1. GENETIC VARIATION OF VIRUSES Part 2 Lecture 3 1

2. Types of Viral Mutation A. Point Mutations Point mutations occur when a single nucleotide is changed. Point mutants with specific characteristics are often selected in the laboratory and may belong to one of the following types: 1.Conditional-lethal mutants 2.Plaque-size mutants 3.Host-range mutants 4.Drug-resistant mutant 5.Enzyme-deficient mutants 6.Hot mutants 2

3. Types of Viral Mutation A. Point Mutations 3

4. 1. Conditional-lethal mutants: ◦ These can multiply under some conditions but not under the conditions that allow the multiplication of wild viruses. For example: a)Temperature-sensitive (t.s.):  Mutants do not replicate well at temperatures between 36 o C and 41 o C; therefore, they are attenuated strains in humans.  Their antigenic make-up is virtually identical to that of the wild strain, and consequently, these mutants are often used for immunization purposes. 4

5. Types of Viral Mutation A. Point Mutations b)Host-dependent mutants:  The mutation results in the replacement of an amino acid codon by a termination (stop) codon.  Such a mutation is called a “nonsense” mutation.  Viruses with these mutations will replicate only in host cells that have mutated tRNA that can recognize the termination codon as coding for a given amino acid.  Although the new amino acid may be different from the original one, in some cases, transcription results in a functional protein 5

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7. Types of Viral Mutation A. Point Mutations 2. Plaque-size mutants: ◦ Mutations here induce changes in the diameter of the zone of viral lysis seen in infected cell monolayers. ◦ The mutation usually affects the capsid protein, allowing easier adsorption to and penetration of permissive cells. ◦ Mutants with larger plaques grow faster in cell culture and are usually more virulent in vivo. 7

8. Types of Viral Mutation A. Point Mutations Plaques: ◦ Many viruses can be isolated as a result of their ability to form discrete visible zones, plaques (areas where cells are killed or altered by the virus infection) in the host cells Plaques formed by a phage in a bacterial culture

9. Types of Viral Mutation A. Point Mutations 9 Wild typeMutant 1Mutant 2Mutant 3

10. Types of Viral Mutation A. Point Mutations 3. Host-range mutants: ◦ These infect cell types or species not infected by the wild type strain. ◦ The basic mutation affects either:  proteins that mediate adsorption to host cells  or genes that modulate the interplay between cellular replication and viral expression. 4. Drug-resistant mutants: ◦ They are not susceptible to antiviral agents successfully used to treat infections caused by the wild type strain. ◦ The mutation usually affects an enzyme that is inhibited by the antiviral drug 10

11. Types of Viral Mutation A. Point Mutations 5. Enzyme-deficient mutants: ◦ Strains that lack an enzyme or have a mutant enzyme. ◦ The mutation may or may not be lethal, depending on whether it affects an enzyme essential for multiplication. 6. Hot mutants: ◦ These grow well at 41 o C and are extremely virulent. 11

12. B. Deletion Mutations Deletion mutants occur when a whole segment of the viral genome is lost. Defective virus particles are examples of deletion mutants. 12

13. INTERACTIONS BETWEEN VIRUSES 13

14. Interactions Between Viral Genomes This type of interaction occurs in multiply infected cells i.e., when many virions gain access to the same cell more or less at the same time. The co-infecting viruses are usually identical or closely related. Because of this multiplicity of infection, the genomes of the infecting particles may interact in a variety of ways: 1.Recombination 2.Reassortment 3.Complementation 14

15. 1. Recombination Recombination is the physical interaction of virus genomes during superinfectrion (i.e., multiple infection) resulting in gene combinations not present in the original infecting viruses. 15

16. 1. Recombination DNA viruses: ◦ Recombination classically involves double- stranded DNA viruses. ◦ Recombination is insignificant when the recombination involves two normal genomes, but recombination may result in the creation of a complete wild-type genome from two mutant genomes, providing that the defect is in different genes (Figure 1) 16

17. 17 Figure 1. Schematic diagram of recombination between two double-stranded DNA molecules. A,B,C and D designate different genes (only recombinants are shown).

