The Structure & Complexity of Virus Genomes more varied than any of those seen in the entire bacterial, plant or a

1. The Structure & Complexity of Virus Genomes • more varied than any of those seen in the entire bacterial, plant or animal kingdoms

2. The Structure & Complexity of Virus Genomes • more varied than any of those seen in the entire bacterial, plant or animal kingdoms • may be single-stranded or double-stranded, & linear, circular or segmented

3. The Structure & Complexity of Virus Genomes • more varied than any of those seen in the entire bacterial, plant or animal kingdoms • may be single-stranded or double-stranded, & linear, circular or segmented • Single-stranded virus genomes may be: • positive (+)sense, i.e. of the same polarity (nucleotide sequence) as mRNA • negative (-)sense • ambisense - a mixture of the two.

4. The Structure & Complexity of Virus Genomes • more varied than any of those seen in the entire bacterial, plant or animal kingdoms • may be single-stranded or double-stranded, & linear, circular or segmented • Single-stranded virus genomes may be: • positive (+)sense, i.e. of the same polarity (nucleotide sequence) as mRNA • negative (-)sense • ambisense - a mixture of the two. • Virus genomes range in size from approximately 3,200 nucleotides (nt) (e.g. Hepadnaviruses) to approximately 800 kilobase pairs (kbp, Mimivirus):

6. Virus genomes may contain in either DNA or RNA. • Viruses are obligate intracellular parasites: • genome must contain information which can be recognized & decoded its host cell • The viral genetic code must match or at least be recognized by the host organism. • Control signals which direct the expression of virus genes must be appropriate to the host.

7. Molecular Genetics: • Questions about any virus genome will usually include the following: • Composition - DNA or RNA, single-stranded or double-stranded, linear or circular. • Size & number of segments. • Terminal structures. • Nucleotide sequence. • Coding capacity - open reading frames. • Regulatory signals - transcription enhancers, promoters & terminators. • Mechanisms for evading host defense systems

8. Direct analysis by electron microscopy, if calibrated with known standards, can be used to estimate the size of nucleic acid molecules. The most important single technique has been gel electrophoresis. It is most common to use agarose gels to separate large nucleic acid molecules (several megabases or kilobases) & polyacrylamide gel electrophoresis (PAGE) to separate smaller pieces (a few hundred bp down to a few nucleotides). The relative simplicity of virus genomes (compared with even the simplest cell) offers a major advantage - the ability to 'rescue' infectious virus from purified or cloned nucleic acids. Infection of cells caused by nucleic acid alone is referred to as transfection:

10. Virus genomes which consist of (+)sense RNA (i.e. the same polarity as mRNA) are infectious when the purified vRNA is applied to cells in the absence of any virus proteins. This is because (+)sense vRNA is essentially mRNA & the first event in a normally-infected cell is to translate the vRNA to make the virus proteins responsible for genome replication. In this case, direct introduction of RNA into cells merely circumvents the earliest stages of the replicative cycle. In most cases, virus genomes which are composed of double-stranded DNA are also infectious. The events which occur here are a little more complex, since the virus genome must first be transcribed by host polymerases to produce mRNA. Using these techniques, virus can be rescued from cloned genomes, including those which have been manipulated in vitro.

11. Types of Viral Genomes: • RNA viruses ds + sense ss - sense ss retroviruses • DNA viruses ds Gapped circle ss

12. RNA Virus Genomes • Positive-Strand RNA Viruses: • The ultimate size of single-stranded RNA genomes is limited by the fragility of RNA & the tendency of long strands to break. In addition, RNA genomes tend to have higher mutation rates than those composed of DNA because they are copied less accurately, which also tends to drive RNA viruses towards smaller genomes. • Single-stranded RNA genomes vary in size from those of Coronaviruses (the largest RNA viruses--cause respiratory infections in humans) at approximately 30kb long to those of bacteriophages such as MS2 & Q at about 3.5kb. • http://chagall.scripps.edu/viper/2ms2.html (MS2)

