BIOL 512 Exam 3

What are the major steps in replicating DNA?

What are the major steps in replicating DNA?

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

What are the mammalian DNA polymerases, and what specific functions do they have?

• 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

What are the 9 components of DNA replication?

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

Does viral DNA replication require viral proteins?

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

What is the major problem with viral DNA replication (of a linear genome)?

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

What are 4 mechanisms DNA viruses use to replicate their genome?

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

What types of RNA polymerases synthesize what types of RNA?

RNA Polymerase I→ RNAs (18S, 28S)

RNA Polymerase II→ mRNA, both cellular and viral

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

What are the parts of a promoter, and how do they help stimulate transcription?

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.

Do viruses make proteins involved in transcription?

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

What are 3 ways cellular mRNAs are processed?

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

Why do several viruses splice their RNAs?

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

• Temporarily regulate viral gene expression

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

What has been discovered by discovering polyomaviruses?

• 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

What is the gene cascade of polyomaviruses?

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

What are the general functions of proteins expressed in each cascade?

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

L proteins comprise the capsid

What is Sp1, and why is it important for polyomavirus transcription?

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

What are the 3 types of SV40 enhancer elements? (polyomaviruses)

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

What are the specific activities found in the SV40 control region? (polyomaviruses)

• Origin of replication

• Packaging

• Enhancer

• Promoters

Essentially everything needed for replication

What is the temporal cascade of adenovirus gene regulation?

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

What factors are involved in the stimulation of adenovirus late gene transcription?

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

Sp1 (cellular)→Binds to Sp1 binding site

TBP and (cellular)→ Binds to TATA Box

How and what proteins control the transport of late adenovirus mRNAs? Do they affect host mRNA transport? If so, how?

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)

How does a host cell try to stop the translation of adenovirus mRNAs, and how does the virus overcome this block?

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

What are the components of a herpes simplex virus (HSV) viral particle?

• Enveloped and a large genome (152 kb)

• Large diameter (225 nm)

• Icosahedral capsid

• Linear dsDNA genome

• Tegument contains viral protein

What is VHS? How does it help viral replication?

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

How is the transcription of HSV IE genes activated?

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

What are the activities of HSV IE protein, ICP4?

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.

What functions do essential HSV E proteins have?

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

• Origin binding protein (UL9)

• Single-stranded DNA binding protein (UL29)

• Viral DNA polymerase (UL30)

• Processivity factor (UL42)

What activities do HSV L proteins have?

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

What is the order of the gene cascade during HSV lytic infection?

IE→ E→ Viral DNA replication→ L proteins

What is the parvovirus B19 virion structure? What is its genome size?

Parvovirus B19 is made up of

• Two proteins (NS1 and VP1/2)

• Linear single strand of DNA

• 5.5 kb genome

What are parvovirus routes of transmission?

• Respiratory (most common)

• Vertical (mother to baby)

• Blood

What is the primary cell type that Parvovirus B19 infects?

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

What are the 5 major diseases that Parvovirus B19 causes?

1. Fifth’s Disease

2. Arthropathy

3. Transient aplastic crisis

4. Persistent anemia

5. Hydrops fetalis and congenital anemia

How is a parvovirus infection diagnosed (what tests/assays are used?)

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)

What are specific treatments against parvovirus B19 infection?

• Fludarabine→ prevents elongation of DNA strands

• Treating symptoms

What types of diseases does adenovirus cause in humans?

• Acute respiratory disease (ARD)

• Febrile upper tract infection

• Pharyngoconjunctival fever

• Acute disease

• Pertussis-like disease

• Pneumonia

• Acute hemorrhagic cystitis

• Epidemic keratoconjunctivitis

• Gastroenteritis

How are adenoviruses transmitted?

• Aerosol

• Close contact

• Oral-fecal route

• Fomites

What are the disease mechanisms of adenovirus, and what is important for recovering from infection?

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

Antibody production is critical for recovery

How does one prevent adenovirus infections, and what are the methods for detection?

Strict handwashing helps to prevent adenovirus infections.

Methods for detection include

• Antibodies

• Adenovirus PCR

What enzyme do all hepadnaviruses encode or express?

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

What is a sign of HBV infection, and what diseases does it cause?

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

How is HBV transmitted?

Through direct contact, blood/body cellular fluids

How and what host cells participate in the development of HBV liver cancer?

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

What 3 types of therapies are available to limit HBV infection in humans?

1. Type 1 Interferons

2. Nucleoside analogue inhibitors

3. Vaccine

What are the parts of a poxvirus virion, and what are the two types of virions?

• 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)

How do poxviruses enter cells?

Uncoating at the plasma membrane and receptor-mediated endocytosis

What are distinct properties of poxviruses?

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

What is the order of the gene cascade for HSV poxviruses?

Early→ viral DNA replication→ intermediate→ late

Describe functions/activities for the poxvirus proteins based on their gene class.

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

What 3 ways can poxviruses exit the cell?

1. Cell lysis

2. Release at the plasma membrane

3. Release at the plasma membrane using an actin tail

What are 4 poxviruses that can infect humans?

1. Monkeypox

2. Cowpox

3. Variola

4. Vaccina

What is variola virus? How is it transmitted?

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

What are the symptoms of smallpox?

Onset symptoms: fever, malaise, headache, bodyaches

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

What role has smallpox played in world history?

Spread by Christopher Colombus and killed off the indigenous population

What is vaccinia virus, and how was it used in relation to smallpox?

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

What was done to eradicate smallpox?

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

What are the major steps in HSV pathogenesis?

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

What is known about HSV latency, and what are hypotheses about how latency is established?

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.

What are stress stimuli for HSV reactivation, and how do these stimuli affect cellular physiology to promote reactivation?

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

What is HSV ICP0, and what activities does it have to promote reactivation?

ICP0 localizes to the antiviral ND10 and directs its degradation.

It impairs the interferon response and activates viral transcription.

What are the factors and temporal events involved in clearing up an HSV-1 infection?

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

What is acyclovir and what are its viral targets?

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.

How does acyclovir stop HSV replication?

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

Why is acyclovir not toxic to cells?

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

Why is acyclovir an effective medication, especially in neurons?

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.

What are the major steps in the HIV life cycle?

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

What is the target of nucleoside analogues for HIV, and how do they work?

Reverse transcriptase, terminates the synthesis of DNA

What are antiviral targets developed for HIV?

Many antiviral drugs target against reverse transcriptase and proteases.

Fusion inhibitor

Entry inhibitor

Integrase inhibitor

What is HAART and why is it an effective suppressive therapy compared to mono-therapy?

Highly Active Anti-Retroviral Therapy

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

What are inhibitors of influenza A replication, their targets, and how do they specifically inhibit IAV?

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.

What are diseases caused by HIV?

HSV 1 causes active lesions and oral-facial infections

HSV-2 causes infections on the genitals

Can also cause pharyngitis, keratitis, hepatitis, esophagitisW

What are routes of HSV transmission?

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

What diagnostic test did Dr. Merino use to identify/verify HSV infection?

Collection of CSF and a positive HSV PCR from CSF

What is the difference between meningitis and encephalitis?

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.

How common is HSV encephalitis in the United States?

Approximately 10-20% of cases are acute encephalitis