virology lec.4 molecular bio/host cell constraints (cont’d)

Virus replication cycles: one-step growth curves

One-step growth experiments:

• a way to study a single replication cycle

• Provide information about events at each step of the infection cycle

• Enders, Welles, and Robbin’s developed cell culture techniques in the 1940s

Viruses are obligate intracellular parasites-

Virus particles are too large to diffuse across the plasma membrane

Key steps of the viral replication cycle

1. Attachment (adsorption)- no specificity

2. Penetration (entry)

3. Uncoating (disassembly and localization)

4. Genome replication and gene expression

5. Assembly

6. Maturation

7. Egress

Step 1: attachment (adsorption) - no specificity

• attach to specific receptor molecules on cell surface (one or more as a receptor)

• Cell surface receptors (essential for all viruses except fungi and plants): proteins, glycoproteins, carbohydrates, glycolipids

- silicon acid as a receptor for influenza virus, known in 1985

• coreceptors (CXCR4/CCR5 for HIV)

Step 2: penetration (entry)

• Cathrine-coated pits

• Endoscopes

• Ph dependent or pH independent

Step 3: Uncoating (disassembly and localization)

• removal or degradation of the capsid to release genome into host cell

Step 4: Genome replication and gene expression

Two events critical to viral infection

• Production of virus structural proteins and enzymes

• Replication of the virus genome (dsDNA, ssDNA,

dsRNA, ssRNA)

dsDNA virus

Most dsDNA viruses use cell’s nucleus for DNA replication and RNA transcription

dsDNA enters nucleus, forms an episome

ssDNA viruses

• must convert linear ssDNA to dsDNA form

• Host cell does not contain any DNA-dependent RNA polymerases to convert ssDNA into mRNA

ss/dsDNA viruses (using an RNA intermediate)

Ex: hepadnavirus- dsDNA virus infect and cause hepatitis

• HBV specifically infects humans and produce spherical virus particles known as Dane particle

• dsDNA enters nucleus, forms an episome

• Virus does not possess integrase activity

Genome may be …

ss or ds, (+) or (-) sense

Type of genome determines what?

First step after Uncoating (translation, transcription or RNA replication)

RNA-dependent RNA polymerase

to synthesize genomes into the host cell

dsRNA viruses

Carry own RNA-dependent RNA polymerase within vision particle

+ssRNA viruses

• translated directly using host cell protein synthesis machinery

• RNA in the virus particle functions as mRNA

• Carry on RNA-dependent RNA polymerase

-ssRNA viruses

• Encode own RNA-dependent RNA polymerases

• Transcribes −ssRNA genome into +ssRNAs

• Also synthesizes viral/progeny genome

+ssRNA genomes using a dsDNA intermediate

Retroviruses

- Unique biology

- Integration of viral complementary DNA (cDNA)

Step 5: assembly

Studied via live-cell imaging technology

• All components of virus assembled into a stable

particle

• Viral factories (viroplasm)

• May serve as a way for viruses to hide from host

cell antiviral defenses

Step 6: maturation

• Stage in life cycle of virus when it becomes infectious

• Viral or cellular proteases often involved

Step 7: egress

• Newly formed viruses released upon lysis

• Some viruses produce an enzyme during replication that destroys cellular receptors so newly assembled virions do not get stuck when exiting cell

RNA viruses mutate more rapidly than DNA viruses. Why and how does it affect it?

RNA polymerases lack proofreading ability and thus make more mistakes than DNA

Keys to antiviral drug development

• Must target a process essential for viral replication

• Must be active against virus without being “toxic” to the host organism

Examples:

• Acyclovir for HSV-1

• Azidothymidine (AZT) for HIV

Sources of novel antivirals

• High-throughput screening (1000 of compounds from

natural sources)

• Bioprospecting adopted to describe searches for new

bioactive compounds in natural sources – for

commercial use

• Reverse pharmacology- screen natural products for

bioactive compounds

• Data mining- retrieve knowledge from ancients texts

(Chinese medicine)

Explain how viruses have been our “eyes” into cells

Viruses have been our “eyes” into cells by hijacking’s cellular functions to optimize viral replication and vision production. Being able to observe virus-host cell interactions in real time helps to learn more about cell structure and how the virus interacts with cellular proteins.