Viruses - I - Supplementary Material PDF

**Viruses** I. **GENERAL INTRODUCTION** Viruses infect all cellular life forms: eukaryotes (vertebrate animals, invertebrate animals, plants, fungi) and prokaryotes (bacteria and archaea). The viruses that infect prokaryotes are often referred to as bacteriophages, or phages for short. Viruses ar...

**Viruses** I. **GENERAL INTRODUCTION** Viruses infect all cellular life forms: eukaryotes (vertebrate animals, invertebrate animals, plants, fungi) and prokaryotes (bacteria and archaea). The viruses that infect prokaryotes are often referred to as bacteriophages, or phages for short. Viruses are important agents of many human diseases, ranging from the trivial (e.g. common colds) to the lethal (e.g. rabies), and viruses also play roles in the development of several types of cancer. As well as causing individuals to suffer, virus diseases can also affect the well-being of societies. Smallpox had a great impact in the past and AIDS is having a great impact today. Viruses are quite diverse. Unlike all other biological entities, some viruses, like poliovirus, have RNA genomes and some, like herpesvirus, have DNA genomes. Further, some viruses (like influenza virus) have single-stranded genomes, while others (like smallpox) have double-stranded genomes. Their structures and replication strategies are equally diverse. Viruses, do, however, share a few features: First, they generally are quite small, with a diameter of less than 200 nanometers (nm). Second, they can replicate only within a host cell. Third, no known virus contains ribosomes, a necessary component of a cell\'s protein-making translational machinery. Viruses differ from cells in the way in which they multiply. A new cell is always formed directly from a pre-existing cell, but a new virion is never formed directly from a pre-existing virion. New virions are formed by a process of replication, which takes place inside a host cell and involves the synthesis of components followed by their assembly into virions. Viruses are therefore obligate parasites of cells, and are dependent on their hosts for most of their requirements, including building-blocks such as amino acids and nucleosides; protein-synthesizing machinery (ribosomes); energy, in the form of ATP. A virus modifies the intracellular environment of its host in order to enhance the efficiency of the replication process. Modifications might include production of new membranous structures, reduced expression of cell genes or enhancement of a cell process. **The discovery of viruses** Evidence for the existence of very small infectious agents was first provided in the late 19^th^ century by two scientists working independently: Martinus Beijerinck in Holland and Dimitri Ivanovski in Russia. They made extracts from diseased plants, which we now know were infected with tobacco mosaic virus, and passed the extracts through fine filters. The filtrates contained an agent that was able to infect new plants, but no bacteria could be cultured from the filtrates. The agent remained infective through several transfers to new plants, eliminating the possibility of a toxin. Beijerinck called the agent a 'virus' and the term has been in use ever since. At around the same time, Friedrich Löffler and Paul Frosch transmitted foot and mouth disease from animal to animal in inoculum that had been highly diluted. **Are viruses living or non-living?** There is an ongoing debate as to whether viruses are living or nonliving; the view taken depends on how life is defined. Viruses have genes and when they infect cells these genes are replicated, so in this sense viruses are living. They are, however, very different to cellular life forms. When viruses are outside their host cells they exist as virus particles (virions), which are inert, and could be described as nonliving, but viable bacterial spores are inert and are not considered to be nonliving. According to a stringent definition of life, they are nonliving. Not everyone, though, necessarily agrees with this conclusion. Perhaps viruses represent a different type of organism on the tree of life --- the capsid-encoding organisms. **Origin of viruses** The origins and evolution of many cellular organisms can be inferred from fossils, but there is very little fossil record of viruses! There are at least four theories which seek to explain the origin of viruses, which are not necessarily mutually exclusive: - **The Regressive Hypothesis - Remnant of cellular organisms** - viruses are degenerate life-forms which have lost many functions that other organisms possess & have only retained the genetic information essential to their parasitic way of life. Poxviruses may have arisen from prokaryotic cells by this mechanism. - **The Progressive Hypothesis - Escaped nucleic acids** - viruses are sub-cellular, functional assemblies of macromolecules which have escaped their origins inside cells. This is the most feasible theory for the evolution of most if not all viruses. - **The Symbiontic Hypothesis - Intracellular microorganisms -** It is assumed that the ancestors of mitochondria and chloroplast adopted parasitic or mutually beneficial modes of life in host cells, and that over time they became increasingly dependent on their hosts, losing the ability to perform various functions, and losing the genes