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1 number 4 Done by Nedaa Bani Ata Corrected by Rama Nada Doctor Ashraf

2 Genome replication and gene expression Remember the steps of viral replication from the last lecture: Attachment, Adsorption, Penetration, and Uncoating. We talked previously about: Components of Glycoproteins in different viruses. Variations in the receptors and variations in the need of viruses to the receptors for example a single receptor, receptor with co receptor or multiple receptors. Penetration and its types, which are: 1-Receptor mediated endocytosis in both naked and enveloped viruses 2-Fusion in enveloped viruses only 3-Translocation Then we talked in the next step about uncoating and we said that it is the least studied step in virus replication There are a few questions that we must answer: Where does it occur? For small DNA viruses it occurs in the nucleus while for all other viruses; RNA and large DNA viruses it occurs mainly in the cytoplasm. Eventually, we reach the 5 th step which is macromolecules synthesis stage. It includes two steps occurring simultaneously: 1- Replication of viral genome 2- Protein synthesis of the viruses. When does the process of protein synthesis occur? 1-Early (nonstructural proteins and enzymes that are needed in viral replication) 2-Late (structural: capsid and Glycoproteins needed for assembly ) Some viruses such as HIV synthesize a long polyprotein which is further cleaved.

3 David Baltimore classification of viruses: (Originally, this classification included only six groups, but it has since been extended to include the hepadnaviruses and caulimoviruses). 1-DS DNA 2-SS DNA (parvovirus only) 3-DS RNA (rotavirus only) 4-SS RNA +VE SENSE 5-SS RNA VE SENSE 6-SSRNA +VE SENSE with reverse transcriptase enzyme (HIV only) 7-partial DS DNA with reverse transcriptase enzyme (the only genome of partial DS DNA is hepatitis B). so, 4 of them are applied for 4 specific viruses while the remaining 3 are applied to other viruses. 1.DS DNA: Topoisomerase enzyme relieves the super coiled structure of DNA then helicase enzyme forms the replication fork. The replication direction is from 5' to 3' So, here we have a replication fork; a Y- shaped are formed by the unzipping of the two strands, forming a strand and its complementary strand (one is going to be the leading strand and the other is a lagging strand). On the leading strand replication occurs from the starting point to the ending point in a single continuous step moving in a regular 5' to 3' direction using the enzyme DNA polymerase.

4 Since the leading and the lagging strand are bi-directional, the lagging strand requires the addition of nucleotides from a 3' to 5' direction, which is not applicable since DNA polymerase only works in a 5' to 3' direction. Consequentially, fragments called Okazaki fragments will form, which are connected to each other using a ligase enzyme. Here we have a problem that is called the end problem where the site of the last primer is going to be left at the end of replication; this will lead to shortening of the new copies of the complementary DNA. How do cells solve this problem? Through the action of a telomerase enzyme; this adds repetitive noncoding sequences at the end forming a Telomere. So basically, telomerase is the enzyme that replaces the gap at the end of the lagging strand by repetitive noncoding sequences of nucleotides. **Note: Virology sheets are written from section 2, so refer to sheet number 2 for the mechanism details. * Remember the name of the enzymes: helicase, topoisomerase, telomerase, ligase, DNA polymerase.

5 2. SS DNA: Replication occurs in the nucleus, involving the formation of a double-stranded intermediate which serves as a template for the synthesis of single-stranded progeny DNA, involving the formation of a (-)sense strand, which serves as a template for (+)strand RNA and DNA synthesis. Examples: phage M13, chicken anemia virus, maize streak virus 3. DS RNA: DS means that we have positive and negative strands (no lagging or leading strand). These viruses have segmented genomes. Each genome segment is transcribed separately to produce monocistronic mrnas; monocistronic means a type of messenger RNA that can encode only one polypeptide per RNA molecule. In eukaryotic cells virtually all messenger RNAs are monocistronic. Theoretically speaking the positive strand is going to separate from the negative strand and each one is going to be complemented as a template. The positive strand is going to be complemented by a negative strand by the action of RNA polymerase and vice versa. Protein synthesis: the negative strand is neglected, and the positive strand serves as mrna for protein synthesis, that travels to the ribosome and is translated to a protein. Examples: Reoviruses, Rotavirues 4. SS RNA with positive sense: Protein synthesis: the positive sense SS RNA goes to the ribosome and is translated to a protein directly where it serves as a mrna molecule, which is where the name positive sense comes from. Genome replication: the positive sense is complemented by a negative strand, and then the complementary negative strand serves as a template for RNA polymerase to make new copies of the positive sense that will travel

6 to the ribosome to synthesize proteins, meaning that the only function of the newly synthesized negative sense is to serve as a template to further synthesize positive sense molecules. Examples on this type could be divided into majorly two types: a) Polycistronic mrna e.g. Picornaviruses; Hepatitis A. Genome RNA = mrna. Means naked RNA is infectious, no virion particle associated polymerase. Translation results in the formation of a polyprotein product, which is subsequently cleaved to form the mature proteins. Polycistronic: a type of messenger RNA that can encode more than one polypeptide separately within the same RNA molecule. Bacterial messenger RNA is generally polycistronic. b) Complex Transcription e.g. Togaviruses. Two or more rounds of translation are necessary to produce the genomic RNA. 5. SS RNA with negative sense: Protein synthesis: the negative strand is complemented by a positive one, and then the positive strand goes to the ribosome to synthesize proteins. Genome replication: positive strands will be synthesized using a negative template, and these newly synthesized positive strands will serve as a template to synthesize more negative strands, which will again serve as templates to produce more positive strand molecules to encourage further translation of proteins in the ribosomes. We can divide this type of replication into two categories: a) Segmented e.g. Orthomyxoviruses. First step in replication is transcription of the (-)sense RNA genome by the virion RNA-dependent RNA polymerase to produce monocistronic mrnas, which also serve as the template for genome replication. b) Non-segmented e.g. Rhabdoviruses. Replication occurs as above and monocistronic mrnas are produced. Examples: Influenza viruses, Hantaviruses

