2.4B Viral Replication

What are the basic steps of the replication cycle of viruses

1. attach to host cell

2. genome entry and if necessary uncoating (release of genome from capsid)

3. Expression of viral genes and proteins

4. Replication of viral genome

5. Assembly and exit from the host cell

What is host range and tissue tropism of viruses dictated by

Dictated by the interaction between viral attachment proteins and host cell receptor molecules

How are the host cell receptor molecules for viruses

They are often molecules critical for cellular function

Why are host cell receptor molecules often critical for cellular function

Because it is difficult for the host cell to change/down regulate the expression of those molecules because they’re so important so it will significantly disrupt cell function when the virus attaches to those cell receptors.

Where are the viral protein located in bacteriophage

The tail fibers with viral attachment proteins

Where are the viral attachment proteins for enveloped viruses

Attachment spike embedded in viral envelope

Is influenza virus enveloped

Yes

Is adenovirus enveloped

no

Where are viral attachment proteins for non-enveloped viruses

Attachment spike extends from viral capsid

Where are viral attachment proteins located for non-enveloped virus like the poliovirus

Attachment protein is part of viral capsid

How do non-enveloped animal viruses enter an animal cell

Through receptor mediated endocytosis

How does the reovirus enter the cell

It is a non-enveloped animal virus so it enters via receptor mediated endocytosis

In receptor mediated endocytosis, how does a conformational change in capsid occur

There is a drop in endosomal pH that triggers a conformational change in the capsid (due to the formation of the endosome)

How does endocytosis happen with a non-enveloped virus

1. The virus attaches to the cell receptor

2. This initiates endocytosis, and the virus moves into the cytoplasm

3. An endosome forms with the virus inside

4. The nucleocapsid escapes to the cytoplasm and uncoats to release the genome (which is triggered by the endosomal pH which causes a change in capsid)

How do enveloped animal viruses enter the cell

1. The virus attaches to the cell receptor (ex. HIV-1 uses a CD4 receptor on the plasma membrane)

2. A conformational change in the attachment protein and bound receptor initiates membrane fusion

3. The viral envelope is made of the same plasma membrane as the animal cell, so the membranes fuse

4. The nucleocapsid enters the cytoplasm and uncoats to release the genome

What is an anti HIV drug

Fuzeon

How does HIV interact with cells

When the HIV protein binds to the CD4 receptor on the host cell membrane, it causes a conformational change of gp120, allowing the co-receptor to bind.

- Additional conformational change exposes the fusion peptide, facilitating membrane fusion.

Fusion with cell membrane is used by which kind of virus?

Used by envelope animal viruses

e.g: HIV

Steps for fusion with cell membrane

1. Virus(HIV) attaches to the cell receptor

2. Conf. change in the attachment of protein and bound receptor initiates membrane fusion

3. Viral envelope fuses with plasma mem.

   • Because the viral envelope is made of host cell mem., the mem. in virus is same as host cell.

4. The nucleocapsid enters the cytoplasm and uncoats to release genome.

Ex. HIV

Role of gp120

Binding of viral envelope to CD4 alters the conf. of gp120 allowing binding to co-receptor.

How does Fuzeon work

Fuzeon is an anti HIV drug that targets the viral protein gp41 and prevents fusion.

- After binding, a fusion peptide in gp41 becomes exposed and interacts with the host cell membrane

- In the absence of Fuezon, gp41 proteins fold, bringing the viral envelope and plasma membrane together allowing fusion.

- In the presence of Fuezon, gp41 folding is blocked and fusion is inhibited

how does influenza enter animal cells

Influenza is an enveloped animal viruses

- It's viral proteins are hemagglutinin (HA)

when this protein makes contact with the host cell receptor, the fusion peptide is exposed and the virus is endocytosed into the cell cytoplasm forming an endosome

- The acidic pH inside of the endosome, due to the entrance of H into the endosome exposes the viral membrane fusion peptide. The viral envelope fuses with endosomal mem. with the help of fusion peptide instead of the cell mem.