that encode those functions. Perhaps a similar evolutionary process continued further, leading to greater degeneracy and loss of functions such as protein synthesis, until the intracellular intruder was no longer a cell or an organelle, but had become a virus. A virus that may have arisen in this way is the mimivirus. The 1.2 Mb genome of this virus encodes a wide variety of proteins, including enzymes for polysaccharide synthesis and proteins involved in translation; it also encodes six tRNAs. - **The Virus-First Hypothesis -- Cell-independent origin** - viruses evolved on a parallel course to cellular organisms from the self-replicating molecules believed to have existed in the primitive prebiotic \'RNA World\'. Once cells had evolved perhaps some were parasitized by some of these RNA molecules, which somehow acquired capsid protein genes. **Thief viruses** Some viruses (e.g. herpesviruses and poxviruses) have recently stoled several genes from the host genome. **Classification and nomenclature of viruses** By 1966 it was decided that some order had to be brought to the business of naming viruses and classifying them into groups, and the International Committee on Taxonomy of Viruses (ICTV) was formed. The ICTV lays down the rules for the nomenclature and classification of viruses, and it considers proposals for new taxonomic groups and virus names. For a long time virologists were reluctant to use the taxonomic groups such as family, subfamily, genus and species that have long been used to classify living organisms, but taxonomic groups of viruses have gradually been accepted and are now established. Some virus families have been grouped into orders, but higher taxonomic groupings, such as class and phylum, are not used. Now that technologies for sequencing virus genomes and for determining genome organization are readily available, the modern approach to virus classification is based on comparisons of genome sequences and organizations. The degree of similarity between virus genomes can be assessed using computer programs, and can be represented in diagrams known as phylogenetic trees because they show the likely phylogeny (evolutionary development) of the viruses. The **Baltimore classification**, developed by David Baltimore, groups viruses into families depending on their type of genome (DNA, RNA, single-stranded (ss), double-stranded (ds) etc.) and their method of replication. Classifying viruses according to their genome means that those in a given category will all behave in much the same way, which offers some indication of how to proceed with further research. In short: - I: dsDNA viruses (e.g. Adenoviruses, Herpesviruses, Poxviruses) - II: ssDNA viruses (+)sense DNA (e.g. Parvoviruses) - III: dsRNA viruses (e.g. Reoviruses) - IV: (+)ssRNA viruses (e.g. Picornaviruses, Togaviruses) - V: (-)ssRNA viruses e.g. Orthomyxoviruses, Rhabdoviruses) - VI: ssRNA viruses (+)sense RNA with DNA intermediate in life-cycle (e.g. Retroviruses) - VII: dsDNA-RT viruses (e.g. Hepadnaviruses) **Viruses and cancer** A virus that is able to cause cancer is known as an oncogenic virus. Evidence that a virus is oncogenic includes the regular presence in the tumor cells of virus DNA, which might be all or a part of the virus genome. In some types of tumor the virus DNA is integrated into a cell chromosome, while in other types it is present as multiple copies of covalently closed circular DNA. Several animal viruses are thought to cause some forms of cancer, including the Epstein-Barr virus, certain human papillomaviruses, the hepatitis B virus, and some retroviruses. Oncogenic viruses will be discussed in more detail in the lecture on Cancer. **Viruses, vaccines, antiviral drugs** This issue will be discussed in more detail in the lecture titled "Biotechnology and Recombinant Gene Technology". II. **STRUCTURE OF VIRUSES** Outside their host cells, viruses survive as virus particles, also known as virions. The virion is a gene delivery system; it contains the virus genome, and its functions are to protect the genome and to aid its entry into a host cell, where it can be replicated and packaged into new virions. The genome is packaged in a protein structure known as a capsid. Many viruses also have a lipid component, generally present at the surface of the virion forming an envelope, which also contains proteins with roles in aiding entry into host cells. A few viruses form protective protein occlusion bodies around their virions. **Virus genomes** A virion contains the genome of a virus in the form of one or more molecules of nucleic acid. For any one virus the genome is composed of either RNA or DNA. Each nucleic acid molecule is either single-stranded (ss) or double-stranded (ds), giving four categories of virus genome: dsDNA, ssDNA, dsRNA and ssRNA. The dsDNA viruses encode their genes in the same kind of molecule as animals, plants, bacteria and other cellular organisms, while the other three types of genome are unique to viruses. It interesting to note that most fungal viruses have dsRNA genomes, most plant viruses have ssRNA genomes and most prokaryotic viruses have dsDNA genomes. The reasons for these distributions presumably concern diverse origins of the viruses in these very different host types. A further categorization of a virus nucleic acid can be made on the basis of