes with the same set of genes but arranged in different order. (A hallmark of T4). circularly permuted genomes are also terminally redundant: some DNA sequences are duplicated on both ends of DNA molecule as a result of the mechanism that generated them. T4 genome are replicated as a unit then several genomic units are recombined end to end to form aa long DNA molecule (concatemer). The concatemer is not cut at specific sequence when the genome is packaged into capsid; headful package: a linear segment of DNA long enough to fill a phage head are generated (common among bacteriophages)

8.10.3 Retroviruses and Reverse Transcriptase

Contain RNA genome, enveloped viruses. ("retro" means "backward" --> transfer RNA to DNA). Uses reverse transcriptase enzyme. 1st viruses shown to cause cancer. (HIV, a retrovirus causes AIDS) Also include integrase, and retroviral-specific protease. Contains 2 identical single-stranded RNAs of the plus sense. Genome contains gag (structural proteins), pro (reverse transcriptase and integrase), and env (envelope proteins). Replication: virion enters the host cell, enveloped is removed, reverse transcription begins in the nucleocapsid. A single strand of DNA is produced & then reverse transcriptase uses this as template to make complementary strand --> double-stranded DNA as final products. the latter is released from the nucleocapsid, enters the host nucleus along with the integrase protein which facilitates the incorporation of the retroviral DNA into the host genome --> become provirus. Remains in host genome indefinitely and proviral DNA can be transcribed by the host RNA polymerase to form copies of the retroviral RNA genome and mRNA. Nucleocapsids are assembled and contains 2 copies of the RNA genome, enveloped as they bud.

8.8.1 The Replication Cycle of a Temperate Phage

During lysogeny, the temperate virus genome can either: - integrated into the bacterial chromosome (lambda) - or exist in the cytoplasm as plasmid (P1). Viral DNA is called prophage in either case: gets replicated along with host cell as long as phage virulent pathways are repressed. Repressor protein: a phage-encoded protein that maintain the lysogenic state. If this protein is inactivated, the prophage can be induced into the lytic stage. Can be induced when cell is stressed to free itself before cell dies.

8.2 Structure of the Virion

In the virion of a naked virus, only nucleic acid and protein are present; the entire unit is called the nucleocapsid. Enveloped viruses have one or more lipoprotein layers surrounding the nucleocapsid. The nucleocapsid is arranged in a symmetric fashion, with the icosahedron being a common morphology. Although virus particles are metabolically inert, one or more key enzymes are present within the virion in some viruses. size: most are smaller than prokaryotic cell: ranging 0.02 to 0.3 um (20-300 nanometer, nm) (largest virus may be sized as smallest bacterial cells; smallest virus may be sized as ribosome)

I- The Nature of Viruses

Infection is when the virion or its genome has gained entry into a suitable host cell. Viruses are not cell; they posses a nucleic acid genome (RNA/DNA) that encodes for replication functions and an extracellular form (virion).

8.8.2 Bacteriophage Lambda

Infects. E. coli, double-stranded DNA, has head and tail. At the 5' end of each DNA strand of the linear lambda genome is a single-stranded region 12 nucleotides long: cohesive ends, complementary in base sequence --> form cos site and cyclize (circularize) the genome. Integration of lambda DNA into the host: lambda genome cyclizes at its cohesive ends. A site specific endonuclease creates staggered ends of phage and host DNA. Lambda genome integrates using lambda integrates (a phage-encoded enzyme that recognize the phage and bacterial gnome attachment site & facilitate integration) and gaps are closed by DNA ligase. If enters the lytic pathway, the concatemers are synthesized by rolling circle replication. Then the genome is cut at its cos site & resulting genomes packaged into lambda phage heads. Transduction: lambda can also package a few chromosomal genes from its lysed host in newly synthesized virions and then transfer them to another host.

8.5.1 Attachment

It is host specific. Virion protein on external surface that interact with specific host cell surface components called receptors. In absence of its specific receptor, virus cannot attach to cell & cannot infect. If receptor is altered (eg. by mutation), the host become resistant to virus infection. Viral receptors on host cell are its component such as proteins, carbohydrates, glycoproteins, lipids, or lipoproteins, or cell structures made from these macromolecules. Appendages such as flagella & pili are also common receptors for bacterial viruses. T4 recognize lipopolysaccharides (LPS) of E. coli.

8.2.3 Enveloped Viruses

Most enveloped viruses infect animal cells because their cytoplasmic membrane is directly exposed to the environment (no cell wall). Typically, the entire virion enters animal cell with the envelope by fusing with the host membrane. Then exit more easily as they draped host membrane material as they pass out of the host cell (taking host cytoplasmic membrane and proteins).

II- Bacteriophage Life Cycles

Much understanding of lytic virus replication comes from the study of bacteriophages infecting Escherichia coli.

8.9.2 Head-andTail Bacteriophages

Ones with double-stranded DNA genomes have been used as models for virus replication. T1-T7: 1st series of tailed phages used. (T refers to tail). T4 has the larger genome than other T phages, & along with phage lambda is the best studied of all bacteriophages.

8.8 Temperate Bacteriophages and Lysogeny

Some bacteriophages are temperate, meaning that they can initiate lytic events or integrate into the host genome as a prophage. This initiates a state called lysogeny in which the virus does not destroy the cell. A well-studied lysogenic virus of Escherichia coli is phage lambda; this phage uses a complex regulatory system to govern whether the lytic or lysogenic state is initiated following infection.

