Replication of DNA Viruses

Question 1

After genomic DNA has been released into the cell, what is the first major biosynthetic process?

Answer 1

DNA transcription. This is because at least one viral protein is required for viral DNA replication

Question 2

SV 40

Answer 2

A DNA virus- the DNA is double stranded and circular, ready for transcription. It is a non-enveloped and spherical virus.

Question 3

SV 40 replication cycle (6)

Answer 3

1. As soon as the genome is released in the nucleus of the cell, it gets chromatinized into histone nucleosome-like structures
2. Then, the DNA undergoes transcription, which follows a cascade
3. Only a portion of the genome is transcribed- these are called early genes. One early gene is large T antigen. They regulate transcription and DNA synthesis
4. Delayed DNA replication
5. Late structural proteins are produced for the assembly of the virus particle
6. Trafficking of viral proteins between cellular compartments

Question 4

How does SV 40 enter the cell?

Answer 4

It fuses with the plasma membrane and is trafficked through caveolin mediated endocytosis. It releases its genome into the host cell’s nucleus

Question 5

Large T antigen

Answer 5

The early gene promoter transcribes large T antigen, which may undergo splicing to produce large T and small T antigen. Large T antigen is shuttled back into the nucleus because it also encoded the nuclear localization signal

Question 6

Processes facilitated by large T antigen (2)

Answer 6

Occurs once the antigen is in the nucleus
1. DNA replication of the whole genome
2. Large T antigen also facilitates transcription on the multiple genomic DNA copies that have been produced during genetic replication
3. Further transcribes genes associated with late promoters

Question 7

Transcription cascade

Answer 7

Early promoters are transcribed first, then late promoters, which produce late genes

Question 8

Why are Hepadnaviruses not ready for transcription?

Answer 8

Hepatitis B virus- has double stranded, circular genome that is gapped. When the virus particle is being assembled, it packages RNA, nucleotides, and reverse transcriptase. The gapped genome results because the virus runs out of nucleotides before it can finish replication. The virus uses the host cell DNA machinery to fill the gap

Question 9

Why are parvoviruses not ready for transcription?

Answer 9

The genome is made of single stranded DNA, with repetitive, inverted repeats at the ends. DNA synthesis can begin at the 3’ end of one of the repeats to fill the gap

Question 10

Why are retroviruses not ready for transcription?

Answer 10

Retroviruses enter the cell as an RNA molecule, although their replicating form is DNA. The virus packages reverse transcriptase, which facilitates reverse transcription of RNA into DNA, which is transported to the nucleus and integrated into the host cell genome.

Question 11

Types of RNA polymerases present in eukaryotic cells

Answer 11

RNA polymerases 1, 2, and 3

Question 12

RNA polymerase 1

Answer 12

Produces pre-mRNA molecules, which produces ribosomal RNA

Question 13

RNA polymerase 2

Answer 13

Produces pre-mRNAs, primary micro RNAs, and small nuclear RNAs (non coding). In viruses, it transcribes pre-mRNA and mRNA. May also transcribe non coding RNAs

Question 14

RNA polymerase 3

Answer 14

Produces tRNAs, 5S ribosomal RNA, and U6 snRNA. Involved in the assembly of ribosomes.

Question 15

Which viruses do not replicate in the nucleus?

Answer 15

Pox virus and some giant viruses. They replicate in the cytoplasm, and therefore must bring their own machinery instead of using the host cell machinery

Question 16

Overview of viral/cellular gene transcription and RNA processing (4)

Answer 16

1. DNA undergoes transcription and the transcript is capped. Guanine transferase and triphosphatases are involved in capping, which is a co-transcriptional modification
2. Transcription produces pre-mRNA, which contains introns.
3. Pre-mRNA undergoes polyA addition and splicing to make mature mRNA. For some retroviruses, immature mRNA is still sent to the nucleus
4. Mature mRNA is trafficked into the nucleus for translation

Question 17

Promoters

Answer 17

Present on the 5’ side of any gene and contains certain signature sequences

Question 18

2 methods of transcriptional regulation

Answer 18

Transcription factors and promoters

Question 19

Initiator sequence

Answer 19

The sequence where transcription initiates. The initiator and TATA sequence form the core promoter

Question 20

TATA sequence

Answer 20

A highly conserved region in promoters. Has a DNA consensus site that attracts a variety of TATA sequence binding proteins. The initiator and TATA sequence form the core promoter

Question 21

Local regulatory sequences

Answer 21

Where specific transcription factor binding sites exist. They bind to modulate the rate of transcription from the promoter

Question 22

Which regions does the promoter consist of?

Answer 22

1. Local regulatory sequences
2. Core promoter- TATA and initiator

Question 23

Transcriptional control region components (3)

Answer 23

1. Distant regulatory sequences
2. Local regulatory sequences
3. Core promoter

Question 24

Distant regulatory sequences

Answer 24

Also known as enhancers or silencers. Proteins that bind to these sequences can still form long-range interactions with the core promoter, and modulate transcription using this promoter

Question 25

Transcriptional control regions in adenovirus

Answer 25

Uses the Ad2 major late promoter- this promoter transcribes the late genes. Downstream, there are sites for transcription factors like Cbf and Usf1 to bind. There is also an Ad2 early promoter

Question 26

Transcriptional control regions in SV40

Answer 26

The SV40 early promoter induces transcription of the early genes. Contains multiple binding sites for transcription factor SPI

Question 27

Initiation of transcription by RNA polymerase 2

Answer 27

1. Necessary binding proteins are recruited to the site in a sequential manner. TATA binding proteins bind to DNA. RNA polymerase 2 is then recruited 2. RNA polymerase separates 2 DNA strands, uses one of them as a template, and initiates transcription

Question 28

Types of regulatory proteins (3)