whether the molecule is linear, with free 5' and 3' ends, or circular, as a result of the strand(s) being covalently closed. Virus genomes span a large range of sizes. Porcine circovirus (ssDNA) and hepatitis delta virus (ssRNA) each have a genome of about 1.7 kilobases (kb), while at the other end of the scale there are viruses with dsDNA genomes comprised of over 1000 kilobase pairs (kbp). The largest virus genomes, such as that of the mimivirus, are larger than the smallest genomes of cellular organisms, such as some mycoplasmas. Most virus genomes consist of a single molecule of nucleic acid, but the genes of some viruses are encoded in two or more nucleic acid molecules. These segmented genomes are much more common amongst RNA viruses than DNA viruses. Examples of ssRNA viruses with segmented genomes are the influenza viruses, which package the segments in one virion, and brome mosaic virus, which packages the segments in separate virions. Most dsRNA viruses, such as members of the family *Reoviridae*, have segmented genomes. The possession of a segmented genome provides a virus with the possibility of new gene combinations, and hence a potential for more rapid evolution. For those viruses with the segments packaged in separate virions, however, there may be a price to pay for this advantage. A new cell becomes infected only if all genome segments enter the cell, which means that at least one of each of the virion categories must infect. The genomes of many viruses contain sequences that are repeated. These sequences include promoters, enhancers, origins of replication and other elements that are involved in the control of events in virus replication. Many linear virus genomes have repeat sequences at the ends (termini), in which case the sequences are known as terminal repeats. If the repeats are in the same orientation they are known as direct terminal repeats (DTRs), whereas if they are in the opposite orientation they are known as inverted terminal repeats (ITRs). **Capsids** In a virion the virus genome is enclosed in a protein coat, known as a capsid. For some viruses the genome and the capsid constitute the virion, while for other viruses there are additional components. There may be an envelope at the surface of the virion, in which case there may be protein (matrix) between the envelope and the capsid. Virus genomes removed from their capsids are more susceptible to inactivation, so a major function of the capsid is undoubtedly the protection of the genome. A second major function of many capsids is to recognize and attach to a host cell in which the virus can be replicated. For many viruses the capsid and the genome that it encloses constitute the virion (referred to as a nucleocapsid = nucleic acid plus capsid). For other viruses a lipid envelope, and sometimes another layer of protein, surrounds this structure. Capsids are constructed from many molecules of one or a few species of protein. The individual protein molecules are asymmetrical, but they are organized to form symmetrical structures. For the vast majority of viruses the capsid symmetry is either helical or icosahedral. **(1)** **Helical symmetry** The capsids of many ssRNA viruses have helical symmetry; the RNA is coiled in the form of a helix and many copies of the same protein species are arranged around the coil. For many ssRNA viruses, such as measles and influenza viruses, the helical nucleic acid coated with protein forms a nucleocapsid, which is inside an envelope. The virions of a few DNA viruses, such as the filamentous phages, also have helical symmetry. **(2)** **Icosahedral symmetry** An icosahedron has 20 equilateral triangles arranged around the face of a sphere. The capsids of some viruses, such as papillomaviruses, are clearly constructed from discrete structures. These structures are called capsomeres and each is built from several identical protein molecules. **(3) Conical and rod-shaped capsids** HIV-1 and baculoviruses have capsids that are conical and rod shaped, respectively. Inside each capsid is a copy of the virus genome coated in a highly basic protein. Both of these viruses have enveloped virions. **Virion membranes (envelopes)** Many viruses have a lipid membrane component. In most of these viruses the membrane is at the virion surface and is associated with one or more species of virus protein. This lipid--protein structure is known as an envelope and it encloses the nucleocapsid. The virions of most enveloped viruses, such as herpesviruses, are spherical or roughly spherical, but other shapes exist. Some viruses have a membrane located not at the virion surface, but within the capsid. Many animal viruses are enveloped, including all those with helical symmetry, e.g. influenza viruses, and a significant number of those with icosahedral symmetry, e.g. herpesviruses. Enveloped virions are much less common amongst the viruses that infect plants and they are extremely rare amongst the viruses that infect prokaryotes. Associated with the membranes of an enveloped virus are one or more species of protein. Most of these proteins are integral membrane proteins and most are glycosylated. Many of the glycoproteins in virion envelopes are present as multimers. Some surface glycoproteins of enveloped viruses perform the function of fusing the virion membrane to a cell membrane