BIOL 512 Exam 3

1. Single-stranded DNA is template for synthesis

2. Each strand is produced via semi-conservative replication

3. Replication begins at the origin and ends at the termini of the template

4. DNA synthesis is catalyzed by DNA-dependent DNA Polymerase

5. Requires a primer with a free 3’ OH end

• Alpha→ assists and priming and binding Okazaki fragments, slow at making DNA

• Delta→ Leading and lagging strand DNA synthesis

• Epsilon→ Leading and lagging strand DNA synthesis

1. Origin binding proteins identify the origin of replication

2. Helicase unwinds DNA

3. Single-stranded binding proteins prevent unwanted binding

4. Primase makes the primer

5. DNA-Dependent DNA Polymerase

6. Processivity factor

7. RNAase gets rid of RNA primer

8. Ligase

9. Topoisomerase I & II

Yes, the replication of DNA-containing viruses requires the expression of at least one viral protein

There is no mechanism for filling in the gap at the end of each DNA strand

1. Bi-directional DNA replication

   1. DNA polymerase adds on both directions in circular genome, the DNA (old and new strand) are intertwined, topoisomerase II separates the strands. Similar to E. coli or plasmid DNA rep.

2. Rolling Circle DNA replication

   1. DNA forms a loop and moves along the linear strand. Results in no gaps at the end of DNA. Occurs in HSV-1 and similar to the bacteria phage- gamma phage

3. Displacement synthesis

   1. The new strand simply “displaces” or takes the place of the non-template DNA strand. Seen in adenoviruses

4. Rolling hairpin DNA replication

   1. Inverted terminal repeats form hairpins and elongate and unfold. Seen in parvoviruses

RNA Polymerase I→ RNAs (18S, 28S)

RNA Polymerase II→ mRNA, both cellular and viral

RNA Polymerase III→ tRNA, 5srRNA in cellular, adenovirus vaRNA

Promoters contain 3 distinct regions

1. Distant regulatory regions

2. Local regulatory sequences

3. Core promoter

The core promoter contains the TATA box and the +1 site where the initiator sequence is located. It aids in the assembly of proteins in complexes.

Yes, some make transcriptional regulatory proteins which either directly or indirectly bind to DNA/RNA

Several encode viral proteins that regulate transcription at various stages of the viral life-cycle

1. G-cap→ 5’ cap aligns mRNA on the ribosome by the addition of GTP in opposite orientation, then a methyl group added in a 5’ to 5’ linkage. Protects the 5’ end of the mRNA from degradation and allows binding to the ribosome.

2. Polyadenylation (poly-A tail)→ addition of AMP molecules to the 3’ end of RNA. Poly-A tail stabilizes mRNA and increases translation efficiency.

3. Splicing→ removal of introns from pre-mRNA

• Increases coding capacity from a limited genome (more proteins)

• Temporarily regulate viral gene expression

• Control of gene expression balance (spliced vs. unspliced)

• Structure of supercoiled DNA

• Identification of eukaryotic origins of DNA replication

• Model for mammalian DNA replication

• The organization of eukaryotic promoters

• Alternative splicing

• Identification of a nuclear localization signal of a protein

• Insights into cell cycle regulation and the functions of tumor suppressor genes

Early (E) transcription→ viral DNA replication→ Late (L) transcription

E transcripts are involved in the cell cycle and viral DNA replication

L proteins comprise the capsid

Sp1= Stimulatory Protein 1

• Large family of transcription factors

• Binds as a monomer to DNA

• Binds to the consensus sequence (G-C rich)

• The first transcription factor identified that bound to a specific DNA sequence

1. Cooperative binding→ Tef-1 in Element A

2. Interactive: Tef-1 and Tef-2 in Element B

3. Oligomerization→ NF-Kappa B in Element B

• Origin of replication

• Packaging

• Enhancer

• Promoters

Essentially everything needed for replication

Intermediate early (IE)→ Early→ viral DNA replication→ Intermediate→ Late

13S-E1A→ Binds to Sp1 and TATA binding protien

Sp1 (cellular)→Binds to Sp1 binding site

TBP and (cellular)→ Binds to TATA Box

E1B→ Complexes with a cellular and viral protein for L transcript transport

E1B + E4orf6→ Stops cellular transcripts from leaving the nucleus (gets rid of competition, don’t want cellular transcripts, want viral transcripts)