- The viral nucleocapsids are now released into the cytoplasm.

how does viral uncoating happen?

Mammalian viruses must "uncoat" their geome - ie disassemble their capsid to release genteic material once inside the cell

-Ex. Poliovirus binds to its receptor, poliovorus receptor (PVR) on the host cell membrane binds and poliovirus receptor attches to the capsid.

Viral uncoating in poliovirus

Poliovirus binds to its receptor, poliovorus receptor (PVR) on the host cell membrane binds and poliovirus receptor attches to the capsid.

A conformational change occurs and the VP4 protein is exposed and lost

Capsid VP1 protein forms a channel in the membrane by inserting itself into the plasma membrane.

The RNA of the poliovirus can enter into the cytoplasm of the host cell through the channel

This occurs at the plasma membrane invaginations or in very early endocytic vesicles

how do bacteriophages enter the cell

Bacteriophages do not actually enter the host cell because of the cell wall and inject their genome instead.

1. The tail fibers attach to receptors on the plasma membrane

2. A conformational change in the tail fibers brings the base of the tail in contact with the host cell surface

3. The rearrangement of tail proteins allows the inner core tube proteins to extend down into the cell wall

4. Contact with the plasma membrane initiates the transfer of DNA through a pore formed in the lipid bilayer allowing the DNA to enter the cell

How do plant viruses enter cells

Unlike animal viruses, plant viruses do not recognize specific cellular receptors on their host cells but rather enter through damage to the cuticle

Why do plant cells often require damage to enter cells

Due to tough cell wall structures, plant viruses often require damage induced by insects to infect the plant cells

Once the viral genome is in the host, what must happen

1. Translation of the viral RNA to make viral proteins

2. Replication of the viral genome for packaging into new virions

What dictates how viral replication will happen

- The type of viral genome (DNA/RNA; double stranded/single stranded, positive/negative) dictates in part how viral replication will happen

What is mRNA by definition

+RNA also sense RNA

what is positive RNA / Sense RNA

It’s RNA that can be directly translated into a functional protein

What is negative RNA

aka the antisense RNA/complementary to the + RNA sequence

what is + DNA

If the RNA version of the DNA sequence can be directly translated into protein, it is + DNA or the sense strand (aka coding strand)

• basically the DNA sequence that matches the mRNA but. will have T’s instead of U’s.

How to know which DNA strand is which

The DNA strand whose codons directly match those of the mRNA strand (besides substituting T's and U's) is the DNA coding strand or the sense strand (+) DNA strand. this strand matches the mRNA strand exactly. (coding strand)

The - DNA strand or the antisense strand also called the template strand is the strand always needed to transcribe +RNA.

So the complementary strand is - DNA or the antisense strand aka template strand because it serves as the template to make mRNA.

What is the rationale behind the baltimore classification system

Knowing the genome type of a virus already provides information about how the virus will generate mRNA and replicate its genome and gives you insight into its life-cycle even if you know nothing else about the virus.

In the baltimore system, what are groups based on?

There are 7 groups

Classified by genome type

• DNA or RNA ss or ds

Classified by route to mRNA

• DNA intermediate or RNA intermediate

Group 1 of the baltimore classification system

Group I is double stranded DNA

• (ex. Human herpesvirus)

- Because this DNA is double stranded, the negative sense DNA strand can be directly transcribed into the positive mRNA .

It will use its own or the host DNA polymerases for replication. It depends on the viral species which is used.

Pretty much, can be transcribed directly into positive mRNA with its own DNA polymerase or host DNA polymerase

DNA Virus so Simplest Virus

Group II of the baltimore classification system

Single stranded DNA

• (Ex parvoviruses)

- Because it only has one strand of DNA, this is a positive sense stand DNA. mRNA cannot be directly transcribed from +DNA, it must be transcribed from negative DNA.