13. Purified (+)sense vRNA is directly infectious when applied to susceptible host cells in the absence of any virus proteins (although it is about one million times less infectious than virus particles). • There is an untranslated region (UTR) at the 5' end of the genome which does not encode any proteins & a shorter UTR at the 3' end. These regions are functionally important in virus replication & are thus conserved in spite of the pressure to reduce genome size. • Both ends of (+)stranded eukaryotic virus genomes are often modified, the 5' end by a small, covalently attached protein or a methylated nucleotide 'cap' structure & the 3' end by polyadenylation. These signals allow vRNA to be recognised by host cells & to function as mRNA.

14. e.g. Norwalk? New, uncharacterized Polio, Rhino

15. Negative-Strand RNA Viruses: • Viruses with negative-sense RNA genomes are a little more diverse than positive-stranded viruses. Possibly because of the difficulties of expression, they tend to have larger genomes encoding more genetic information. Because of this, segmentation is a common though not universal feature of such viruses.

16. Negative-sense RNA genomes are not infectious as purified RNA. Virus particles all contain a virus-specific polymerase. The first event when the virus genome enters the cell is that the (-)sense genome is copied by the polymerase, forming either (+)sense transcripts which are used directly as mRNA, or a double-stranded molecule known either as the replicative intermediate (RI) or replicative form (RF), which serves as a template for further rounds of mRNA synthesis.

17. Ambisense Genome Organization: • Some RNA viruses are not strictly 'negative-sense' but ambisense, since they are part (-)sense & part (+)sense: Hanta La Crosse Lymphocyte Choriomenengitis Virus

18. DNA Virus Genomes • 'Small' DNA Genomes: • Bacteriophages have been extensively studied as examples of DNA virus genomes. Although they vary considerably in size, in general terms they tend to be relatively small.

19. The structure of the filamentous bacteriophage M13 genome has been studied in great detail & modified extensively for use as a vector for DNA sequencing and cloning. The genome of this virus is: • circular • single-stranded DNA • approximately 7,200 nucleotides long • Unlike other virion structures, the filamentous M13 capsid can be lengthened by the addition of further protein subunits. The genome size of this virus can also be increased by the addition of extra sequences in the non-essential intergenic region without the penalty of becoming incapable of being packaged into the capsid. This is very unusual. In other viruses, the packaging constraints are much more rigid, e.g. in phage lambda, only DNA of between approximately 95% - 110% (approximately 46kbp - 54kbp) of the normal genome size (49kbp) can be packaged into the virus particle.

20. As further examples of small DNA genomes, consider those of two families of animal viruses, the parvoviruses & polyomaviruses: • Parvovirus genomes are: • linear • non-segmented • (+)sense • single-stranded DNA • about 5kb long

21. These are very small genomes, & even the replication-competent parvoviruses contain only two genes: • rep, which encodes proteins involved in transcription & • cap, which encodes the coat proteins. • The ends of the genome have palindromic sequences of about 115 nt, which form 'hairpins'. These structures are essential for the initiation of genome replication.

22. The genomes of polyomaviruses consist of double-stranded, circular DNA molecules, approximately 5kbp in size: Harmful to immunocompromized people.

23. The entire nucleotide sequence of all the viruses in the family is known & the architecture of the polyomavirus genome (i.e. number & arrangement of genes & function of the regulatory signals & systems) has been studied in great detail at a molecular level. Within the particles, the virus DNA is associated with four cellular histones. The genomic organization of these viruses has evolved to pack maximal information (6 genes) into minimal space (5kbp). This has been achieved by the use of both strands of the genome DNA & overlapping genes.

