Moleculer biology of viruses_033456_110725.pptx

It includes everything about how Viruses replicates inside their hosts.

1. Molecular Biology Genomes and genetics

2. The replication of viral DNA The basic mechanism whereby a new strand of DNA is synthesized in a cell is the same, regardless of whether the DNA is of cellular or viral origin.

3. Locations of virus genome replication in eukaryotic cells when viruses infect eukaryotic cells the genomes of some are delivered to the cytoplasm and some are conveyed to the nucleus. The destination of a virus genome, and hence the location in which it is replicated, varies with the type of genome

4. Locations of virus genome replication in eukaryotic cells The genomes of most DNA viruses are replicated in the nucleus, but those of some dsDNA viruses are replicated in the cytoplasm. The genomes of most RNA viruses are replicated in the cytoplasm, but those of the minus-strand RNA viruses with segmented genomes are replicated in the nucleus. The retroviruses and pararetroviruses are special cases: each replicates RNA to DNA in the cytoplasm and DNA to RNA in the nucleus.

5. Initiation of genome replication Each virus genome has a specific sequence where nucleic acid replication is initiated. This sequence is recognized by the proteins that initiate replication Nucleic acid replication requires priming, which is the first reaction of a nucleotide with an –OH group on a molecule at the initiation site. Replication of the genomes of many RNA viruses (including rotaviruses, and rhabdoviruses ) initiates when the first nucleotide of the new strand base pairs with nucleotide in the viral RNA. The initial nucleotide effectively acts as a primer for RNA replication when its 3 –OH group becomes linked to the second nucleotide.

6. Initiation of genome replication Some ssDNA viruses, such as parvoviruses, use self-priming. At the 3 end of the DNA there are regions with complementary sequences that can base pair The –OH group of the nucleotide at the 3 end forms a linkage with the first nucleotide, then DNA synthesis proceeds by a rather complex process to ensure that the whole genome is copied. In order to initiate the replication of many DNA genomes, and some RNA genomes, a molecule of RNA or protein is required to act as a primer.

7. RNA and protein primers Synthesis of cell DNA commences after a region of the double helix has been unwound by a helicase and after a primase has synthesized short sequences of RNA complementary to regions of the DNA. These RNAs act as primers; one is required for the leading strand, while multiple primers must be synthesized for the Okazaki fragments of the lagging strand. The first nucleotide of a new sequence of DNA is linked to the 3 –OH group of the primer RNA.

8. RNA and protein primers Some DNA viruses also use RNA primers during the replication of their genomes. Some viruses, such as polyomaviruses , use the cell primase to synthesize their RNA primers, while others, such as herpesviruses and phage T7, encode their own primases .

9. RNA and protein primers For some viruses the primer for initiation of nucleic acid replication is the –OH group on a serine or tyrosine residue in a protein. DNA viruses that use protein primers include some animal viruses (e.g. adenoviruses) and some phages (e.g. tectiviruses ). RNA viruses that use protein primers include some animal viruses (e.g. picornaviruses ) and some plant viruses (e.g. luteoviruses ).

10. RNA and protein primers Hepadnaviruses are DNA viruses that use a protein primer to initiate (−) DNA synthesis and an RNA primer to initiate (+) DNA synthesis. Protein primers (and the RNA primers of hepadnaviruses ) are not removed once their role is performed and they are found linked to the 5 ends of the genomes in virions

11. Polymerases The key enzymes involved in virus genome replication are DNA polymerases and RNA polymerases. Many viruses encode their own polymerase, but some use a host cell enzyme A DNA virus requires a DNA-dependent DNA polymerase. Amongst the DNA viruses that replicate in the nuclei of eukaryotic cells, viruses with small genomes (e.g. papillomaviruses) use the cell enzyme, while viruses with large genomes (e.g. herpesviruses ) encode their own enzyme. Those DNA viruses that replicate in the cytoplasm must encode their own enzyme.

12. Polymerases The enzyme that replicates the genome of an RNA virus is often referred to as a replicase ; for many RNA viruses this is the same enzyme as that used for transcription The retroviruses and the pararetroviruses encode reverse transcriptases to transcribe from RNA to DNA, and use the host cell RNA polymerase II to transcribe from DNA to RNA. Many viral polymerases form complexes with other viral and/or cell proteins to produce the active enzyme. Some of these additional proteins are processivity factors, for example an Escherichia coli thioredoxin molecule functions as a processivity factor for the DNA polymerase of phage T7.

13. DNA replication The viruses of Class I ( dsDNA ) and Class II ( ssDNA ) replicate their genomes via dsDNA . The ssDNA viruses first synthesize a complementary strand to convert the genome into dsDNA . Each viral DNA has at least one specific sequence ( ori ; replication origin) where replication is initiated. The proteins that initiate DNA replication bind to this site, and amongst these proteins are a helicase (unwinds the double helix at that site); a ssDNA binding protein (keeps the two strands apart); a DNA polymerase.

