Section 6. Junaid Malek, M.D.

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1 Section 6 Junaid Malek, M.D.

2 The Golgi and gp160 gp160 transported from ER to the Golgi in coated vesicles These coated vesicles fuse to the cis portion of the Golgi and deposit their cargo in the cisternae gp160 glycosylated in the Golgi When gp160 reaches the trans-golgi, it is cleaved by a protease into gp41 and gp120

3 The Golgi and gp160 As gp120 and gp41 prepare to leave the Golgi, they are sorted into secretory vesicles These vesicles are targeted through the regulated secretory pathway to the cell membrane

4 The Golgi: Recap First major function of Golgi is glycosylation of proteins: sugars are added, modified and removed Second major function of Golgi is to sort proteins to different destinations

5 Viral Assembly We now have gp120 and gp41 packaged into secretory vesicles Gag - made in cytoplasm and modified by addition of fatty acid group This fatty acid group targets Gag to the cell membrane One domain of the protein recruits the viral genome through interactions with the viral RNA

6 Viral Assembly Gag protein thought to play a role in deforming the plasma membrane to help promote fission This releases viral particles complete with a membrane (derived from the plasma membrane of the host cell) as well as the glycoproteins gp41 and gp120

7 Q: How is the membrane deformed to form a vesicle? A: Coats! (a.k.a. protein cages) - The shape and the chemical properties of the coat proteins help pay the energy cost of deforming the membrane. The coat proteins help neutralize the negative charge on the phospholipids head groups. Some coat proteins have intrinsic curvature, which facilitates the curving of the membrane.

8 Q: How are the correct cargoes enriched in the proper vesicle? How are proteins that should remain in the donor compartment excluded? A: Coats help ensure that the correct cargo get packaged into the proper vesicle by interacting with proteins that are to be enriched in the vesicle. These interactions may be direct or may be mediated through other proteins.

9 Q: How do vesicles know what compartment to fuse with? What catalyses this fusion process? A: SNARES!!!

10 SNARES Specificity in targeting ensured by pairing between v-snares and t-snares Vesicles display v-snares (vesicle SNAREs) on their surface that identify them according to their origin and type of cargo Target membranes display complementary t-snares (target SNAREs) that recognize the v-snares

11 SNARES Favorable structural change of SNARE pairing is used to overcome the unfavorable process of membrane fusion The pairing of the v-snare with the t- SNARE forms a stable bundle of 4 alphahelices that forces the water molecules from the interface and brings charged lipid head groups together Analogous to structural change in gp41 that drives membrane fusion

12 SNARES

13 Retrograde Traffic Remember that transport in a cell is a twoway process and not just one-way Retrograde traffic allows for mis-sorted proteins to be sent back to the correct compartment Retrograde traffic also ensures that the balance of lipids and the relative size of the compartments is maintained

14 Steady State vs. Equilibrium If a system is in equilibrium, the forward and reverse rates of conversion between states are equal, so the relative populations of states remain constant As there is no net conversion to any state, G for the conversion between states at equilibrium = 0 Example: A B C. If states A, B and C are in equilibrium, for every molecule that goes from A to B, there is a molecule that goes from B to A. Same applies for B to C and C to B. Result: no net movement of molecules in or out of system.

15 Steady State vs. Equilibrium If a system is at steady state, the forward and reverse rates of conversion between states are NOT equal, but the relative populations of states still remains constant The reason for this is that the rate of conversion to a particular state is equal to the rate of conversion from this state Note that these conversion processes are not the forward/reverse of each other. Each process can have its own G 0, so long as the rates of the two processes are equal.

16 Steady State vs. Equilibrium Example: A B C. If A, B and C are at steady state, the population of state B remains constant. For every molecule that goes from A to B, a molecule goes from B to C Therefore, the population of B never changes, but there is in fact net movement from A C Note that steady state does not tell you anything about the G of the individual conversion processes or the relationship between the rates of forward and reverse reactions for the individual conversions

17 Co-option and HIV HIV, like all viruses, require other oranisms/ cells to survive and replicate In order for this to occur, HIV must co-opt cellular machinery and processes Listeria is another example of co-option One key difference to remember is that Listeria is a bacterium and does not require a host cell for replication

18 DNA Nomenclature If two strands of dsdna are labelled Strand A and Strand B where Strand A is used to transcribe RNA, then Strands A and B are called... Strand A Coding Strand Non-Template Strand Sense Strand Strand B Non-Coding Strand Template Strand Anti-Sense Strand

19 Prokaryotic Transcription Prototers located at -10 and -35 upstream from the transcriptional start site (position +1) Transcription factors Specific sigma factor(s) bind to the -10 and -35 sites and recruit RNA polymerase RNA Polymerase Catalyzes the polymerization of new RNA strands