24. 'Large' DNA Genomes • There are a number of virus groups which have double-stranded DNA genomes of considerable size & complexity. In many respects, these viruses are genetically very similar to the host cells which they infect. Two examples of such viruses are the adenovirus & herpesvirus families:

25. Herpesvirus genomes: • The herpesviruses are a large family containing more than 100 different members, at least one for most animal species which have been examined to date, including seven human herpesviruses (including simplex, varicella and cytomegalovirus. • Herpesviruses have very large genomes composed of up to 230kbp linear, double-stranded DNA. The different members of the family are widely separated in terms of genomic sequence & proteins, but all are similar in terms of structure & genome organization. • Some herpesvirus genomes consist of two covalently joined sections, a unique long (UL) & a unique short (US) region, each bounded by inverted repeats.

28. Adenovirus genomes: • The genomes of adenoviruses consist of linear, double-stranded DNA of 30-38kbp. These viruses contain 30-40 genes. The terminal sequences of each DNA strand are inverted repeats of 100-140bp & therefore, the denatured single strands can form 'panhandle' structures. These structures are important in DNA replication.

30. Adenovirus infections are very common, most are asymptomatic. Most people have been infected with at least 1 type at age 15. Virus can be isolated from the majority of tonsils/adenoids surgically removed, indicating latent infections. It is not known how long the virus can persist in the body, or whether it is capable of reactivation after long periods, causing disease (it is hard to isolate this occult virus as it may be present in only a few cells). It is known that virus is reactivated during immunosuppression, e.g. in AIDS patients. Adenoviruses have been the workhorse of molbio: 1st identified oncogenic DNA virus 1st demonstration of RNA splicing 1st soluble DNA replication in vitro 1st mapping of ORFs 1st experiment showing that MHC expression is under viral control

31. Segmented & Multipartite Virus Genomes • Segmented virus genomes are those which are divided into two or more physically separate molecules of nucleic acid, all of which are then packaged into a single virus particle. • Multipartite genomes are those which are segmented & where each genome segment is packaged into a separate virus particle. These discrete particles are structurally similar & may contain the same component proteins, but often differ in size depending on the length of the genome segment packaged.

32. There are many examples of segmented virus genomes, including many human, animal & plant pathogens such as orthomyxoviruses (Flu), reoviruses (respiratory enteric orphan viruses: rotaviruses cause infantile diarrhea) & bunyaviruses (Hanta). There are rather fewer examples of multipartite viruses, all of which infect plants. These include: • bipartite viruses (which have two genome segments/virus particles) • tripartite viruses (three genome segments/virus particles)

33. Separating the genome segments into different particles removes the requirement for accurate sorting, but introduces a new problem in that all of the discrete virus particles must be taken up by a single host cell to establish a productive infection. This is perhaps the reason why multipartite viruses are only found in plants. Many of the sources of infection by plant viruses, such as inoculation by sap-sucking insects or after physical damage to tissues, results in a large input of infectious virus particles, providing the opportunity for infection of an initial cell by more than one particle.

34. Retroviruses Cross-sectional schematic diagram of HIV virion. Each virion expresses 72 glycoprotein projections composed of gp120 (orange) and gp41 (light blue). Gp41 is a transmembrane molecule that crosses the lipid bilayer of the envelope. Gp120 is noncovalently associated with gp41 and serves as the viral receptor for CD4 on host cells. The viral envelope also contains some host-cell membrane proteins such as class I and class II MHC molecules. Within the envelope is the viral core, or nucleocapsid, which includes a layer of a protein called p17 (green) and an inner layer protein called p24 (yellow). The HIV genome consists of two copies of ssRNA, which are associated with two molecules of reverse transcriptase p64 (light red) and nucleoid proteins p10, a protease (red), and p32, an integrase (dark blue).

36. SUMMARY: • There is more genetic diversity among viruses than in all the rest of the Animal, Plant & Bacterial kingdoms, all of whose genomes consist of d/s DNA. • The expression of virus genetic information is dependent on the structure of the genome of the particular virus concerned, but in every case, the genome must be recognized & expressed using the mechanisms of the host cell.