7 6. SS RNA +VE SENSE with reverse transcriptase *HIV /RETROVIRUSES, RETROVIRIDIAE FAMILY: RETROVIRIDIAE SUBFAMILY: LEUTEVARIANDAE GENUS: HIV TYPE: HIV1, HIV2 -Genome: positive sense SS RNA. HIV is unique as it has two copies of its genome (called: diploid). HIV replication: 1. HIV Glycoproteins bind to the receptor on the T-cells surface that is called CD4 and co receptor called CCR5. 2. Entry of the virus via a fusion mechanism, this mechanism starts with fusion of the viral envelope with the cell membrane, then small pore formation followed by large pore formation, then entry of nucleocapsid and dissociation of viral proteins in the cytoplasm. *Note: no injection of genetic material in viruses except for bacteriophages. Genome replication: positive sense SSRNA will serve as a template to the enzyme reverse transcriptase (complex of enzymes: more than one enzyme): 1-The first enzyme is reverse transcriptase that is going to act on positive sense SS RNA to give a complementary DNA strand, at this stage you get both an RNA and DNA intermediate. 2-RNA will dissociate from DNA and is broken down by Ribonuclease H which is another component of the reverse transcriptase complex. 3-DNA polymerase - like enzyme which is another component of reverse transcriptase is going to make DS DNA -Then the DNA strand goes to the nucleus and integrates with the cellular genome by an enzyme called integrase which is going to produce sticky ends. Integrase is going to take nucleotides from the viral DNA and

8 nucleotides from cellular DNA, so it is going to act on the genomic DNA (bacterial DNA) and make complementary sticky ends there and exert their viral DNA to genomic DNA. It s very useful to watch this video: At this stage we called the inserted viral DNA provirus. -Inserted viral DNA becomes part of the genomic DNA and undergoes TRANSCRIPTION which gives mrna which combines the viral DNA and the cellular genome. -mrna will exit the nucleus and will be translated by a ribosome to give a specific protein. HIV protein synthesis: The whole mrna will be translated entirely at the ribosome, so all HIV proteins remain as a connected series (polyprotein). RNA replication of the virus: The transcribed DNA will give mrna for protein synthesis and part of the mrna will be the genome of the newly formed viruses. So, the transcription process will result in the protein synthesis and the genome synthesis for the new copies of HIV This will be followed by the assembly of two copies of HIV genome with structural /nonstructural protein. Maturation will happen next: Polyprotein is the nonfunctional form of the virus, it becomes functional by cleavage via viable enzyme proteases that will give individual proteins; every protein is encoded by a single gene, so each gene is going to be encoded as a protein produced via a viral protease. This will be followed by the release of the viruses.

9 7. Partial DS DNA via reverse transcriptase: It is circular DNA found in hepatitis B, composed of complete external circle and a missing segment internal circle. -All DNA viruses replicate in the nucleus except poxvirus, so hepatitis B replicates in the nucleus; its genome will not be integrated with the cellular genome, so it acts as an extra genome. -Protein synthesis: transcription of viral DNA produces mrna which will exit the nucleus and is translated in a ribosome to synthesize a protein. **Genome replication: -DNA polymerase will act on the partial strand to make it complete, then the genome goes to the nucleus, its going to be extra chromosomal circular double stranded DNA there. -Transcription is going to occur for cellular and viral genome mrna (positive sense) that is produced from DNA transcription will act as a template for reverse transcriptase enzyme (which give an DNA-RNA intermediate) to synthesize DNA (negative sense), the complete ring, then this negative DNA ring will serve as a template to synthesize a partial positive template, DNA polymerase will continue to synthesize a template until it reaches a certain point till it stops for that reason the template ring will be partial. Note: here we say positive and negative to distinguish between the complete ring (negative/synthesized first) and the partial template ring (positive/synthesized secondly). Note: In the case of SS RNA positive sense with reverse transcriptase the enzyme reverse transcriptase functions in the first steps (after the SSRNA enters the nucleus), while in the case of DS DNA with reverse transcriptase the enzyme reverse transcriptase serves in the last steps (after transcription). Replication Challenges for DNA Viruses:

10 1-Access to nucleus (small DNA viruses are uncoated in the nucleus, as they are small they enter the nucleus through nuclear pores with their capsid). 2-Competing for nucleotides (the viral genome will compete with the cellular genome for the nucleotides to replicate and synthesize a template). **If there are no nucleotides the virus can t replicate. 3- Cell cycle control in eukaryotes - S phase dependent materials for some Viruses (Parvo). S phase is the synthetic phase of the cell during which the cellular machinery is ready to synthesize (replicate) the cellular DNA. There are some viruses such as parvovirus that totally depend on cellular machinery, so it waits until the cell become in the S phase to use its enzymes and machinery in replication, other viruses don t wait for the cell to remain in the S phase to replicate, so that the cellular machinery and enzymes are available all the time. Continuous S phase means the cell is in continuous replication so there is a high chance for mutations to occur, and the replication here is uncontrolled (tumor), so the question here is: Is there a relation between viruses and tumors? Yes, there are DNA viruses associated with tumors, such as human papilloma virus which is associated with cervical cancer (this cancer occurs after years of infection with papilloma virus).