8.2.1 Virion Structure

Structures are diverse in size, shape, and chemical composition. But always have capsid, made of capsomeres (individual protein molecules). Information required for proper folding & assembly of viral proteins into capsomere and capsids (self-assembly, spontaneous) is in viral genome. some require assistance from host cell.

8.8.3 Lysis or Lysogeny?

The "genetic switch": Lysis or lysogeny depends on the levels of 2 key repressor proteins that can accumulate in the cell following infection: the lambda repressor (cI protein) and a second repressor called Cro. The 1st repressor accumulation will control the outcome of the infection. cI expression leads to accumulation and repression of Cro --> lysogenic pathway. Cro expression represses cII expression, a protein whose function is to activate the synthesis of cI --> lytic pathway.

8.5 Attachment and Entry of Bacteriophage T4

The attachment of a virion to a host cell is a highly specific process. Recognition proteins on the virus recognize specific receptors on the host cell. Sometimes the entire virion enters the host cell, whereas in other cases, as with most bacteriophages, only the viral genome enters.

Nucleocapsid

The complex of nucleic acid and proteins of a virus

Reverse transcriptase

The retroviral enzyme that can produce RNA from an RNA template

Capsomere

The subunit of a capsid; are rranged in precise and highly repetitive pattern around the nucleic acid. Small size of viral genomes restricts the number of distinct viral proteins that can be encoded --> few viruses have only a single kind of protein in the capsid.

Lytic pathway

The type of virus infection that leads to virus replication and destruction of the host cell

8.3 Overview of the Virus Life Cycle

The virus replication cycle can be divided into five major stages: - attachment (absorption), - penetration (uptake of the entire virion or injection of the nucleic acid only), - protein and nucleic acid synthesis, - assemble and packaging, and - release of new virions from cell. Growth response of virus replication is a one-step growth curve (named because virions # shows no increase during the replication cycle until cells burst & release their newly synthesized virions): Following absorption, infectious virions cannot be detected in the growth medium (eclipse phase - early enzymes, nucleic acid, & protein coats). Latent period includes eclipse and early maturation phases: viral nucleic acid replicates & protein synthesis occurs. During maturation period, virus nucleic acid & protein are assembled into mature virions & then released (either as cell lysis or by budding or excretion). Burst size: number of virions released per cell.

8.10 An Overview of Animal Viruses

There are animal viruses with all known modes of viral genome replication. Many animal viruses are enveloped, picking up portions of host membranes as they leave the cell. Viral infection of animal host cells can result in cell lysis, but latent or persistent infections are also common, an few animal viruses can cause cancer. Retroviruses like the AIDS virus are RNA viruses that employ the enzyme reverse transcriptase to replicate their RNA genome through a DNA intermediate. The DNA can integrate into the host chromosome where it can later be transcribed to yield viral mRNA and genomic RNA. 2 key differences between bacterial & animal viruses are: - the entire virion of animal viruses (rather than just the nucleic acid) enters the host cell - eukaryotic cells contain a nucleus, where many animal viruses replicate.

Overlapping genes

Two or more genes in which part or all of one gene is embedded in the other

Concatemer

Two or more linear nucleic acid molecules joined covalently in tandem

8.1.2 Viral Genomes

Viral genomes consist of either DNA or RNA, single or double stranded, and linear or circular. RNA may be plus sense (like mRNA) or minus sense (like complementary to mRNA). Genome size: (bacterial) 145 kbp (coding about 170 gene) to 1.25 Mbp (350) Can be classified based on the infected host type and genome structure:bacterial viruses, archaeal viruses, animal viruses, plant viruses, protozoan viruses, etc. Bacterial viruses are called bacteriophages (or phage). Been studied as model systems for molecular biology and genetics of virus replication. Animal viruses have been studied extensively as well because of their frequent medical importance.

8.1.1 Viral Structure and Activities

Virion consists of a protein shell - capsid. Most are naked (no further layers), many are enveloped (protein and lipid). Nucleocapsid: nucleic acid form plus capsid protein. Once inside host cell, virus may participate in virulent infection (replicate and lyse cell) or lysogenic infection (genome replicate and become part cell).

8.2.2 Virus Symmetry

Viruses are highly symmetric. 2 primary viral forms are recognized: helical (rod-shape symmetry), and icosahedral (spherical symmetry). Others have complex structure: icosahedral head & helical tail bacteriophage (eg. T4)

8.4 Culturing, Detecting, and Counting Viruses

Viruses can replicate only in their correct host cells. Bacterial viruses have proved useful as model systems because their host cells are easy to grow and manipulate in culture. Many animal viruses can be grown in cultured animal cells. Viruses can be quantified (titered) by a plaque assay. Plaques are clearings that develop on lawns of host cells, and in analogy to bacterial colonies, arise from the viral infection of a single cell.

8.2.4 Enzymes Inside Virions

Viruses do not carry out metabolic processes & thus are metabolically inert. But carry enzymes in the virion that have important roles in infection such as enzymes that resembles lysozyme (assists with transferring nucleic acid to host cytoplasm, or to lyse cell) RNA viruses carry their own RNA replicases (to replicate viral RNA genome & produce viral-specific mRNA) - important because cells cannot make RNA from RNA template. Retroviruses are unusual RNA animal viruses that replicate by making DNA from RNA template using RNA-dependent DNA polymerase (reverse transcriptase) - something cells cannot do.