during the infection process. Most virion membranes are derived from host cell membranes that undergo modification before incorporation into virions. For example, the HIV-1 envelope is derived from the plasma membrane of the host cell, but the virus envelope contains more cholesterol and sphingomyelin, and less phosphatidylcholine and phosphatidylinositol. The envelope of herpesviruses is derived from the membrane of the transport vesicle of Golgi apparatus. Many enveloped viruses, including influenza viruses and retroviruses, have a layer of protein between the envelope and the nucleocapsid. This protein is often called a matrix protein. Viruses without envelopes are called naked viruses. III. **VIRAL INFECTION** The aim of a virus is to replicate itself, and in order to achieve this aim it needs to enter a host cell, make copies of itself and get the new copies out of the cell. In general the process of virus replication can be broken down into seven steps: 1\. Attachment of a virion to a cell 2\. Entry into the cell 3\. Transcription of virus genes into messenger RNA molecules (mRNAs) 4\. Translation of virus mRNAs into virus proteins 5\. Genome replication 6\. Assembly of proteins and genomes into virions 7\. Exit of the virions from the cell However, not all of the seven steps are relevant to all viruses, the steps do not always occur in the same order and some viruses have an additional step. For many viruses, transcription, translation, genome replication, virion assembly and exit can all be in progress at the same time. In this chapter, we will discuss only the replication of animal viruses. **Attachment of viruses to the cells** A virion attaches via one or more of its surface proteins to specific molecules on the surface of a host cell. These cellular molecules are known as receptors and the recognition of a receptor by a virion is highly specific, like a key fitting in its lock. It has been found that some viruses need to bind to a second type of cell surface molecule (a co-receptor) in order to infect a cell. Receptors and co-receptors are cell surface molecules, usually glycoproteins, with a wide range of functions that include acting as receptors for chemokines and growth factors or mediating cell-to-cell contact and adhesion. The virus attachment sites of naked viruses are on the capsid surface, sometimes within depressions (e.g. poliovirus) and sometimes on ridges (e.g. foot and mouth disease virus). The virus attachment sites of some naked viruses are on specialized structures, such as the fibers and knobs of adenoviruses and the spikes of rotaviruses, while the virus attachment sites of enveloped viruses are on the surface glycoproteins. endocytosis -- fusion; exocytosis - fusion **Entry of viruses into cells** After binding to receptors animal viruses must cross the plasma membrane to gain entry to the host cell. They may do this either at the cell surface or they may cross the membrane of an endosome, which is a vesicle formed by part of the plasma membrane pinching off into the cytoplasm. This process (endocytosis) is used by cells for a variety of functions, including nutrient uptake and defence against pathogens. There are a number of endocytic mechanisms, including clathrin-mediated endocytosis and caveolin-mediated endocytosis; most animal viruses hi-jack one or more of these mechanisms in order to gain access to their host cells. If a virion binds to a region of the plasma membrane coated with clathrin or caveolin these protein molecules force the membrane to bend around the virion. Many viruses, such as adenoviruses, are endocytosed at clathrin-coated regions of the plasma membrane. The virions end up in clathrin-coated endosomes, from which the clathrin is soon lost. Some viruses, such as simian virus 40, are endocytosed at caveolin-coated regions of the plasma membrane and the virions end up in caveolin-coated endosomes. An endosome may fuse with other vesicles such as lysosomes, which have a pH of 4.8--5.0. The pH may be further lowered by a process that pumps hydrogen ions across the membrane. This acidification of the environment of the virion is important for those enveloped viruses that need to carry out acid-triggered fusion of the envelope with the vesicle membrane. ***(1) Entry of naked viruses*** It is possible that some naked viruses deliver their genomes into their host cells through a pore formed in the plasma membrane, but for most naked viruses irreversible attachment of the virion to the cell surface leads to endocytosis. The plasma membrane 'flows' around the virion, more receptors bind, and eventually the virion is completely enclosed in membrane, which pinches off as an endosome. The endosome contents, however, are part of the external environment and the virus is not yet in the cytoplasm. The mechanisms by which virions, or their genomes, are released from endosomes are not fully understood. ***(2) Entry of enveloped viruses*** There are then two processes whereby infection of the cell may occur: either fusion of the virion envelope with the plasma membrane, or endocytosis followed by fusion of the virion envelope with the endosome membrane. Both processes involve the fusion of the virion envelope with a cell membrane, either the plasma membrane or a vesicle membrane. Lipid bilayers do not fuse spontaneously and each enveloped