Answer 28

1. DNA binding proteins- bind directly to DNA in a sequence specific manner 2. Co-activators- bind to other proteins bound to DNA, rather than to DNA directly 3. Chromatin modulators

Question 29

Chromatin modulators

Answer 29

Modulate the 3D organization and packaging of the genome. Can modify histones and the DNA itself. When chromatin is closed, transcription initiation factors can't access the promoters, repressing transcription. Examples- HMT and HDAC (repressive), HAT (activating)

Question 30

Enhancer binding proteins

Answer 30

These proteins use their DNA binding domains to bind to the enhancer, and use other domains to bind to TATA. In doing so, they form a loop near the initiation complex and can modulate transcription

Question 31

NF-KB signaling

Answer 31

Activated by a variety of viruses. HIV and EBV both have signaling cascades that converge on NF-KB signaling. NFKB will then bind to the appropriate sequence on promoters. Other viruses secrete proteins that directly modulate NFKB signaling

Question 32

Why is NFKB signaling important for viruses?

Answer 32

Many DNA viruses have a NFKB binding site in their promoters

Question 33

HIV promoter

Answer 33

Has long term repeats on both ends. The 5' LTRs function as promoters. The promoter also contains NFKB binding sites that NFKB will bind to when it is activated and improve transcription

Question 34

Strategies for viral DNA transcription (2)

Answer 34

1. Host proteins plus one viral protein 2. Host proteins plus more than 1 viral proteins involved in viral DNA transcription- larger viruses

Question 35

Host proteins + 1 viral protein transcription strategy

Answer 35

The viral proteins transcribe late proteins and regulate viral DNA transcription. Examples- Parvovirus, Papillomaviruses, Polyomavirus, Hepadnaviruses, Retroviruses

Question 36

2 mechanisms of viral proteins regulating transcription

Answer 36

1. Autoregulatory 2. Cascade regulation

Question 37

Autoregulatory transcription regulation

Answer 37

The viral promoters are recognized by host cell machinery as soon as the virus enters the cell. This allows the first transcription to occur and viral protein to be produced before anything else. Autoregulatory proteins control their own transcription by turning off or inducing expression based on their concentrations

Question 38

Cascade regulation

Answer 38

A gene is expressed to produce a protein, which will regulate the next step. It regulates the transcription of the next gene promoter, therefore regulating the production of the corresponding protein product. The second protein cannot be produced unless the first protein has been produced

Question 39

Regulation of gene transcription in SV40

Answer 39

Gene transcription is divided into early and late genes. One of the the early genes is large T antigen, which goes on to help transcribe the late promoters and regulate the synthesis of viral DNA. When present in high concentrations, large T cell antigen can autoregulate its own expression. This antigen is also an oncoprotein because it can mediate uncontrolled cell proliferation

Question 40

Production of large T antigen

Answer 40

Large T antigen is produced in the cytoplasm, then shuttled into the nucleus, where it mediates transcription and production of the late genes

Question 41

Adenovirus replication cycle stages (3)

Answer 41

1. Immediate early 2. Early 3. Late stage- marked by onset of DNA replication and transcription of late genes

Question 42

Antirepression

Answer 42

Adenovirus- Late genes are repressed until a certain point in the replication cycle. The early proteins induce expression of these genes

Question 43

Adenovirus replication proteins

Answer 43

1. EIA- immediate early protein. Produced by and recognized by host cell transcription machinery. Drives the transcription of early genes 2. E2- early gene. Goes on to drive the expression of late genes 3. L4- late genes. Produce structural proteins. IVa2 is also produced during this stage, and stabilizes the production of E1A proteins

Question 44

Adenovirus E1A stimulation of transcription (4)

Answer 44

1. Immediate early proteins are expressed, but early proteins are repressed by the binding by E2f and retinoblastoma protein 2. Retinoblastoma recruits histone deacetylases to keep the chromatin in closes conformation and repress transcription 3. When E1A has been produced from the immediate early promoter, it binds to retinoblastoma in the repressor complex. It then kicks out retinoblastoma from the binding site 4. Once retinoblastoma is gone, there is no more repression, and the early promoter can be transcribed

Question 45

Adenovirus replication cycle (5)

Answer 45

1. After the partially destabilized capsid docks on the nuclear pore region, the virus' double stranded DNA genome is released 2. The genome can be directly utilized by the host machinery to transcribe the first protein- immediate early genes 3. EI genes can go back into the nucleus and mediate the transcription of early genes 4. Early genes allow DNA replication and transcription of the late proteins 5. Late structural proteins form the virus particle, where the genomic copies can be packaged

Question 46

HSV DNA transcription (6)

Answer 46

1. When the viral particle is uncoating its genome in the cell, it is also uncoating VP16 2. VP16 has a nuclear localization signal, so it brings the DNA to the nucleus 3. Once in the nucleus, VP16 will bind to the immediate early promoter and transcribe it to produce 2 proteins- ICP4 and ICP0 4. ICP4 undergoes autoregulation- it can repress the immediate early promoter once enough protein has been produced 5. ICP0 has a cascade mechanism- it induces the expression of both early and late promoters 6. Once the late proteins are expressed, DNA replication can begin

Question 47

VP16

Answer 47

For HSV, even to make immediate early proteins, it needs to package a viral protein inside the virus particle.

Question 48

HSV sequence of gene expression (4)

Answer 48

1. The virus fuses at the plasma membrane and releases the capsid 2. The capsid docks on the nuclear pore, releasing the viral genome and VP16. Due to the nuclear localization signal, VP16 can meet the genome in the nucleus 3. VP16 binds to the immediate early promoter, transcribes it, and produces alpha proteins ICP4 and ICP0 4. These proteins induce additional transcription and make beta proteins, which carry out DNA replication