18. 1. Recombination RNA viruses: ◦ When copying (+) strands into (-) strands to be used as templates for replication, the RNA polymerase may “jump” from one (+) strand to another, producing a hybrid (-) RNA template (Figure 2). ◦ A similar mechanism may contribute to the genetic diversity of HIV. ◦ The genome of HIV is made up of two strands of (+) RNA. 18

19. 1. Recombination In the process of transcribing DNA from RNA, the reverse transcriptase often “jumps” from one RNA strand to the other. Such jumps are inconsequent if the strands are identical; however, if two different mutant strains of HIV manage to infect the same cell, the progeny virions will contain non-identical RNA strands. Under these conditions, the polymerase “jumps” may cause the emergence of recombinant genomes. 19

20. 20 Figure 2. Production of hybrid (recombinant) RNA molecule by RNA polymerase jump (A, B, C and D represent different genes). RNA polymerase RNA Replicase Progeny genome

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22. 2. Reassortment This mechanism applies only to viruses with segmented genomes such as, influenza virus. For example, when a host cell is co-infected with two different strains of influenza virus, that have mutated in different segments of their genomes, a wild-type virus may be formed by reconstituting a normal set of genome segments, using segments from both infecting mutants (Figure 3). Reassortment is believed to be the major cause of antigenic shift in influenza virus 22

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24. 24 Figure 3. Reassortment between two influenza viruses.

25. 3. Complementation: This is a process that allows defective viruses to replicate and spread. a)If a cell is co-infected by two viruses for example, one that lacks the gene that codes for an essential polymerase and another that lacks the gene that codes for the glycoprotein spikes. ◦ The defective viruses may assist each other in producing infective progeny by coding for the structures necessary to assemble non-defective particles between the two of them. ◦ However the packaged genomes remain defective. 25

26. 3. Complementation: b)Alternatively, if a defective virus (for example, one lacking one or several genes essential for its full replication) co-infects a cell with a nondefective virus of very similar structure, the defective genome may be packaged into a viral particle whose structural components are coded by the non-defective virus. ◦ This is the way in which defective tumor-causing retroviruses propagate and spread in nature 26

27. 3. Complementation: c) Phenotypic mixing and pseudotype formation are special examples of complementation that may take place when two closely related viruses (e.g., poliovirus I and II) coinfect a cell. ◦ Phenotypic mixing:  In the process of assembly of progeny virions, hybrid capsids constituted by subunits coded by the two different viruses may be generated. ◦ Pseudotype formation:  At the time of encapsulating progeny nucleic acid, viral genomes of one virus may be encapsulated in the capsids of the second virus, or vice versa.  The genetic process resulting in pseudotype formation is known as phenotypic masking. 27

28. 28 IT INVOLVES NO ALTERATION IN GENETIC MATERIAL, the progeny of such virions will be determined by which parental genome is packaged and not by the nature of the envelope Pseudotype formation

29. 3. Complementation:  Phenotypic masking is reversed when a single pseudotype infects a host cell.  That is, if the particle carries a type II genome inside a type I capsid, the progeny will necessarily carry type II capsids and genomes, because all progeny components will be coded by a type II genome (Figure 4). 29

30. 30 Figure 4. Pseudotype formation. Notice that pseudotypes return to their normal states after single infection of a host cell. Red and blue colors represent different genomes and different capsid

31. Defective interfering (DI) particles Viral particles that contain defective genomes can not multiply on their own. Their successful replication depends on simultaneous infection with infectious “helper” virus. However, the defective particles often interfere with the replication of the “helper” virus, hence the designation “defective interfering particles”. High multiplicity of infection at the single-cell level favors the formation of DI particles. 31

32. Unusual Infectious Agents Genomes of nondefective viruses range in size from 2,400,000 bp of double-stranded DNA (Pandoravirus salinus) to 1,759 bp of single-stranded DNA (porcine circovirus). Are even smaller viral genomes possible? Viroids, satellites, and prions provide answers to these questions. The adjective “subviral” was coined, in part, because these agents did not fit into the standard taxonomy schemes for viruses. The Subviral RNA Database lists 2,923 nucleotide sequences for viroids and satellites. No prion sequences will be found in this database, because these infectious agents are, remarkably, devoid of nucleic acid. 32

33. Viroids A small circular RNA molecules, rod-like secondary structure, there is no capsid or envelope. They are associated with certain plant diseases and transmitted by seeds or pollens. Viroid RNA does not code for any known proteins. Some viroids are ribozymes, having RNA enzyme properties which allow self-cleavage and ligation of unit-size genomes from larger replication intermediates. It has been proposed that viroids are “escaped introns” 33

34. Satellite (virusoids) They are viroid-like molecules but somewhat larger. They depend on other virus replication for multiplication” hence-satellite”. They are packaged into virus capsid as passenger. When a satellite encodes the coat protein in which its nucleic acid is encapsidated, it is referred to as a satellite virus. 34

35. Quasispecies They are closely-related”but nonidentical” mutant and recombinant self-replicating RNA viral genomes. Quasispecies are subjected to continuous genetic variation, competition and selection. The structure and dynamics of replicating RNA enable virus populations to persist in their host and cause disease. 35