virus has a specialized glycoprotein responsible for membrane fusion. **Four virus uncoating strategies (A) Some enveloped viruses, such** as HIV (and herpesviruses), fuse with the host cell plasma membrane (in a receptor-mediated manner) to release their genome (blue) and capsid proteins (orange) into the cytosol. **(B)** Other enveloped viruses, such as influenza virus, first bind to cell-surface receptors, triggering receptor-mediated endocytosis. When the endosome acidifies, the virus envelope fuses with the endosomal membrane, releasing the viral genome (blue) and capsid proteins (orange) into the cytosol. **(C)** Poliovirus, a nonenveloped virus, binds to a receptor (green) on the host cell surface and then forms a pore (not shown) in the host cell membrane to extrude its RNA genome (blue). **(D)** Adenovirus, another nonenveloped virus, uses a more complicated strategy. It induces receptor-mediated endocytosis and then disrupts the endosomal membrane, releasing part of the capsid and its DNA genome into the cytosol. The trimmed-down virus eventually docks onto a nuclear pore and releases its DNA (red) directly into the nucleus. **Intracellular transport** Once in the cell the virus, or at least its genome, may have to be delivered to a particular location, such as the nucleus. For some viruses the destination is reached using one of the transport systems of the cell, such as the microtubules. Most RNA viruses of eukaryotes replicate in the cytoplasm; the majority encode all the enzymes for replication of their genomes and they have no requirement for the enzymes of the nucleus. The influenza viruses, however, are exceptions as they require the cell splicing machinery, so their genomes must be delivered into the nucleus. Retroviruses too are RNA viruses that replicate their genomes in the nucleus. They copy their genomes to DNA in the cytoplasm, then most retroviruses must wait in the cytoplasm until mitosis begins. During mitosis the nuclear envelope is temporarily broken down and the virus DNA (with associated proteins) is able to enter the nuclear compartment. These viruses therefore can replicate only in cells that are dividing. The DNA (with associated proteins) of a group of retroviruses, however, can be transported into an intact nucleus. This group (the lentiviruses, which includes HIV) can therefore replicate in non-dividing cells. Some DNA viruses, such as iridoviruses and poxviruses, replicate in the cytoplasm of eukaryotic cells, but most DNA viruses replicate in the nucleus. For these viruses (and the influenza viruses and the lentiviruses) the virus genome must be transported to the nuclear envelope and then across it. The structural proteins of some of these viruses have sequences that allow them to attach to microtubules. A number of viruses (including herpesviruses, adenoviruses, parvoviruses and retroviruses) exploit this transport system to take their nucleocapsid, or a structure derived from it, from the periphery of the cytoplasm to a location close to the nucleus. The nuclear pores act as gatekeepers, controlling the transport of materials in and out of the nucleus. Small nucleocapsids/virions, such as the parvovirus virion, can pass through the nuclear pore complex, but larger viruses must either shed some of their load to form slimmer structures or uncoat at a nuclear pore, i.e. the complete or partial removal of the capsid to release the virus genome. **Modified Central Dogma** In 1958 Francis Crick proposed a 'central dogma of molecular biology'. James Watson, Crick's collaborator in deducing the structure of DNA, made significant contributions to the formulation of the dogma, which stated that the flow of genetic information is always from DNA to RNA and then to protein, with genetic information transmitted from one generation to the next through copying from DNA to DNA. Increasing understanding of how viruses replicate their genomes necessitated some modifications to this dogma in 1970; many viruses have RNA genomes that are copied to RNA, and some viruses copy from RNA to DNA. **Transcription and translation** IRES are often used by viruses as a means of shutting down translation in the host cell so that the cell\'s translational machinery will operate to translate viral mRNA. The virus accomplishes this by cleaving eIF-4G so that it cannot interact with eIF-4E. Interaction between these two initiation factors are necessary for mRNA 5\'cap to 3\'poly-A-tail loop formation, which is usually a necessary event for initiation of translation. The virus may even use the eIF-4G to aid in initiation of IRES-mediated translation. Bicistronic and polycistronic mRNAs: Most eukaryotic cell and virus mRNAs have one ORF, but there are a number of virus mRNAs that have two or more ORFs. Some of these bicistronic and polycistronic mRNAs are functionally monocistronic, but some structurally bicistronic mRNAs are functionally bicistronic. A difference in the rate of translation of the two ORFs provides a mechanism for expressing two genes at different levels. In many bicistronic mRNAs the ORFs overlap; in others there is an ORF within an ORF. (1) One mechanism to read the second ORF involves leaky scanning; a 40S ribosomal subunit may overlook the ORF 1 start codon and initiate translation at the start of ORF 2. The ORFs for the two proteins are in