The cell activates the PKR pathway

• PKR can inactivate eIF2 by phosphorylating it. No eIF2→ no transcription initiation complex→ no transcripts

Adenovirus blocks PKR activity

• Adenovirus proteins can bind to PKR and inhibit its activity

• eIF2 can bind to the transcript and then initiate translation

• Enveloped and a large genome (152 kb)

• Large diameter (225 nm)

• Icosahedral capsid

• Linear dsDNA genome

• Tegument contains viral protein

Virion Host Shutoff (VHS) is a tegument protein that shuts off host protein synthesis

The tegument protein VP16 complexes with two factors, Oct-1 and Hcf to enhance transcription of IE genes

ICP4 is a major transcription factor of HSV-1

• Stimulates transcription of E and L promoters

• When expressed at high levels, it down-regulates the expression of its own gene and other IE genes by binding to DNA sites near transcription initiation site.

• Helicase/primase activity (UL5, UL8, and UL52)

• Origin binding protein (UL9)

• Single-stranded DNA binding protein (UL29)

• Viral DNA polymerase (UL30)

• Processivity factor (UL42)

L genes are involved in egress, assembly, and structural components of the virion

IE→ E→ Viral DNA replication→ L proteins

Parvovirus B19 is made up of

• Two proteins (NS1 and VP1/2)

• Linear single strand of DNA

• 5.5 kb genome

• Respiratory (most common)

• Vertical (mother to baby)

• Blood

Typically infects the bone marrow erythroid progenitor cells (immature erythrocytes)

1. Fifth’s Disease

2. Arthropathy

3. Transient aplastic crisis

4. Persistent anemia

5. Hydrops fetalis and congenital anemia

1. Erythema infectiosum→ prodromal symptoms rash

2. Arthropathy→ symmetric joint pain in hands/feet

3. Erythrocyte aplasia→ Blood testing for viral load, B19 antibodies, hemoglobin

4. Intrauterine/Vertical infection→ hydrops fetalis, swollen liver, swollen belly)

• Fludarabine→ prevents elongation of DNA strands

• Treating symptoms

• Acute respiratory disease (ARD)

• Febrile upper tract infection

• Pharyngoconjunctival fever

• Acute disease

• Pertussis-like disease

• Pneumonia

• Acute hemorrhagic cystitis

• Epidemic keratoconjunctivitis

• Gastroenteritis

• Aerosol

• Close contact

• Oral-fecal route

• Fomites

The virus infects mucoepithelial cells of respiratory tract and persists in lymphoid tissue

Antibody production is critical for recovery

Strict handwashing helps to prevent adenovirus infections.

Methods for detection include

• Antibodies

• Adenovirus PCR

A reverse transcriptase enzyme that converts RNA to DNA and packages DNA into their virions

Causes hepatitis, cirrhosis (scarring of the liver), and hepatocellular carcinoma.

Through direct contact, blood/body cellular fluids

Hepatocytes are weakened, CD8+ cells don’t help much either

1. Type 1 Interferons

2. Nucleoside analogue inhibitors

3. Vaccine

• Linear double stranded DNA genome (130-380 kb)

• Large brick-shaped virion

• Enveloped virus

• Two lateral bodies which contain virion associated enzymes and proteins

There are two types of infectious virus particles

1. Intracellular mature virion

2. Extracellular enveloped virion (contains a lipid envelope)

Uncoating at the plasma membrane and receptor-mediated endocytosis

1. Viral replication occurs in the cytoplasm

2. All enzymes are required for the production of viral mRNAs and viral DNA replication must be encoded by the virus

3. Linear double stranded DNA with inverted terminal repeats that form terminal loops

Early→ viral DNA replication→ intermediate→ late

Early genes

• Play a role in DNA replication

• Intermediate transcription factors

• RNA polymerase

• Growth factors

• Evading the immune response

Intermediate genes

• Late transcription factors

Late genes

• Encode proteins involved in virion assembly and egress, structural components of the virion and E transcription factors

