Viral Replication and Enzymes

Eukaryotic gene expression is regulated at many stages.

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Require host cell nucleus and host DNA polymerase

Replicate in host cell cytoplasm with viral polymerases

In Groups: How do different viruses replicate in host cell?

To understand how virus will interact with the host during replication, always consider these two questions….

• How are viral mRNAs produced from the viral genome?
• Viruses will always hijack the host cell ribosomes for protein�production so the key question is how are viral mRNAs produced�
• What serves as the template(s) for viral genome replication?
• Viral genome replication will always require a polymerase but the source and mechanism of action of different polymerases is dependent on viral genome structure and composition

We want to explore how this works for DNA and RNA viruses (dsRNA, +ssRNA, -ssRNA and retroviruses)....

Host and viral DNA polymerases

Most DNA polymerases have high fidelity, making a mistake only 1 in a billion nucleotides on average (for eukaryotic polymerases).

DNA polymerases require Mg²⁺ ions that serve as active site cofactors as well as triphosphate DNA nucleotides for replication.

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Viral DNA Polymerase

Bacterial DNA Polymerase

Human DNA Polymerase

Active site of DNA polymerase

Observe how R-groups help stabilize reactants/substrate in the catalytic site (active site) of an enzyme

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Observe how Mg²⁺ (metal ion) is a cofactor in the enzyme active site to help stabilize reactants/substrates in precise orientation needed for new bond formation.

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Observe how partial charges (dipole, polarity) on P and O plays a role in new bond formation (black arrow) and orientation stabilization

Host cell RNA polymerase

Most RNA polymerases have lower fidelity, making a mistake on average 1 in every 100,000 nucleotides.

Why is there a significant difference in the fidelity rate of RNA polymerases versus DNA polymerases?

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RNA processing enzymes (in eukaryotes)

Poly-adenine tailing enzyme on the 3’ end of eukaryotic mRNAs

5’ guanine capping enzyme

dsRNA (antisense-stranded) Viral Replication

Viral RNA-dependent RNA polymerase

SARS-CoV-2 proteins nsp7 and 8 combine together together with nsp12 to form the functional RNA-dependent RNA polymerase

Viral RNA-dependent RNA polymerase

SARS-CoV proteins nsp7 and 8 combine together as a ring with quaternary structure that is thought to encircle a piece of the viral RNA genome to begin replication in the host cell.

RNA-dependent RNA polymerase inhibitors

Remdesivir was originally developed in hopes that it would prevent Ebola and/or influenza viral replication.

Remdesivir is a modified natural nucleotide triphosphate that, once-added to the growing RNA strand, blocks further RNA strand elongation.

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https://pdb101.rcsb.org/motm/249

Retrovirus biosynthesis

Early events

• The virus is uncoated and then using an enzyme known as reverse transcriptase, the ssRNA genome is converted into cDNA and then proviral dsDNA in a multi-step process (having often begun in the capsid)
• The proviral dsDNA is then imported into the host nucleus along with the protein, integrase, that then permanently inserts the viral dsDNA into the host chromosomal DNA (beginning of proviral latency)

Late events

• The proviral region is then slowly transcribed by host RNA polymerase II to make viral proteins and replicate the viral genome
• This components are exported and assembled in cytoplasm and slowly but steadily released through budding to spread mature virions

HIV biosynthesis enzymes

HIV reverse transcriptase

What are the main functions of the reverse transcriptase enzyme?

HIV reverse transcriptase inhibitors

Reverse transcriptase has relatively low fidelity (makes mistakes up to about one in every 2,000 bases that it copies)

This allows the virus to evolve in response to inhibitors.

Shown here is the reverse transcriptase inhibitor, nevirapine.

Does this inhibitor seem to work competitively or non-competitively?

HIV integrase

HIV integrase is designed to splice (or integrate) viral DNA into the host cell nuclear DNA so that the virus can permanently replicate every time the host cell divides.

What are the consequences of viral DNA integration into the host cell?

HIV antiretroviral therapy

Retroviral treatment for HIV involves a cocktail of drugs known as HAART that together inhibit viral….

• Fusion (gp120, gp41 or CCR5)
• Reverse Transcription (RT)
• Integration via Integrase (IN)
• Maturation via protease (PR)

Multi-drug HAART HIV-prevention therapy

How do viruses cause disease?

Viruses damage host cells/tissues by...

• reducing gene expression capacity
• depleting cellular resources
• causing cell lysis or apoptosis
• promoting tumorigenesis
• creating damaging immunological response

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Viral genome size vs. mutation rate

Genetic drift - the process by which viruses mutate (and therefore adapt/evolve) with some predictability due to their polymerase error rate

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Genetic shift - the process by which genetic recombination can lead to viral genetic changes (either by a crossing over mechanism or genome segment reassortment for viruses with segmented genomes)

miRNA defense against viruses�

RNA interference (siRNA/miRNA)� defense against viruses�

RNA Interference pathways

2020 Nobel Prize in Chemistry - CRISPR/Cas system

Emmanuelle Charpentier, Ph.D.

Umeå University, Sweden

Max Planck Institute, Germany

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Jennifer Doudna, Ph.D.

UC Berkeley

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CRISPR-Cas defenses against viruses

CRISPR-Cas gene expression (in bacteria)

CRISPR-Cas loading and targeting (in bacteria)

CRISPR-Cas9 system (in bacteria)

Full complex contains:

1. Cas protein (Cas9)
2. Guide RNA (single synthetic or tracrRNA:crRNA hybrid)
3. Target DNA