14. DNA replication Viral dsDNA is generally replicated by a process similar to that used by cells to copy their genomes. Fewer proteins are involved in bacterial systems than in eukaryotic systems; for example, the helicase– primase of phage T7 is a single protein molecule, while that of herpes simplex virus is a complex of three protein species

15. DNA replication 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

16. DNA replication 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 1. Theta and 2. sigma These terms refer to the shapes depicted in diagrams of the replicating molecules, which resemble the Greek letters θ ( theta) and σ ( sigma)

17. DNA replication 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 .

18. Double-stranded RNA replication Double-stranded RNA, like dsDNA , must be unwound with a helicase in order for the molecule to be replicated. Some dsRNA viruses, e.g. Pseudomonas phage ϕ6 ( ϕ = Greek letter phi), replicate their genomes by a semi-conservative mechanism, similar to dsDNAreplication ; each of the double-stranded progeny molecules is made up of a parental strand and a daughter strand. Other dsRNA viruses, including members of the family Reoviridae replicate by a mechanism designated as conservative because the double-stranded molecule of the infecting genome is conserved

19. Single-stranded RNA replication The ssRNA genomes of viruses in Classes IV and V are replicated by synthesis of complementary strands of RNA that are then used as templates for synthesis of new copies of the genome. The synthesis of each RNA molecule requires the recruitment of an RNA-dependent RNA polymerase to the 3 end of the template, therefore both plus- and minus-strand RNA must have a binding site for the enzyme at the 3 end. An interesting point to note here is that all class IV viruses of eukaryotes replicate their RNA in association with cytoplasmic membranes. For many groups of viruses, including picornaviruses

20. Single-stranded RNA replication these membranes are derived mainly from the endoplasmic reticulum, but other membranous structures are used, including endosomes (by togaviruses ) and chloroplasts (by tombusviruses ). Viral proteins, including the RNA polymerases, are bound to the membranes. During the replication of ssRNA both (+) and (−) strands of RNA accumulate in the infected cell, but not in equal amounts. Plus-strand RNA viruses accumulate an excess of (+) RNA over (−) RNA, and for minusstrand RNA viruses the reverse is true.

21. Reverse transcription Some RNA viruses replicate their genomes via a DNA intermediate, while some DNA viruses replicate their genomes via an RNA intermediate. Both of these modes of genome replication involve reverse transcription, which has two major steps: synthesis of (−) DNA from a (+) RNA template followed by synthesis of a second DNA strand. Both steps are catalysed by a reverse transcriptase that is encoded by the virus. Reverse transcription takes place within a viral structure in the cytoplasm of the infected cell. No viruses of prokaryotes are known to carry out reverse transcription.

22. The central dogma DNA Self-replication loop Transcription RNA Translation Protein The classic view of the central dogma of biology states that: “The coded genetic information hard-wired into DNA is transcribed into individual transportable cassettes, composed of messenger RNA (mRNA); each mRNA cassette contains the program for synthesis of a particular protein (or small number of proteins)."

23. Protein synthesis

24. Viral transcription We have seen how there are four main categories of virus genome: dsDNA , ssDNA , dsRNA and ssRNA Because of distinct modes of transcription within the dsDNA and ssRNA categories a total of seven classes of viruses can be recognized This division of the viruses into classes based on genome type and mode of transcription was first suggested by David Baltimore and this scheme of virus classification is named after him. He initially proposed six classes.

25. Viral transcription In the summary of the scheme depicted in Figure 6.1 most of the nucleic acid strands are labelled (+) or (−). This labelling is relative to the virus mRNA, which is always designated (+). A nucleic acid strand that has the same sequence as mRNA is labelled (+) and a nucleic acid strand that has the sequence complementary to the mRNA is labelled (−).

26. Viral transcription The viruses with (+) RNA genomes (Classes IV and VI) have the same sequence as the virus mRNA. When these viruses infect cells, however, only the Class IV genomes can function as mRNA. These viruses are commonly referred to as plus-strand (or positive-strand) RNA viruses. The Class V viruses are commonly referred to as minus-strand (or negativestrand ) RNA viruses. Class VI viruses must first reverse transcribe their ssRNA genomes to dsDNA before mRNA can be transcribed. Because they carry out transcription in reverse (RNA to DNA)

27. Viral transcription Class VI viruses are known as retroviruses. The ability of some DNA viruses to carry out reverse transcription was discovered later; these viruses became known as pararetroviruses and Class VII was formed to accommodate them.