1. Cell lysis

2. Release at the plasma membrane

3. Release at the plasma membrane using an actin tail

1. Monkeypox

2. Cowpox

3. Variola

4. Vaccina

Variola virus is the causative agent of smallpox

Is spread by

• Direct contact with infected bodily fluids

• Contaminated objects, or fomites

• Aerosol entering upper respiratory tract

Onset symptoms: fever, malaise, headache, bodyaches

• Rash develops in mouth, sores break open, large amounts of virus are released

Spread by Christopher Colombus and killed off the indigenous population

The virus used in the smallpox vaccine is related to smallpox virus, but causes milder disease.

The WHO started a global smallpox eradication project in 1959 and the eradication program took off. The last case of smallpox was in Somalia in October 1977

HSV is latent in neurons

1. Stress causes the viral particles to migrate along a sensory neuron to a site where a cold sore develops

2. Goes away, becomes latent again

3. Stress reactivates viral production

We know that there are no activators, repressors are present, it initiates an immune response.

Latency-associated transcripts (LATs) express microRNAs that suppress apoptosis, allowing the virus to become latent.

1. DNA damage response→ causes thymine dimers to form and body wants to get rid of them.

2. Heat shock response→ Unfolded proteins either refold or degrade.

3. Immunosuppression→ Elevated glucocortocoid levels and a long term fight or flight response

4. Hormonal changes→ menstruation and androgen receptors

ICP0 localizes to the antiviral ND10 and directs its degradation.

It impairs the interferon response and activates viral transcription.

On day 1

• MHC class I expression by HSV-1 is suppressed

• CD4+ T-cells and Natural Killer Cells move to site of infection

On day 2

• CD4+ and NK cells release cytokine gamma-interferon which makes the MHC I receptor reattach to the surface

Acyclovir suppresses HSV lytic infection and viral DNA replication. It is a nucleoside analogue of Guanine and targets thymidine kinase. It is phosphorylated by HSV TK more efficiently than cellular TK.

Acyclovir terminates the nucleotide chain. It’s sugar lacks C1-C4, so there is no 3’ OH group to add nucleotides to.

It is phosphorylated by HSV TK more efficiently than cellular TK.

Actively dividing cells make TK to replicate their DNA, however, terminally differentiated neurons DO NOT replicate their DNA, so cellular TK is not expressed in neurons.

1. HIV attachment to host cell

2. HIV fusion with host cell membrane

3. Production of HIV DNA

4. Integration of viral DNA into host

5. Transcription into RNA

6. Translation into viral proteins

7. Migration to cell surface and assembly

8. Viral budding

9. Mature HIV produced

Reverse transcriptase, terminates the synthesis of DNA

Many antiviral drugs target against reverse transcriptase and proteases.

Fusion inhibitor

Entry inhibitor

Integrase inhibitor

Highly Active Anti-Retroviral Therapy

Combines 3 or more anti-viral medications and do not see death of CD4+ helper T-cells

Mantadine and Rimantadine inhibit influenza A replication, block the flow of protons through a channel and prevent initiating infection.

Relenza and Tamiflu: Neuraminidase inhibitors prevent the receptors from binding to neuraminidase which prevents virion release.

HSV 1 causes active lesions and oral-facial infections

HSV-2 causes infections on the genitals

Can also cause pharyngitis, keratitis, hepatitis, esophagitisW

Direct contact with active lesions or with people who are shedding HSV from mucocutaneous surfaces

Collection of CSF and a positive HSV PCR from CSF

Encephalitis is the inflammation of the brain parenchyma and is hallmarked by fever, headache, and an altered level of consciousness

Meningitis is the swelling on the meninges however the cerebral function remains normal.

Approximately 10-20% of cases are acute encephalitis