Cell S&F Lecture 31

The HIV Lifecycle

1. Binding and Entry: Virus uses receptors to attach to cell surface and fuses its way inside

2. Reverse Transcription: Virus converts viral RNA to DNA for reading

3. Nuclear Translocation: Viral DNA moves into the nucleus

4. Integration: Viral DNA incorporates into the host's genome, allowing replication with host DNA.

5. Transcription: Viral DNA is transcribed into mRNA, which will be translated into viral proteins.

6. Regulation: Cell processes these blueprints and prepares them for building

7. Translation: viral proteins produced

8. Assembly and release

Describe the two-step process of Reverse Transcription in HIV.

1. First-strand (minus strand) DNA is built using the viral RNA template.

2. Second-strand (plus strand) DNA is built to create a double-stranded DNA molecule.

What must happen to the virus between Step 2 (DNA synthesis) and Step 3 (Transcription)?

Integration. The newly formed double-stranded viral DNA must be inserted into the host cell's genome. This is the point of no return for the infection.

What is the goal of Step 3 (Transcription) in the HIV life cycle?

The host cell "reads" the integrated viral DNA to produce Progeny HIV RNA. These are the genetic copies used to assemble new viruses.

How does the cell’s APOBEC3G "break" HIV?

It performs Deamination (converting C to U) on the viral minus-strand DNA during reverse transcription. This leads to G → A hypermutations that make the virus non-functional.

How does the HIV protein Vif counteract the host’s APOBEC3G defense?

Vif targets APOBEC3G for proteasomal degradation. It tags APOBEC3G with Ubiquitin, causing the cell to destroy its own defense protein before it can be packaged into new viruses.

Why are HIV particles produced without Vif usually non-infectious?

Without Vif to destroy it, APOBEC3G gets packaged into the budding virus. When that virus tries to infect a new cell, APOBEC3G will mutate the viral genome into a "hypermutated" defective state.

miRNA Biogenesis

the multi-step process your cells use to "cut and paste" a tiny piece of RNA until it is ready to turn off specific genes.

3 Step miRNA Biogenesis steps

1. Nucleus(Initial Cut) : Pol II starts with the DNA and creates a long, floppy strand called Pri-miRNA. Drosha (the first set of "scissors") trims it into a shorter, hairpin-shaped loop called Pre-miRNA.

2. The Exit(Doorway) : Exportin 5 and RanGTP act as a security escort, carrying the Pre-miRNA out of the nucleus and into the main body of the cell (the cytoplasm)

3. The Cytoplasm(Final Trim): Dicer (the second set of "scissors") cuts off the loop of the hairpin. You are left with a tiny, double-stranded miRNA (about 22 units long) that is ready to stop genes from making proteins.

Drosha and Dicer

Drosha: Initial trim in the nucleus

Dicer: Final Trim in the cytoplasm

Exportin 5

A protein that transports Pre-miRNA from the nucleus to the cytoplasm, working alongside RanGTP.

Which of the following enzyme does not use a template for adding nucleotides

1. DNA polymerase (DNA replication)

2. RNA polymerase (transcription)

3. Reverse Transcriptase

4. Poly A polymerase

5. Primase

Poly A polymerase

Which of these two are most similar?

1. DNA polymerase (DNA replication)

2. RNA polymerase (transcription)

3. Reverse Transcriptase

4. Poly A polymerase

5. Primase

RNA Polymerase and Primase

From mRNA to Protein

The nucleotide sequence of a gene, through mRNA, determines the sequence of amino acids in a polypeptide chain

Exons and Introns

Exons are coding sequences that remain in mRNA after splicing, while introns are non-coding sequences that are removed.

What markers define the beginning and end of the Coding Sequence?

It begins at the Start Codon (AUG) and ends at a Stop Codon (UAG, UAA, or UGA). Everything outside these markers is "untranslated."

What are the two protective structures added to the 5' and 3' ends of mRNA?

A 5' cap and a poly-A tail.

What type of code is the genetic code?

A triplet code

An extraterrestrial life form has a genetic code system similar to

ours except there are FIVE different nucleotides and the codons are

doublets instead of triplets. What is the maximum number of

different amino acids that could be accommodated by this genetic

code?