28. Viral transcription During the infectious cycles of viruses with DNA genomes, viral messenger RNA (mRNA) synthesis must precede production of proteins. In most cases, this step is accomplished by the host cell enzyme that produces cellular mRNA, RNA polymerase II This enzyme also transcribes the proviral DNA of retroviruses. The signals that control expression of the genes of these viruses are similar to those of cellular genes. In fact, much of our understanding of the mechanisms of cellular transcription stems from study of viral DNA templates.

29. Viral transcription In general, enzymes and regulatory proteins needed in smaller quantities are made during the initial period of infection, whereas structural proteins of virus particles are made only after viral DNA synthesis begins. Such orderly gene expression is primarily the result of transcriptional regulation by viral proteins. This pattern is quite different from the continual expression of all viral genes that is characteristic of the infectious cycles of many RNA viruses

30. Some Viral Genomes Must Be Converted to Templates Suitable for Transcription There is considerable variation in the reactions needed to produce templates that can be recognized by the cellular machinery. Some viral genomes are double-stranded DNA molecules that can be transcribed as soon as they reach the nucleus. Other viral DNA genomes must be converted from the form in which they enter the cell to double-stranded molecules that serve as transcriptional templates. The hepadnaviral genome is an incomplete circular DNA molecule with a large gap in one strand that is repaired by cellular enzymes to form a fully double-stranded DNA molecule

31. Some Viral Genomes Must Be Converted to Templates Suitable for Transcription Similarly, single-stranded genomes such as that of the adenovirus-associated virus, a parvovirus, are converted to double-stranded molecules by a cellular DNA polymerase. The prerequisites for expression of retroviral genetic information are even more demanding, for the RNA genome must be both converted into viral DNA and integrated into the cellular genome. Reverse transcription creates an appropriate double-stranded DNA template that includes the signals needed for its recognition by components of the cellular transcriptional machinery

32. Promoters and enhancers A promoter is  a region of DNA where transcription of a gene is initiated . Promoters are a vital component of expression vectors because they control the binding of RNA polymerase to DNA Enhancers contain sequences that bind transcription factors and these interactions may increase the rate of transcription starts by RNA polymerase II

33. Transcription by RNA Polymerase II Accurate initiation of transcription by RNA polymerase II is directed by specific DNA sequences located near the site of initiation and called the promoter . The promoter and the additional DNA sequences that govern transcription make up the transcriptional control region .

34. Transcription by RNA Polymerase II Biochemical studies using model transcriptional control regions, such as the adenoviral major late promoter, established that initiation of transcription is a multistep process. The initiation reactions include promoter recognition, unwinding of the duplex DNA around the initiation site to form an open initiation complex, And movement of the transcribing complex away from the promoter ( promoter clearance

35. Transcription by RNA Polymerase II T A T A A T G Downstream Coding Strand 5` 3` Initiation Site Attachment Site Upstream

36. Transcription by RNA Polymerase II Core promoters of viral and cellular genes contain all the information necessary for recognition of the site of initiation and assembly of precisely organized preinitiation complexes . These assemblies contain RNA polymerase II and a common set of general initiation proteins required for accurate and precise initiation. A hallmark of many core RNA polymerase II promoters is the presence of a TA-rich TATA sequence 20 to 35 bp upstream of the site of initiation which is recognized by the TATA-binding protein Short sequences, termed initiators , which specify accurate (but ineffi cient ) initiation of transcription in the absence of any other promoter sequences, are also commonly found

37. Many of the interactions among components of the transcriptional machinery take place before a promoter is encountered: RNA polymerase II is present in cells in extremely large assemblies that contain the initiation proteins, as well as others that are essential for transcription or its regulation. Such assemblies, termed holoenzymes , appear to be poised to initiate transcription as soon as they are recruited to a promoter

38. Regulation of RNA Polymerase II Transcription Numerous patterns of gene expression are necessary for eukaryotic life: some RNA polymerase II transcription units must be expressed in all cells, whereas others are transcribed only during specific developmental stages or in specialized differentiated cells. Many others must be maintained in an almost silent state, from which they can be activated rapidly in response to specific stimuli, and to which they can be returned readily. Transcription of viral genes is also regulated during the infectious cycles of most of the viruses

39. Regulation of RNA Polymerase II Transcription Large quantities of viral proteins for assembly of progeny virions must be made within a finite (and often short) infectious cycle. Consequently, some viral genes must be transcribed at higher rates than others. In many cases, viral genes are transcribed in a specific and stereotyped temporal sequence. Such regulated transcription is achieved in part by means of cellular control mechanisms, for example, cellular proteins that repress transcription. In general, however, viral proteins are critical components of the circuits that establish orderly transcription of viral genes.