2. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Section R Viruses R1 Introduction to Viruses R2 Bacteriophages R3 DNA Viruses R4 RNA Viruses

3. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences R1 Introduction to Viruses Viruses Virus genomes Replication strategies Virus Virulence

4. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Viruses-I Definition: Viruses are extremely small (20-300 nm) parasites, Incapable of replication, transcription or translation outside of a host cell. Viruses of bacteria are called bacteriophages. Virus particles Nucleic acid genome Protein coat / capsid Outer envelope Nonstructural proteins For transcription or replication soon after infection Bi-layer lipoprotein, derived from host cell membrane Nucleocapsid

5. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Capsids of Some Viruses (A) Tomato bushy stunt virus; (B) poliovirus ; (C) simian virus 40 (SV40); (D) satellite tobacco necrosis virus.

6. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences (A) Phage T4, (B) Potato virus X (C) Adenovirus (D) Influenza virus A B C D The Coats of Viruses

7. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Virus genomes Genome features and classifications: Virus genomes Types of nucleic acid Strand construction Defined relative to the mRNA sequence Size Replication enzyme source RNA or DNA Double-stranded or Single-stranded Positive, negative or ambi-sense Small (1kb ~) Large (~300kb) Viral enzymes and Cellular enzymes. Shapes Linear or Circular

8. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Schematic drawings of several types of viral genomes

9. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Replication strategies Viral replication strategies depend largely on the type of nucleic acid and size of genome: Large DNA viruses: e.g. herpesvirus, often encode their own polymerases; Small DNA viruses: e.g. SV40, may use the host cellular DNA polymerases; RNA viruses require: virus-encoded RNA-dependent polymerases for their replication; RNA retroviruses: use an RNA-dependent DNA polymerase (reverse transcriptase) to replicate via a DNA intermediate.

10. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Virus virulence Virulence is the capacity to cause disease. The Virulence mechanisms of viruses fall into six categories: 1. Damage to cellular metabolism (e.g. competition for enzymes and nucleotides, or growth factors essential for virus replication). 2. Damage to the cell membrane during transmission between cells (e.g. lysis by many bacteriophages or cell fusion by herpes viruses). 3. Disease signs helps the transmission between hosts (e.g. sneezing caused by common cold viruses). 4. Immune evasion of the host‘s immune system, for example by rapid mutation. 5. Harmful immune responses directed at viral antigens (e.g. hepatitis B virus) or cross-reactive responses leading to autoimmune disease. 6. Transformation of cells and tumor formation (e.g. SV40).

11. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences R2 Bacteriophages General properties Lytic and lysogenic inferction Bacteriophage M13 Bacteriophage Transposable phage

12. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences General properties Features: Phages are viruses which infect bacteria. Their genomes can be of RNA or DNA; Their size is from around 2.5 to 150 kb; They can have simple lytic life cycles or more complex life cycles involving integration in the host genome. Functions: Bacteriophages have played an important role in the research history of both virology and molecular biology; They have been studied intensively as model viruses.

13. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Lytic and lysogenic infection Lytic infection: (e.g. phage M13 infection) In lytic infection, the phages are released from the cell by lysis, but some phages (e.g. M13) release without lysis of the host cell. Their DNA replication in the cytosol independently; They replicate very quickly: infection, replication, assembly and release by lysis of the host cell may all occur within 20 minutes; Lysogenic infection: (e.g. phage Mu infection) In lysogenic infection, phages integrate their genomes into that of the host DNA, and may be stably inherited through several generations before returning to lytic infection. Another group of phages replicate while integrated into the host DNA via a combination. of replication and transposition Alternative infection: (e.g. bacteriophage ). Other phages alternate between a lylic phase of infection, and a lysogenic phase.

14. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences RF Bacteriophage M13 Genome features: Size is small (6.4 kb); Single-stranded; Circular genome; DNA; Positive-sense. The genome has 10 tightly packed genes and two terminators. g3p g6p g7p g8p g9p Host enzymes T O Infection: M13 particles attach specifically to E.coli sex pili (encoded by a plasmid called F factor), through a minor coat protein (g3p). Binding of g3p induces a structural change in the major capsid ( 衣 壳 ) protein. This causes the whole particle to shorten, injecting the viral DNA into the host cell.

15. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Bacteriophage M13 Replication: Host enzymes convert the viral ssDNA into dsDNA replicative form (RF). Normal dsDNA replication produce multiple copies of the RF. ssDNA making: If RF replication involves elongation of the 3'- OH group of a nick made in the (+) strand by a viral endo- nuclease (the product of gene 2), rather than RNA priming, the (+) ssDNAs are made by continuous replication of each RF. Assembly and release: The packaging precursors are transported to the cell membrane and there, the DNA binds to the major capsid protein. At the same time, new virions are extruded from the cell's surface without lysis. M13-infected cells continue to grow and divide (even if at a low rate), giving rise to generations of cells, each of which is also infected and continually releasing M13 phage.

16. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Bacteriophage M13 Why phage M13 is an ideal cloning vector? RF likes plasmid: The double-stranded, circular RF can be handled in the laboratory just like a plasmid; No strict limit for insert: The lack of any strict limit on genome and particle size means that the genome will tolerate the insertion of relatively large fragments of foreign DNA; ssDNA: The genome is single-stranded makes viral DNA an ideal template for DNA sequencing; Non-lytic nature of the cell: makes it very easy to isolate large amounts of pure viral DNA.

17. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Bacteriophage The phage binds to membrane of E. coli, and the viral dsDNA is injected by the tail into the cell. In the cell, the linear dsDNA rapidly bind their cos ends producing a nicked circular genome, then which is repaired by cellular DNA ligase. Construction of the virion An head Is icosahedral, containing the 48.5 kb linear dsDNA genome A tailLong and flexible Infection:

18. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Bacteriophage Within the infected cell, the phage may either undergo lytic or lysogenic life cycles. In the lysogenic life cycle, the phage DNA becomes integrated as a prophage in the host cell's genome. Lytic life Lysogenic life UV

19. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Bacteriophage Phage has 61 genes which are expressed at different times after infection, and they can be divided into three classes. 1. Immediate-early genes: [N  pL] and [pR  Cro] 2. Delayed-early genes: [att, int, gam, cIII, red, N  pL] and [pR  cro, cII, O, P, Q] [cI  pcI] makes phage into lysogenic life cycle 3. Late genes: produces the structural proteins necessary for the assembly of new virus particles and lysis of the cell. A W B C D E F Z UV G H M L K I J b2 att int xis gam red cIII N cI cro cII O P Q S R pL pR

20. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Cycling amplification 5‘ 3‘ 3’ Bacteriophage 5’

21. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Bacteriophage mu Definition: Phage mu is one of the transposable phages that have lytic and lysogenic life cycles. The name “Mu” stands for “mutation”, because it may cause insert mutation in host genome. transpoase Mu Host DNA New copy

22. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences R3 DNA Viruses DNA genomes: replication and transcription Small DNA viruses (SV40) Large DNA viruses ( Herpesviruses ) Herpes simplex virus-1

23. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences DNA genomes: replication and transcription DNA virus genomes: –Can be double-stranded or single-stranded. Replication and transcription : –Almost all eukaryotic DNA viruses replicate in the host cell's nucleus and make use of host cellular replication and transcription as well as translation. Life cycles: –Large dsDNA viruses: often have more complex life cycles, including temporal ( 时序 ) control of transcription, translation and replication of both the virus and the cell. –Small DNA viruses: their genomes may be much more dependent on the host cell for replication.

24. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Life cycle of DNA virus 病毒

25. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences SV40: is one of the smallest viruses. It is belong to papovavirus, and well studied, because it is a tumorigenic virus. Genome: SV40 has a 5 kb, double-stranded circular genome, which is supercoiled and packaged with cell-derived histones within a 45 nm, icosahedral virus particle. Overlap genes: In order to pack five genes into so small a genome, the genes are found on both strands and overlap each other. Small DNA viruses (SV40) 5’ 3’5’ BglI 3’ Ori EcoR I t T VP2 VP3 VP1 T(t) = tumor

26. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences R4 RNA Viruses RNA genomes: general features Classification of animal virus SARS coronaviruses Retroviruses Life cycle of retroviruses Oncogenic retroviruses Retroviral genome structure and expression

27. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences General features Viral RNA genomes may be: single-stranded or double-stranded, positive sense or negative sense, replications have a wide variety of mechanisms. All, however, rely on virus-encoded RNA-dependent pol, the inaccuracy of which in terms of making complementary RNA is much higher than that of DNA-dependent pol. Evolution: This feature affects the evolution of RNA viruses by increasing their ability to adapt, but limits their size. Guasi-species: Some RNA viruses mutate so rapidly that they exist as guasi-species, that is to say as populations of different genomes (often replicating through complementa-tion), within any individual host, and can only be molecularly defined in terms of a majority or average sequence.

28. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences + mRNA DNA viruses RNA viruses Class Genome IIIIIIIVVVI Classification of animal virus + _ + _ or _ + + _ _ _ + _ + _ +

29. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Life cycle of SARS virus 1. (+) ssRNA invades cell and translates its RNA polymerase; 2. With the RNA pol the (+)ssRNA transcribes its (-) ssRNA, which is the template; 3. (-)ssRNA transcribe several mRNA, which then are expressed as proteins; 4. (-)ssRNA replicates (+)ssRNAs with RNA dependant RNA pol; 5. The virus particles are assembled, and release from the cells. 6. All of the processes of the life cycle of SARS virus are taken place in the cytoplasm.

30. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Retroviruses 1. Retroviruses have a ssRNA genome. 2. Two copies of the sense ssRNA genome are within the viral particle. 3. When they infect a cell, the ssRNA is converted into a dsDNA copy by the RT (class VI). 4. Replication and transcription occur from this dsDNA intermediate, i.e. the pro-virus. 5. which is integrated into the host cell genome by a viral integrase enzyme. 6. Retroviruses vary in complexity. At one extreme there are HIVs.

31. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences 5’3’ gagpolenvv-onc +RNA 3’5’ gagpolenvv-onc - DNA 3’5’ gagpolenvv-onc - DNA 5’3’ gagpolenvv-onc +DNA 3’5’ gagpolenvv-onc 5’3’ gagpolenvv-onc Host DNA Host DNA RT Host DNA pol Virus Integase Host RNA pol. II U3 R U5 Oncogenic retroviruses LTR

32. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences Retroviral genome expression gag: proteins of the icosahedral capsid; pol: RT, RNase H, integrase and protease; env: the envelope proteins. v-onc: protein of regulation of cell division. 3’5’ gagpolenvv-onc 5’3’ gagpolenvv-onc U3 R U5 U3: Strong promoter; R: RT binding site; U5: RNA binding Site LTR

33. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences HIV Genome

34. Section R: Bacteriophages and VirusesYang Xu, College of Life Sciences That’s all for Section R