Viral Replication Notes

How Viruses Multiply: Viral Replication

Intended Learning Outcomes

By the end of this session, you should be able to:

• Explain how a virus takes over a cell and uses its resources for its own purposes.

• Describe the replication strategy of a DNA virus.

• Describe and explain the replication strategy of an RNA virus, appreciating that some have positive-sense genomes while others have negative-sense genomes.

• Discuss the unique replication strategy of some viruses.

Properties of Viruses

1. Infect animals, plants, bacteria.

2. Size - too small for light microscope (submicroscopic).

3. Classified by morphology, genome & replication.

4. Obligatory intracellular replication.

Viruses lack the genes and enzymes necessary for energy production, so they are unable to replicate on their own. Replication depends on living host cells and is directed by the viral genome to produce the virus components.

Viral Growth Curve

When one virion (one virus particle) infects a cell, it can replicate in approximately 10 hours to produce hundreds of virions within that cell. This amplification explains how viruses spread rapidly from cell to cell. The eclipse period is also shown in the viral growth curve.

Stages of Virus Replication

1. Attachment

2. Penetration

3. Uncoating

4. Synthesis of viral components

5. Assembly

6. Release

1. Attachment (Adsorption)

Occurs to specific receptor sites on the surface of the susceptible host cell. These interactions determine viral host range (e.g., human viruses and plant viruses) and tissue specificity or tropism (e.g. hepatotropic viruses and neurotropic viruses).

2. Penetration

Non-enveloped viruses: Endocytosis

Examples:

• Poliovirus

• Adenovirus

Enveloped viruses: Fusion

Examples:

• HIV

• Herpes viruses

Fusion process:

1. Receptor binding.

2. Virus with host membrane fusion.

3. Release of virion contents into host cytoplasm.

Enveloped viruses can also penetrate by endocytosis and fusion. Examples include Influenza and Hepatitis C virus.

Methods of virus entry:

• Fusion: HIV, Herpesviruses

• Endocytosis + Fusion (low pH): Influenza virus

• Endocytosis: Poliovirus, Adenovirus

3. Uncoating

The nucleic acid is released from the capsid.

4. Synthesis of Viral Components

viral nucleic acid --> Specific messenger RNAs --> viral components according to the type of viral nucleic acid, involving transcription and translation.

Expression & replication of viral genome:

• ‘Early’ viral genes transcribed pre replication

  • Regulate cell NA & protein synthesis

  • Regulate expression of viral genome

  • Code for viral enzymes required for replication of viral nucleic acid

• ‘Late’ viral genes transcribed post replication for synthesis of structural proteins

Replication of DNA viruses:

$1$. Synthesis of early proteins: Transcription of early genes (fraction of the viral genome transcribed prior to initiation of viral DNA synthesis) Translation Parental DNA --> Early mRNAs --> Early proteins
$2$. Replication of virus DNA
$3$. Synthesis of late proteins: Transcription of late genes (fraction of the viral genome transcribed after initiation of viral DNA synthesis). Replication of DNA viruses --> Progeny DNA --> Late mRNAs --> Late proteins
$4$. Assembly of nucleocapsids

5. Assembly

Nucleic acids are enclosed within the protein coats to form mature viruses (virions).
This occurs in:

• Nucleus of host cell, e.g., herpes viruses

• Cytoplasm, e.g., polioviruses

6. Release

New viruses are released either by:
a- Lysis of host cell in non-enveloped viruses
b- Budding through the cell membrane in enveloped viruses

Example: Adenovirus

1. Adsorption

2. Penetration

3. Uncoating: Viral DNA in the nucleus

4. Synthesis of early mRNA

5. Early Translation: Synthesis of early proteins (enzymes such as DNA polymerase)

6. DNA Synthesis and Late Transcription: Synthesis of progeny viral DNA and late mRNA

7. Late Translation: Synthesis of late proteins (capsid proteins)

8. Assembly

9. Release of Virus and Lysis of Infected Cell

General Viral Replication

Infecting virus (enveloped or non-enveloped) --> attachment (receptor, host cell, nucleus) --> penetration --> uncoating --> replication (synthesis of viral messenger RNA via host machinery, synthesis of viral protein for new capsids, synthesis of viral nucleic acid) --> assembly (capsids from around nucleic acid) --> release (by budding, forming envelope; or by cytolysis if no envelope) --> capsid shed

Viral Growth Curve

Shows eclipse period, virus yield, and the relationship between viral nucleic acid and virions per cell over time (hours).