So it must first copy the + sense DNA into - sense DNA to generate a complementary strand which can be done by the host DNA polymerase. Once the - DNA is made, it can be transcribed into + mRNA with host RNA polymerase.

This is because u cannot go from + DNA to + mRNA, there must be a -DNA strand to use as complementary strand to make + mRNA.

Can use hosts or its own polymerases encoded in the viral genome.

Group III in the baltimore classification system

Group III includes double stranded RNA viruses

• (ex. reovirsuses)

- Requires an RNA dependent RNA polymerase to make mRNA and genomic RNA(using RNA as a template to make more RNA). This molecule will use the negative sense RNA of the double strand as a template to make more copies of the + mRNA.

Host RNA pol will use DNA as a template, so host RNA pol can’t copy RNA into RNA.

Group IV of the baltimore classification system

Group IV consists of viruses with + single stranded RNA

• (ex. poliovirus)

Positive RNA can be directly translated by the ribosome into proteins, but in order to make more copies of the + RNA, you must have the complementary - RNA strand to serve as a tenplate. You cannot make + RNA from + RNA. So, this virus uses RNA dependent RNA polymerase to make a template for mRNA and genome replication by making - RNA.

So it must transcribe + RNA into - RNA and then they can use the - RNA to make many copies of the + RNA (which can be translated into proteins)

Group V in the baltimore classification system

Group V consists of - single stranded RNA

• (ex. influenza virus).

These require RNA dependent RNA polymerases to make mRNA and replicate its genome. But these can be directly transcribed into + mRNA molecules that can be translated into protein later.

Needs to package and encode their RNA pol because it’s negative

Group VI in the baltimore classification system

Group VI includes retroviruses

• (ex. HIV).

Has a DNA intermediate. This group has a single stranded +RNA genome, but it packages its own reverse transcriptase molecule to make dsDNA.

So it first transcribes its ss + RNA into - antisense - DNA.

antisense -DNA is copied into + sense DNA making dsDNA and then this DNA is integrated into the host genome. Once the viral DNA is integrated into the host genome, it is transcribes into mRNA with the host polymerases.

• Technically doesn’t have to package RNA pol because it’s going in as +RNA but HIV does.

Goup VII in the baltimore system

It is double stranded DNA pararetrovirus (has a dsDNA genome, with an RNA intermediate)

• ex is Hepatitis B virus.

Many pararetroviruses are plant viruses because plants have reverse transcriptase encoded in their genome. Animals do not, so animal pararetroviruses must encode their own reverse transcriptase. So it requires plant host reverse transcriptase to make dsDNA. Once the DNA is transcribed into mRNA, the mRNA serves as a template to make more copies of DNA using the reverse transcriptase. so it goes from DNA to RNA back to DNA.

If it is a plant virus it can use the host reverse transcriptase but if it is an animal virus the viral genome must encode its own reverse transcriptase.

RNA viruses and animal retroviruses or pararetroviruses must encode their

Viral polymerase within the viral genome, meaning that there is a gene for this protein encoded in the viral genome.

• Host cells do not have RNA dependent RNA pol. and animal cells do not have the reverse transcriptase to copy RNA to DNA.

How do some viruses require their viral polymerase

Some viruses require their viral polymerase immediately upon infection and must package the polymerase protein inside the capsid. the viral polymerase is STILL encoded in the viral genome. It is just also packaged as a protein.

Groups I and II in terms of viral replication

Groups I and II are DNA viruses.

- DNA viruses must replicate in the nucleus if they use host DNA/RNA polymerases

The virus contains the capsid with DNA inside wrapped in an envelope. The envelope fuses with the plasma membrane of the host, releasing the capsid + DNA into the cytoplasm(DNA is still enveloped). The capsid releases the DNA (uncoating) which enters into the nucleus. The viral DNA can be replicated by host DNA dependent DNA pol . into more viral DNA or transcribed into mRNA by the hosts DNA dependent RNA pol. It is then translated into viral proteins (like the capsid) outside of the nucleus by host ribosomes.