20 Prokaryotic Transcription Termination of Transcription At specific sequences (termination sequences), the newly synthesized RNA will fold onto itself due to selfcomplementarity This creates a hairpin structure that helps the newly synthesized RNA push off RNA polymerase from the RNA/DNA hybrid

21 Eukaryotic Transcription Similar to prokaryotic transcription, but several components are more complex Promoters The regulatory domain consists of a core promoter sequence that has a binding site for the RNA polymerase complex as well as regions of DNA that have binding sites for transcription factors

22 Eukaryotic Transcription Transcription factors Similar to Sigma factor, but insead fo one protein being used, many proteins bind and assemble to perform same function They recruit and activate RNA polymerase to a specific gene RNA polymerase performs the same function as bacterial polymerase mrna transcription uses RNA Pol II

23 Reverse Transcriptase To co-opt cellular machinery, HIV uses reverse transcriptase (RT) to convert RNA genome into DNA Once HIV has made a DNA copy of its RNA genome, it inserts itself into the cell s genome using a protein called integrase This allows for the HIV genome to be replicated with the host DNA, and also allows for transcription of new HIV RNA The newly synthesized HIV RNA can be packaged into new virus particles and helps complete the viral lifecycle

24 AZT First available drug to combat HIV Nucleoside analog that inhibits the function of RT Chain terminator Does not appreciably affect human cells due to error correction functions of human DNA polymerase

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26 HIV Genome Extremely compact Multiple genes have overlapping regions This is seldom if ever seen in human DNA Some genes encode multiple proteins Pol encodes RT, Integrase and Protease Important genes to know include Gag, Pol, Rev and Tat

27 TAT and Transcription TAT enhances the rate of HIV transcription from the reverse transcribed, host genome integrated HIV DNA RNA Polymerase will have some limited affinity to bind and transcribe from the integrated HIV DNA However to improve the rate and fidelity of transcription, RNA polymerase needs to be activated via phosphorylation

28 TAT and Transcription Phosphorylation is normally performed by transcription factors binding to specific promoter sites upstream of the gene to be transcribed The reverse transcribed HIV does not carry specific promoter sites, and also may not necessarily co-opt host promoters depending upon where it inserts into the host genome

29 TAT and Transcription HIV has developed an alternate method to phosphorylate and activate RNA polymerase using TAT The 5 end of the nascent mrna transcript has a specific sequence known as the TAT activating region (TAR) that forms a hairpin loop that the protein TAT is able to bind to TAT then recruits CDK9 and CyclinT, which phosphorylate and activate RNA polymerase Activated RNA polymerase will then polymerize the remainder of the HIV RNA much more efficiently and rapidly

30 Q: Why doesn t AZT affect the host cell s ability to replicate DNA? A: The reason is twofold: (1) DNA polymerase doesn t have as high an affinity for AZT compared to RT (2) DNA polymerase has proofreading capabilities to remove AZT from replicating DNA while RT lacks that function

31 TAT and TAR While transcription occurs at specific locations dictated by the presence of promoters and enhancers, proviral integration occurs somewhat randomly However, the virus integrates preferentially in actively transcribed genes

32 TAT and TAR HIV proviral DNA has a weak promoter that can also attract transcription factors and RNA polymerase RNA polymerase will bind and begin transcription, but unless something else happens, the transcription will be slow, and non-processive

33 TAT and TAR TAR is a sequence near the 5 end of the nascent HIV RNA transcript that forms a hairpin loop that TAT recognizes and binds During the slow (non-processive) phase of transcription, the TAR sequence is transcribed and made available to TAT TAT can then bind and recruit Cyclin T and CDK9, which phosphorylate RNA Pol II to enhance the polymerases processivity Thus TAT increases the amount of HIV RNA transcribed by enhancing processivity of transcription

34 What do CycT and CDK9 normally do? They enhance the processivity of RNA transcription However it is unclear how they are recruited to nascent RNAs undergoing transcription

35 How do you get the first TAT molecule? Though transcription is at first slow and non-processive, every so often you will get a full-length transcript Once that first full-length transcript is made, it will be translated into TAT and REV The first TAT molecule will subsequently enhance the transcription of additional HIV RNAs which will encode more TAT

36 Q: Based on the position of TAR, do you think HIV has genes encoded on both strands or only one strand of proviral DNA? A: Probably only one. The TAR sequence would only be transcribed in one direction from the proviral DNA, and thus transcription in only one direction would be enhanced. This makes it unlikely, but not impossible, that HIV would place genes on the reverse strand.

37 Differences in Genome Human E. coli HIV size 3.3 x 10 9 bp 4.6 X bp Proteins per gene 1 1 or more 1 or more Gene organization random random + operons? Coding sequence Discontinuous Introns/exons Continuous (no introns) Both

38 Important HIV Genes to Know! gag Erin Envelope protein, helps budding env Erin Encodes gp120 and gp41 pol Rob/David Encodes reverse transcriptase, integrase, and protease tat Rob Enhances transcription rev Rob Nuclear export