Viral Nucleic Acid Replication Strategy

Viral Nucleic Acids

Viruses can have DNA or RNA as their genetic material.

Types of Viral Nucleic Acids

• DNA: ssDNA, dsDNA

• RNA: ssRNA (+ sense, - sense), dsRNA

Strandness of Genome

• Single-stranded nucleic acid: RNA, DNA

• Double-stranded nucleic acid

DNA Structure and + Sense

$5' \text{ to } 3'$ direction example: ATG, $\text{transcription} \rightarrow$ mRNA, $\text{translation} \rightarrow$ Proteins.
The + sense strand can be directly translated.

Baltimore Classification

All viral genomes lead to mRNA.

• Viral nucleic acid $\downarrow \text{Transcription}$

• Specific messenger RNAs $\downarrow \text{Translation}$

• Viral components

Important Facts and Definitions

• RNA -> RNA: RNA-dependent RNA polymerase

• RNA -> DNA: RNA-dependent DNA polymerase - reverse transcriptase

• DNA -> RNA: DNA-dependent RNA polymerase (Viral or Host)

• Viral Host DNA -> DNA: DNA-dependent DNA polymerase (Host)

Baltimore Classification

Summarized:

1. dsDNA (±): Transcription by cell's DNA-dependent RNA polymerase --> mRNA (+)

2. ssDNA (+ or -): Cell's DNA polymerase -->

3. dsRNA (±): Viral dsRNA-dependent RNA polymerase --> mRNA (+)

4. ssRNA (+): Genome acts as mRNA

5. ssRNA (-): Viral ssRNA-dependent RNA polymerase --> mRNA (+)

6. ss Retrovirus RNA (+): Viral reverse transcriptase -->

7. Partially ds hepadnavirus DNA (±): Viral DNA polymerase -->

DNA Viruses

Double-stranded DNA genome (dsDNA):

Examples:

• Adenoviruses: Sore throat, conjunctivitis, gastroenteritis, hemorrhagic cystitis

• Herpes simplex virus (HSV) types 1 and 2: Cold/genital sores, encephalitis

• Papilloma viruses: Warts, cervical cancer

Single-stranded DNA genome (ssDNA):

• Human parvovirus B19: Erythema infectiosum, aplastic crisis

Replication Details for DNA Viruses

• dsDNA: mRNA translation. Transcription by Host’s DNA-dependent RNA polymerase enzyme (Poxviruses use their own polymerase structural proteins & enzymes) Baltimore group 1

• ssDNA: mRNA translation. Transcription by Host cell DNA-dependent RNA polymerase enzyme Structural proteins & enzymes ds DNA DNA viruses (2) Parvoviruses only example ssDNA Host cell DNA polymerase Baltimore group 2

Most DNA viruses, with one exception, replicate in the nucleus and use the host cell DNA-dependent RNA polymerase to synthesize their mRNA. The poxviruses are the exception because they replicate in the cytoplasm, where they do not have access to the host cell RNA polymerase. They therefore carry their own polymerase within the virus particle.

RNA Viruses

Single stranded (positive sense) Examples of $+$ve ss RNA viruses:

• Rhinoviruses: Common cold

• Enteroviruses: Meningitis, paralysis

• Hepatitis A virus: Hepatitis

• Hepatitis C virus: Hepatitis, cirrhosis, liver cancer

• Noroviruses: Gastroenteritis

Single stranded (negative sense) Examples of $-$ve ss RNA viruses:

• Influenza A virus, Influenza B virus: Influenza

• Measles virus: Measles

• Mumps virus: Mumps

• Rabies: Rabies

dsRNA Viruses

• dsRNA viruses, (e.g., rotavirus $\rightarrow$ infantile gastroenteritis)

• One strand is first transcribed by viral RNA-dependent RNA polymerase into mRNA RNA viruses: ds RNA Baltimore group 3

ssRNA Viruses (Positive Sense Versus Negative Sense)

Positive sense (+ve polarity) Baltimore group 4

• RNA acts directly as mRNA. In host cell:

• Immediately translated by host ribosomes to protein.

Negative sense (- ve polarity) Baltimore group 5

• No immediate translation

• 1st transcribed to +ve sense

• HOW?

Positive Sense

• RNA acts directly as mRNA In host cell:

• Immediately translated by host ribosomes to protein.

Negative Sense

• No immediate translation.

