Retroviruses and Trans(retro)posons

Transposons Mobile DNA (elements) in the genome Jumping genes DNA fragments (elements) with the capacity to move in the genome Mobile DNA (elements) in the genome Jumping genes Function: structural ~ 50% genomic in mammals. Clasification: DNA transposons Retro (RNA) transposons

Retro (RNA) transposons Mostly in Eukaryotes Yeast Drosophila Mammals (humans y ~8%) Mechanism: (copy and paste) Types: ---Retroviruses like (LTR) ---No related to retroviruses Structure: Inverted repeats LTR (Long Terminal Repeats) (250-600 pb) Sequence coding region-Enzymes Reverse Transcriptase (RT), Integrase (IN) and other proteins.

Transposons DNA fragments (elements) with the capacity to move in the genome Mobile DNA (elements) in the genome Jumping genes Function: 1-structural Function: 2-Alterations in the patterns of gene expressions

Introduction Retrovirus life cycle --Retrovirus (two copies of RNA packaged in a capsid) attaches to cell membrane (Env) and fuses (Env), allowing entry of RNA into cell. --RNA is reverse transcribed into dsDNA and integrated into the host genome as a provirus. --The host transcribes the provirus, making all the proteins necessary for assembly of the virus which buds out of the cell as the complete retrovirus. Figure 22.1

The Retrovirus Life Cycle Involves Transposition-Like Events 1-A retrovirus has two copies of its genome of single-stranded RNA. 2-An integrated provirus is a double-stranded DNA sequence. 3-Polyproteins are initially produced and then processed (proteases) to give all the viral protein products necessary for retrovirus reproduction.

A retrovirus generates a provirus by reverse transcription of the retroviral genome. ---Retroviral RNA (plus strand) is reverse transcribed (Pol – reverse transcriptase) into ssDNA (minus strand). ---The complementary strand of DNA (plus strand) which adds LTRs (long terminal repeats) to the 5’ and 3’ end, the linear dsDNA is inserted into the host genome (Pol-integrase). ---Transcription of the provirus (integrated retrovirus sequence) into RNA (plus strand) by the host RNA polymerase. Figure 22.2 v vm

Retroviral Genes Codes for Polyproteins A typical retrovirus has three genes: gag, pol, and env. Gag and Pol requires either stop codon suppression or a frame-shift. This reduces the amount of Pol produced. Env is translated from a separate viral mRNA that is generated by splicing. Each of the three protein products are processed by proteases to give multiple Proteins. Full length viral mRNA is packaged as the viral genome. The terminal region of the viral mRNA contains specific sequence (U and R segments) mRNA is capped 5’ and polyadenylated 3’ Figure 22.3 U3 U5 vmRNA

Retrovirus encoded polyproteins that must be processed vmRNA U5 U3 Retrovirus encoded polyproteins that must be processed Gag - structural proteins in matrix, capsid, and nucleocapsid. Pol – protease, reverse transcriptase, and integrase. Env – surface protein, transmembrane protein for recognition and fusion

What is the source of the viral membrane? Figure 17.4 What is the source of the viral membrane?

Influenza Virus Must “Uncoat” in Cytoplasm H5N1 hemagglutinin (HA) type 5 and neuraminidase type 1 https://www.youtube.com/watch?v=0qCTyKrhVWc

Viral DNA Is Generated by Reverse Transcription A short sequence (R) is repeated at each end of the viral RNA. 1-Reverse Transcription 2-Integration 3-Transcription Figure 22.5 Virus RNA structure U5 – unique sequence at 5’ end (80-100 bp) U3 – unique sequence at 3’ end (170-1350 bp). A short sequence (R): is repeated at each end of the viral RNA, so the 5’ and 3’ ends are R-U5 and U3-R.

Viral DNA is generated by reverse transcription -RT converts RNA to DNA -RT shows DNA polymerase activity -RT has RNAase H activity Reverse transcriptase starts synthesis when a tRNA primer binds to a site 100 to 200 bases from the 5′ end. (Synthesis= 5’ to 3’ direction and requires a double stranded region to initiate) (tRNA is a “viral transcriptional RNA “?”) When the enzyme reaches the end, the 5′-terminal bases of RNA are degraded. This exposes the 3′ end of the DNA product. The exposed 3′ end base pairs with the 3′ terminus of another RNA genome Strand switching event Synthesis continues, generating a product in which the 5′ and 3′ regions are repeated. This gives each end the structure U3-R-U5. Quality and Quantity---Control U3 R U5 Figure 22.6

Quality and Quantity---Control Similar strand switching events occur when reverse transcriptase uses the DNA product to generate a complementary strand. -Synthesis continues, generating a product in which the 5’ and 3’ regions are repeated, giving each end the structure U3-R-U5 (LTR) (Long Term Repeat) -Similar strand switching events occur when reverse transcriptase uses the DNA product to generate a complementary strand. -Strong stop plus strand DNA jumps to 3’ end of cDNA. This adds U3 to 5’ end of dsDNA and completes the formation of the LTR. U3 R U5 U3 R U5 U3 R U5 Quality and Quantity---Control Figure 22.7

Viral DNA Integrates into the Chromosome The organization of proviral DNA in a chromosome is the same as a transposon. The provirus is flanked by short direct repeats of a sequence at the target site. Linear DNA is inserted directly into the host chromosome by the retroviral integrase enzyme. Two base pairs of DNA are lost from each end of the retroviral sequence during the integration reaction.

RNA has direct (inverted) repeats, linear DNA form of virus has Long Terminal Repeats (LTR) and the integrated DNA form (provirus) has LTRs that are shortened by 2 bp And a 4-6 bp repeat of host DNA. Integrase, a viral product, acts on both viral DNA and target-host DNA. -It targets random sites by making staggered cuts at the selected-host DNA sites. -it targets end of the viral DNA converting the blunt ends into the recessed ends by removing bases beyond the CA dinucleotide sequence. -it needs sticky ends -1 to 10 copies can be introduced into host cells. (amplification and rearrangement of DNA) U3 region of each LTR carries a promoter and enhancer ( initiates transcription) Figure 22.9

How are the Replication-defective viruses generated? Figure 22.10 Transducing retro-viruses: a virus variant containing cellular RNA. Replication-defective: a virus variant in which a viral sequence has been replaced. Helper virus: a retro-virus that provides the missing viral function to a replication- defective virus. U5 U3 U3 U5 How are the Replication-defective viruses generated? Why is the addition of v-onc into the viral genome a problem?

Figure 30.13 Replication-defective virus

Homologous recombination Non-Homologous recombination (Non-homologous end joining) Inappropriate NHEJ can lead to gene translocations

http://www.whfreeman.com/catalog/static/whf/kuby/content/anm/kb03an01.htm Non-germline vs germline?