– 10

– 24

– 25

– 64

25

What does it mean that the genetic code is nonoverlapping?

It means each nucleotide (letter) is part of only one codon. The cell reads them in distinct sets of three (1-2-3, 4-5-6) rather than overlapping them (1-2-3, 2-3-4).

In a nonoverlapping triplet code, how many amino acids are produced from a 9-nucleotide sequence?

Three amino acids are produced.

Why is a nonoverlapping code "safer" than an overlapping one?

changing one nucleotide usually only affects one amino acid. In an overlapping code, one single change could alter threeconsecutive amino acids.

Why are there exactly three possible reading frames for any single strand of mRNA?

Each mRNA strand can be read in three different ways, starting from one of the first three nucleotides, and each frame will produce a different sequence of codons.

What is an open reading frame(ORF)

It is a stretch of DNA/RNA without any stop codons. This allows the cell to read through the entire sequence to build a full protein.

Frameshift mutations

are genetic mutations caused by insertions or deletions of nucleotides that change the reading frame of the resulting protein. This can lead to the production of entirely different and nonfunctional proteins.

Sickle cell anemia

caused by a single mutation in the β-hemoglobin gene (HBB)

• Anemia: low number of red blood cells

• Hereditary disease

• Red blood cells of patients are of sickled shape because of abnormal hemoglobin structure

In the HBB gene mutation for sickle cell, what is the specific amino acid change?

Glutamic acid to valine

• caused by a single nucleotide swap (A to T in DNA, A to U in mRNA).

Why does swapping Glu for Val cause hemoglobin proteins to "stick" together?

Glu is water-loving, but Val is water-fearing. The Valine tries to hide from the watery environment of the cell by sticking to other hemoglobin molecules.

Degenerate Coding

Refers to the redundancy in the genetic code, where multiple codons can specify the same amino acid, minimizing the impact of mutations.

Unambiguous coding

While one amino acid can have many codons, one specific codon only ever codes for one specific amino acid.

Which of the following is considered a “silent mutation”?

– A base substitution that introduces a stop codon

prematurely.

– A base insertion that introduces a stop codon

prematurely

– A base substitution that changes the encoded amino

acid from one to another

– A base substitution that does not change the amino

acid encoded

A base substitution that does not change the amino acid encoded.

What is Codon Usage Bias?

The phenomenon where specific codons are used more often than their "synonyms" (other codons that code for the same amino acid) within a particular species.

Translation: The Template (Bacterial)

Contains a specific Ribosome-binding site in the 5' Untranslated Region (UTR).

• Start/Stop: Begins at AUG and ends at a Stop Codon (UAG, UAA, or UGA).

• Structure: Generally simpler; lacks the fancy "decorations" (Cap/Tail) found in more complex cells.

Translation: The Template (Eukaryotic)

Has a 5' Cap and a 3' Poly(A) Tail for protection and ribosome recognition.

• Start/Stop: Also begins at AUG and ends at a Stop Codon.

• Structure: The Coding Sequence is just one part of the full mRNA, sandwiched between two Untranslated Regions (UTRs).

Translation: The Players

mRNA: the template

ribosome: the machine

tRNA: the carrier

Aminoacyl-tRNA synthetase: the feeder

Translation Factors: the facilitators

Translation Overview: What are the three main steps of protein synthesis?

1. Initiation: The "Start." Ribosome subunits, mRNA, and the first tRNA (carrying the first amino acid) meet at the AUG start codon.

2. Elongation: The "Build." tRNAs bring amino acids one by one, and the ribosome joins them into a growing chain.

3. Termination: The "Finish." The ribosome reaches a Stop Codon. A Release Factorenters, and the completed protein is set free.

What happens when the ribosome hits a Stop Codon (UAG, UAA, or UGA)?

No tRNA matches these codons. Instead, a Release Factor (a protein) binds to the ribosome. This triggers the machinery to fall apart and release the Completed Polypeptide (the protein)

In a sequence with multiple AUGs, how does a Eukaryotic ribosome decide where to start?

It uses the Scanning Model. The ribosome starts at the 5' end and picks the first AUG it encounters. Everything before that first AUG is the 5' UTR.