different reading frames, so the proteins that they encode are unrelated. Of course it is essential that the sequence 'makes sense' in both reading frames! (2) Another mechanism for reading a second ORF in an mRNA involves ribosomal frameshifting; a ribosome shifts into a different reading frame towards the end of ORF 1. It therefore does not recognize the ORF 1 stop codon, but continues along the mRNA, reading ORF 2 to produce an elongated version of the ORF 1 protein. Frameshifting occurs when the ribosome moving along the RNA encounters a frameshift signal (a specific sequence) followed by a secondary structure, usually a pseudoknot. (3) IRES sequence between two genes allow a cap-independent translation initiation. **Transport of newly synthesized viral proteins** Virus molecules synthesized in the infected cell must also be transported to particular sites. Virus mRNAs are transported from the nucleus to the cytoplasm, and virus proteins may be transported to various locations, including the nucleus. Many proteins have a sequence of amino acids (a 'post code') that specifies their destination. Proteins destined to be incorporated into membranes have a signal sequence, which is a series of hydrophobic amino acid residues, either at the N terminus or internally. Protein synthesis begins on a free ribosome, but when the signal sequence has been synthesized it directs the polypeptide--ribosome complex to the endoplasmic reticulum, where protein synthesis continues. Many of the proteins synthesized in the rough endoplasmic reticulum are transported via vesicles to the Golgi complex, and most integral membrane proteins become glycosylated in these membrane compartments. From here the glycoproteins may be transported to other membranes, such as the plasma membrane or the nuclear envelope. Progeny virions may bud from these membranes. If the virus replicates in the nucleus then most, if not all, of the virus proteins must be transported into the nucleus. These proteins, like cell proteins that are transported into the nucleus, have a nuclear localization signal. RNAs are also transported within the cell; for example, mRNAs synthesized in the nucleus must be exported through nuclear pores to the cytoplasm. The RNAs are taken to their destinations by proteins. The Rev protein of HIV-1 has both a nuclear localization signal and a nuclear export signal. The nuclear localization signal ensures that Rev is transported into the nucleus, where it binds specifically to HIV-1 RNA. The nuclear export signal ensures that Rev and its RNA cargo are transported from the nucleus to the cytoplasm via a nuclear pore. **Virus genome replication** Generally, DNA viruses copy their genomes directly to DNA and RNA viruses copy their genomes directly to RNA. There are, however, some DNA viruses that replicate their genomes via an RNA intermediate and some RNA viruses that replicate their genomes via a DNA intermediate. Single-stranded genomes are designated as plus or minus depending on their relationship to the virus mRNA. Plus strand genomes have the same sequence as the mRNA (except that in DNA thymine replaces uracil), while minus-strand genomes have the sequence complementary to the mRNA. Single-stranded DNA is converted to dsDNA prior to copying. There are two classes of viruses with (+) RNA genomes. Class IV viruses copy their (+) RNA genomes via a (−) RNA intermediate, while Class VI viruses replicate via a DNA intermediate. The synthesis of DNA from an RNA template (reverse transcription) is also a characteristic of Class VII viruses. Class IV viruses copy their (+) RNA genomes via a (−) RNA intermediate, while Class VI viruses replicate via a DNA intermediate. The synthesis of DNA from an RNA template (reverse transcription) is also a characteristic of Class VII viruses. DNA synthesis takes place near a replication fork. One of the daughter strands is the leading strand and the other is the lagging strand, synthesized as Okazaki fragments, which become joined by a DNA ligase. After a dsDNA molecule has been copied each of the daughter molecules contains a strand of the original molecule. This mode of replication is known as semiconservative, in contrast to the conservative replication of some dsRNA viruses. Some DNA genomes are linear molecules, while some are covalently closed circles. Some of the linear molecules are circularized prior to DNA replication, hence many DNA genomes are replicated as circular molecules, for which there are two modes of replication, known as theta and sigma. These terms refer to the shapes depicted in diagrams of the replicating molecules, which resemble the Greek letters θ (theta) and σ (sigma). The sigma mode of replication is also known as a rolling circle mode. The genomes of some DNA viruses may be replicated by the theta mode of replication early in infection and the sigma mode late in infection. Replication of the DNA of some viruses, such as herpesviruses and phage T4, results in the formation of very large DNA molecules called concatemers. Each concatemer is composed of multiple copies of the virus genome and the concatemers of some viruses are branched. When DNA is packaged during the assembly of a virion an endonuclease cuts a genome length from a concatemer. **Assembly and exit of virions from cells** ***(1) Nucleocapsid assembly*** The assembly