• 1st transcribed to +ve sense.

• HOW?????

• Viral RNA dependant RNA polymerase enzyme

+ve sense ssRNA

structural proteins & enzymes

-ve sense ssRNA

+ve sense ssRNA structural proteins & enzymes translation RNA dep. RNA polymerase translation

Key takeaway

• DNA-Viruses mRNA (by transcriptase enzyme, i.e., host DdRp).

• RNA-Viruses Positive strand (+ssRNA) acts directly as mRNA. Negative strand (-ssRNA) positive RNA strand (mRNA) by viral RNA polymerase (RdRp).

Retroviruses

While most RNA viruses undergo their entire replicative cycle in the cytoplasm, Retroviruses are an exception as they integrate a DNA copy of their genome into the host cell DNA.

Steps

1. BINDING TO CELL SURFACE RECEPTORS

2. ENTRY

3. UNCOATING

4. REPLICATION

5. TRANSCRIPTION

6. TRANSLATION

7. VIRION ASSEMBLY

8. RELEASE

\rightarrow RNA viruses
\rightarrow DNA viruses

RNA Viruses - Retroviruses

+ve ssRNA utilizes unique reverse transcriptase enzyme : ssRNA $\rightarrow$ DNA. Retro = backwards

HIV

• +ve ssRNA

• Reverse transcriptase (Viral RNA dependent DNA polymerase) transcription Structural proteins & enzymes Integration in host cell genome DNA RNA viruses - Retroviruses mRNA

Replication of HIV in Infected Cells

Attachment (Adsorption)

1. gp120 binds tightly to CD4 molecules on the cell surface.

2. Then, binds to a co-receptor.

3. Binding to a co-receptor activates gp41 , triggering fusion between viral envelope & cell membrane.

After Fusion & Uncoating

1. Reverse transcription:
   Reverse transcriptase enzyme uses the virus RNA as a template to build complementary strands of DNA accumulation of proviral, non-integrating HIV DNA.

2. Integration:
   The newly made HIV DNA moves to the cell nucleus, where it is integrated into the host cell DNA with the help of HIV integrase. HIV DNA integrated in the DNA of the cell is called a "provirus".

3. Transcription:
   HIV mRNA is transcribed from the provirus by host cell polymerase.

4. Translation:
   HIV mRNA is transported from the cell nucleus to the cytoplasm, and translated into viral polyproteins and enzymes

5. Assembly and budding (2):
   HIV protease cleaves the polyproteins from immature virions Forming infectious viral particles Released by budding through cell membrane
   Cleavage by the protease enzyme is essential for maturation and production of infectious virus

HIV Replication Cycle

Hepatitis B Virus (Baltimore Group 7)

Partially double-stranded circular DNA genome. The HBV DNA genome is unusual because it is partially double stranded circular DNA and replicates via an RNA intermediate using its own encoded reverse transcriptase (polymerase).

• Synthesizes the missing portion of DNA $\rightarrow$ double-stranded closed-circular DNA (cccDNA).

• Produces genome DNA by reverse transcription with mRNA as the template

Steps

1. Viral entry

2. Uncoating

3. Nuclear import

4. Repair

5. Transcription

6. Translation

7. Encapsidation

8. Assembly & budding

Key Takeaway

Remember HIV and hepatitis B viruses both use Reverse Transcriptase in their infectious cycle and are known collectively as reversiviruses as the infectious cycle involves a step reversing from RNA to DNA

Replication Strategy

DNA viruses replication strategy

• ds DNA viruses

• ss DNA virus

• Partially double-stranded circular DNA virus

RNA viruses replication strategy

• +ve sense ssRNA (any exceptions?)

• -ve sense ssRNA

• dsRNA viruses

Classification of Viruses - Revisited

1. Type of nucleic acid (DNA or RNA)

2. Virus replication strategy

3. Virus morphology (capsid symmetry & envelope)

Baltimore classification - Revisited

Same as previously described Baltimore classification

Suggested Reading

• Chapter 19. Ward K N, McCartney A C, Thakkar B. Notes on Medical Microbiology 2nd edition, Churchill Livingstone, 2009.

• Review of Medical Microbiology and Immunology, Warren Levinson

• Part III: Basic Virology

• Chapter 29: Replication

• Part IV: Clinical Virology

• Chapter 45: Human Immunodeficiency Virus

Here's a table summarizing dsDNA, ssDNA, and RNA viruses: