Unit 9: Viral Replication

Very Important

Viral cells always rely on host cell for translation, so they need to generate useful mRNA

Classification of Viruses

Classification depending on structure, genome, organization, transcription and replication strategies

All viruses must generate mRNA that can be recognized by host cell ribosomes this can also be used to classify viruses

Seven Classes: dsDNA, ssDNA, dsDNA, dsRNA, +ssRNA, -ssRNA using reverse transcriptase

Sequencing

You can use PCR or RT-PCR

This can be used to look at the genes in that things are coding for and determine aspects of the 

But PCR can only be done on DNA 

If a virus’s code is in RNA, then it needs RT-PCR

This works by isolating RNA adding a primer and RT which would make a DNA copy then RT would be denatured, and other enzymes are added then DNA can be obtained 

Recognition of Host Cell

Done via interactions between attachment proteins and host cell receptors

Each virus has a specific range/type of cell that they can infect

Range is dictated by the interaction between viral attachment proteins and host cell receptor molecules 

Host cell receptors are often molecules critical for cellular function and not easily eliminated 

Ex: HIV does CD4 (immune cell), Epstein-Barr does CR2 B-cell activation

Viral Infection

1. Recgonition of host cell

2. Viral entry and uncoating

3. Viral replication (genome replication, gene expression, and protein production)

4. Viral assembly and egress

Strain Specificity in Bacteriophage

Depends on attachment proteins, never go into the bacteria

Bacteriophages have attachment proteins on tail fibers

T2 recognizes lipopolysaccharide on outer membrane (gram -) 

Experiment created a chimera of tail fibers of a bacteriophage that was infections and to a capsid of a noninfectious bacteriophage and infection occurred

Enveloped Viruses

Attache by a specific viral protein (spike) embedded within the envelope

Ex: Hemagglutinin (HA) proteins 

Like influenza (enters through endocytosis) or HIV (but membrane fusion)

Non-Enveloped Viruses

Ex: Adenovirus: Have spikes that extend from capsid 

Ex: Poliovirus: Capsid directly interacts with receptors 

Binding and Entry of Influenza

An enveloped virus that entry is dependent on pH

1. Binind of influenza to hemagglutinin

2. Endocytosis

3. Acidification of Endosome vesicle 

4. Low pH triggers conformational change in HA protein and exposes a fusion peptide

5. Fusion peptide facilitates fusion of viral envelop and endosome membrane

6. Release of genome 

Once enveloped, two plasma membranes separate genome from host cell membrane (receptor protein needs to change conformation) 

Binding and Entry of HIV

Needs two receptors

1. Binding to CD4 to gp120 induces structural change in gp120

2. Binds to co-receptor

3. Additional structural changes expose fusion peptide on surface protein gp41

4. Fusion between viral envelope and plasma membrane

Receptor engagement and membrane fusion can be blocked by specific drugs

Entry of Non-Enveloped Viruses

Occurs via receptor-mediated endocytosis

Ex: Reovirus

Direct penetration of cellular membrane 

1. Virus binds to its receptor on cell surface

2. Endocytosis is triggered

3. Low pH of endosome induces conformational changes within protein of viral capsid

4. Exposure of pore forming domain creates a hole in plasma membrane

Bacteriophages and Injection Genome

Do not enter the cell but inject their genome instead

1. Phage attachment proteins interact with host cell receptor

2. Tail of phage contracts

3. Tail core penetrates cell wall

4. Brings DNA in contact with plasma membrane

5. Phage pilot protein binds phage DNA and assists in allowing DNA to penetrate the plasma membrane 

Plant Viruses

Due to tough cell wall and waxy thick cuticula plant viruses need the help of insects to damage induced by insects to infect the plant

Viral Disassembly (Polio)

Necessary for subsequent replication

Each virus may use a different method, but the viral genome must be inserted into the genome

Uncoating (Capsid disassembly) occurs through conformational changes of capsid proteins or proteolytic processing

Virus is not inserted into cell, but binding followed by insertion of VP4 into cell allows release of genome

Kinetics of Viral Infection

1. Latent phase (genome replication and protein production)

2. Rise phase (Cell lysis and viral release)

3. Plateau phase (virus accumulation reaches a constant titer)

The last two are the only quantifiable places because first step cannot be

Class I

dsDNA viruses

Like living organisms, uses everything from host 

Transcription: DNA dep DNA polymerase and occurs in the nucleus then DNA dep RNA polymerase

Genome replication is done by replicating dsDNA into dsDNA occurs in the nucleus 

Ex: Herpes Virus 

Only exception is poxvirus which replicates in the cytoplasm using viral enzymes so it must bring its own enzymes 

DNA Viruses

Most will replicate and transcribe in the nucleus and utilize host enzymes to operate 

Class II

ssDNA virus

Similar to class I

Transcription and Replication occur in the nucleus 

But since replication must be done in dsDNA, they must use host cell DNA dep DNA polymerase to convert ssDNA to double stranded DNA 

Class VII

dsDNA

Replication of genome uses mRNA and this needs to be reverse transcribed 

Transcription and replication occur in nucleus 

Host cell DNA dep RNA polymerase used for transcription

Virus brings reverse transcriptase 

First step for genome replication is transcription where they take their transcripts and use reverse transcriptase to get it into DNA

Replication of RNA Viruses

Because genome is in RNA but host reproduce in DNA there is a problem

RNA cannot be made into RNA in eukaryotes 

All RNA viruses must employ a virally encoded RNA dep RNA polymerase

Responsible for synthesis of genomic RNA and mRNA (needs to be translated by ribosomes) 

Replication of RNA viruses occur independently of host cell proteins in cytoplasm 

Class III

dsRNA virus

Replication and transcription occurs in the cytoplasm

Uses RNA dep RNA polymerase from virus for transcription and for the first step of replication

Once they have single stranded RNA they use RNA polymerase to convert it back to 

Class IV

+ssRNA

Genome can be used directly with ribosomes in cytoplasm

No transcription needs to be performed, their DNA is already transcribed

When they do genome replication in cytoplasm it requires RNA dep RNA polymerase from the virus this converts it to dsRNA 

Ex: Poliovirus 

Really does not need the whole virus, just needs the RNA

Class V

-ssRNA

Genome serves as the complement to mRNA so it needs viral RNA dep RNA polymerase to replicate the genome and create +ssRNA for transcription

First complementary +ssRNA copy must be generated by RNA dep RNA polymerase 

Risk of RNA damage

Class VI (Retroviruses)

ssRNA utilizes reverse transcriptase

Reverse transcriptase and RNA dep DNA polymerase converts ssRNA into dsDNA which is then integrated into the host genome

Transcription and Replication occur in cytoplasm and nucleus (Replication occurs first in cytoplasm then in nucleus)

Genome replication uses viral RNA dep RNA polymerase, integrase, DNA dep DNA polymerase of the host and DNA dep RNA polymerase of the host

Transcription uses reverse transcriptase, integrase, and DNA dep RNA polymerase from host 

Viral replication of Bacteriophages

Temperate: Phage DNA is integrated into genome or is maintained as plasmids, can remain for a while until environmental phages turn into lytic (this gives the advantage of replicating when genome bacteria are replicating and daughter cells will have infection too)

Virulent/lytic phages: Production of virus and then lyse of cell through production of lysozyme 

Replication of Temperate Lambda phage 

Lambda DNA is linear and must circularize before entering the cell

Integrase enzyme catalyzes integration into host cell

How is lytic or Temperate determined

This is done by promoter and the availability of other proteins

Transcription factors or regulators

In lytic stage there is a reduction of transcription factors and less stress so there is reduced protein levels which cause lysis

In temperate stage there is an increase in transcription factors and increase in stress which causes it to stay 

Viral Assembly

Many viral components are self-assembling making the entire process fast and cheap from an energetic perspective, interaction of polypeptide chains into multimeric protein complexes

Some viral proteins are involved but will not remain in mature viral particle (proteolytic process)

First precursor protein is formed before viral/cellular enzymes modify capsomere structure

Capsid proteins may interact with packaging sequence on viral genome to coalesce around nucleic acid 

How do viruses form and exit

Mechanisms depend on virus type and host cell type

Can be a lysozyme, or lysis of the cell

Enveloped viruses plant viral proteins in the host cell plasma membrane dock to them and bud out