of virions and nucleocapsids of ssRNA viruses with [helical symmetry] involves coating the genome with multiple copies of a protein. The assembly of virions and nucleocapsids of many viruses with [icosahedral symmetry] involves the construction of an empty protein shell, known as a procapsid, or a prohead in the case of a tailed bacteriophage. The procapsid is filled with a copy of the virus genome; during or after this process it may undergo modification to form the mature capsid. The genome enters the procapsid through a channel located at a site that will become one of the vertices of the icosahedron. Any enzymes involved in packaging the genome are located at this site. In the context of packaging virus genomes during virion assembly the question 'How are virus genomes selected from all the cell and virus nucleic acids?' is an intriguing one. In a retrovirus-infected cell, for example, less than one per cent of the RNA is virus genome. In fact retroviruses do package a variety of cell RNAs, including tRNA, which plays a key role when the next host cell is infected. For the most part, however, cell nucleic acids are not packaged. For some viruses it has been shown that this is achieved through the recognition by a virus protein of a specific virus genome sequence, known as a packaging signal. In single-stranded genomes the packaging signal is within a region of secondary structure. Most viruses with single-stranded genomes package either the plus strand or the minus strand, so the packaging signal must be present only in the strand to be packaged. The dsDNA of large icosahedral viruses such as herpesviruses and the tailed phages is packed so tightly that the pressure within the capsid is about ten times greater than the pressure inside a champagne bottle. For some viruses infectious virions can be reassembled from the purified components (protein and nucleic acid), under appropriate conditions of pH and in the presence of certain ions. The viruses that can self-assemble in this way are those with a relatively simple virion composed of a nucleic acid and one or a small number of protein species. Viruses that can self-assemble in a test tube are assumed to undergo self-assembly in the infected cell. Examples are tobacco mosaic virus (helical symmetry) and the ssRNA phages (icosahedral symmetry). Self-assembly is economical because no additional genetic information is needed for the assembly process. The virions of more complex viruses, such as herpesviruses and the tailed phages, do not reassemble from their components in a test tube. The environment within the infected cell is required and the virions are constructed by a process of directed assembly. Directed assembly of icosahedral viruses may involve proteins that are temporarily present while the virion is under construction, but are not present in the mature virion. These proteins are known as scaffolding proteins. Once their job is completed they are removed from the procapsid. ***(2) Formation of virion membranes*** Enveloped virions acquire their membrane envelopes by one of two mechanisms; either they modify a host cell membrane and then nucleocapsids bud through it, or the virus directs synthesis of new membrane (it is very rare: e.g. in baculoviruses), which forms around the nucleocapsids. Most enveloped viruses acquire their envelopes by budding through a membrane of the host cell. For viruses with eukaryotic hosts this membrane is often the plasma membrane; the virions of most retroviruses and rhabdoviruses acquire their envelopes in this way. Regions of membrane through which budding will occur become modified by the insertion of one or more species of virus protein, the vast majority of which are glycoproteins. ***(3) Virion exit from the cell (egress)*** The bacteriophages are normally released from the infected cell when it bursts (lyses), a process that is initiated by the virus. Many phages produce enzymes (lysins, such as lysozymes) that break bonds in the peptidoglycan of the host bacterial cell walls. Animal viruses do not lyse their host cells; instead, progeny virions are released from the cells over a period of time. Obviously the concept of burst size is not relevant for this situation. We have already seen how virions that acquire an envelope from a cell surface membrane leave the cell by budding. Virions that acquire envelopes from internal membranes of the cell exit the cell in other ways. Some are transported to the cell surface in vesicles, which fuse with the plasma membrane to release the virions. Others, such as vaccinia virus, attach via motor proteins to microtubules and use this transport system to reach the cell surface where they are released. **Complicated strategies for viral envelope acquisition** **(A) Herpesvirus** nucleocapsids assemble in the nucleus and then bud through the inner nuclear membrane into the space between the inner and outer nuclear membranes, acquiring a membrane coat. The virus particles then apparently lose this coat when they fuse with the outer nuclear membrane to escape into the cytosol. Subsequently, the nucleocapsids bud into the Golgi apparatus and bud out again on the other side, acquiring two new membrane coats. The virus then buds from the cell with a single membrane when its outer membrane fuses with the plasma membrane. **(B) Poxvirus** assembles in "replication factories "within the cytosol, far away from the plasma membrane. The first structure that assembles contains two membranes, both acquired from the Golgi apparatus by a poorly defined wrapping mechanism. Some of these viral particles are then engulfed by the membranes of a second intracellular membrane-enclosed compartment; these viral particles have a total of four layers of membrane. After fusion at the plasma membrane, the virus escapes from the cell with three membrane layers. **Outcomes of infection and immune response** At the section "viral infection" we have looked at aspects of the virus replication cycle that culminate in the exit of infective progeny virions from an infected cell. When this is the outcome the infection is said to be productive (or lytic). Virions may be released when the host cell lyses, or the cell may survive releasing virions for a period, which may be short, as in the case of HIV infection, or it may be long, as in the case of hepatitis B virus infection. Some virus infections, however, are not productive, for a variety of reasons. An infection may become latent with the virus genome persisting, perhaps for the lifetime of the cell, and perhaps in the daughter cells if the cell divides. An infection may be abortive, in which case neither a latent infection nor a productive infection is established. One cause of an abortive infection is a virus with a mutated genome; the virus is said to be defective. It is unable to undergo a complete replication cycle, unless the cell is also infected with a virus that can provide the missing function(s). A virus that is able to provide functions for a defective virus is known as a helper virus. Some virus infections persist in their hosts for long periods, sometimes for life. In some cases persistent infections are productive, e.g. HIV; in other cases persistence may take the form of periods of latency alternating with periods of productive infection, e.g. many herpesviruses. Long-term infection with some viruses may lead to the development of cancer in the infected host. Over time the organisms that are hosts to viruses have evolved anti-viral defenses, but the viruses have not sat idly by. The relationship between a virus and its host usually involves an arms race and viruses have developed various countermeasures against host defenses. A major factor affecting the outcome of infection is the efficiency of the immune systems of the host. In animals there are both innate and adaptive immune systems. **Glossary** **Endemic**: prevalent to a particular region An **epidemic** (epi- meaning \"upon or above\" and demic- meaning \"people\"), occurs when new cases of a certain disease, in a given human population, and during a given period, substantially exceed what is expected based on recent experience. The disease is not required to be communicable. Examples of epidemics are cancer, heart disease and swine flu. An epidemic may be restricted to one locale, more general, or even global, in which case it is called a pandemic. **Mamavirus** is a very large virus, larger than many bacteria, and can itself be infected by other viruses (sputnic virophage\*). Although, first discovered in the early 1990s, it was merely put in cold storage for more than ten years, since it was originally assumed that it was a bacterium. In 2003, a team of scientists reported that it was indeed a virus, and a member of a supposed family of very large viruses, yet to be discovered. **Mimivirus**: The 1.2 Mb genome of this virus encodes a wide variety of proteins, including enzymes for polysaccharide synthesis and proteins involved in translation; it also encodes six tRNAs. **Natural reservoir**: refers to the long-term host of the pathogen of an infectious disease. It is often the case that hosts do not get the disease carried by the pathogen or it is carried as a subclinical infection and so asymptomatic and non-lethal. Once discovered, natural reservoirs elucidate the complete life cycle of infectious diseases, providing effective prevention and control. A **pandemic** (from freek Greek *pan* \"all\" + *demos* \"people\") is an epidemic of infectious disease that is spreading through human populations across a large region; for instance a continent, or even worldwide. A widespread endemic disease that is stable in terms of how many people are getting sick from it is not a pandemic. Further, flu pandemics exclude seasonal flu, unless the flu of the season is a pandemic. Throughout history there have been a number of pandemics, such as small pox and tuberculosis. More recent pandemics include the HIV pandemic and the 2009 flu pandemic. The **Sputnik virophage** is an icosahedral subviral agent (they are smaller than viruses but have some of their properties) that is 50 nanometres in size. Sputnik has been found to multiply inside of an Amoeba, although the conditions for this are rather unusual. The Subviral agent is unable to multiply itself inside of the host cell on its own, but when the host cell has been infected sputnik harnesses the viral proteins to rapidly produce new copies of itself. Sputnik has a circular double stranded DNA genome which contains genes able to infect all three domains of life: Eukarya, Archaea and Bacteria. It is associated with the mamavirus, which presumably is related to the *mimivirus*