Which enters the nucleus. Now all of the viral DNA that has been replicated is assembled inside of the capsid which leaves the nucleus and buds out from the host cell plasma membrane, taking some of the host plasma membrane with it.

What is an atypical replication cycle of groups I and II viruses

An ex. is poxviruses which replicate their genome in the cytoplasm

- The enveloped virus enters via endocytosis and the viral enveloped membrane fuses with the endosomal membrane releasing the capsid into the cytoplasm which will uncoat and release the dsDNA and RNA pol.

- Now the genome must be transcribed so a viral DNA-dependent- RNA polymerase will transcribe the viral DNA into viral genes. (this polymerase was packaged in the the viral genome because it does not have access to the host polymerase, because this is happening in the cytoplasm. These transcribed genes can be early and late genes. Early genes are transcribed into mRNA which will be translated into proteins for the viral DNA polymerase and proteins for replication. This DNA will then be replicated. Late genes are transcribed into mRNA and then translated into proteins for capsid construction by the DNA polymerase of the viral genome. Now the replicated DNA and the capsid proteins are assembled and the virus exocytosis from the host cell.

Group IV in viral replication

- Since RNA viruses are not dependent on host polymerase, they usually replicate in the cytoplasm

for example, the Poliovirus

- The ssRNA in the + form enters into the cell via endocytosis and is uncoated.

- Then the + sense RNA is immediately translated into RNA-dependent RNA polymerase

- The RNA dependent RNA polymerase can then transcribe the negative sense RNA to make a template to make more positive sense RNA in RNA replication

- These +RNA can be translated into capsid proteins and some packed into new viral particles.

Group V in replication

Group V is - RNA viruses

- When the -RNA viruses enter the cell , the viral capsid is uncoated and the - ssRNA genome is free in the cytoplasm.

- The Viral RNA dependent RNA pol is packaged originally with the viral particle.

- The viral RNA dependent RNA polymerase immediately copies the - RNA into + RNA that can be translated into protein or can be used to make more - RNA.

- If the + sense mRNA is translated into viral proteins these are viral proteins like the capsid and viral RNA dependent RNA polymerase

Group VI work in replication

Group VI are retroviruses

• An example of this is HIV(Envelope Virus)

The virus enters the host cell and uncoats releasing its + sense RNA into the cytoplasm

- It carries its own reverse transcriptase that transcribes +RNA into -DNA.

- Travels to nucleus where +DNA can be made, giving us dsDNA.

- It then uses the integrase enzyme to integrate the viral dsDNA into the host genome now called a provirus (integrated viral DNA)

• While it’s a provirus, the genome can be replicated, but not actively express any proteins.

- The viral DNA can then be transcribed into + mRNA and translated into viral proteins like reverse transcriptase and integrase. Or the DNA viral genome can be replicated into + ssRNA genome.

- The + ssRNA genome and the viral proteins are assembled together into the mature virion which buds out from the host cell.

How much of the human genome is compromised of retroviruses

8% of the human genome is compromised of retroviruses that entered the genome through infections of germline cells and became permanent residents.

What do endogenous HERV DNA sequences contain and what does this mean

Contains copies of the gag (capsid), pol (polymerase), and env (envelope) genes and are flanked by two inverted long-terminal repeats (LTR) of non-coding regions.

Many viral components are ____ making the entire process fast and cheap from an energetic perspective

self-assembling

How can capsid proteins be used in viral assembly

Capsid proteins may interact with packaging sequences on viral genome to coalesce around the nucleic acid (ex. tobacco mosaic virus)

How are empty capsids in viral assembly

Empty capsids may partially form, then have viral genomes inserted into them (ex. poliovirus)

Does additional processing of viral proteins ever need to happen

Sometimes additional processing of viral proteins needs to occur (such as proteolytic cleavage) to generate an infectious particle (ex. HIV)

The mechanism of viral exit depends on

Depends on virus type and host cell type

How do enveloped viruses exit

They place viral proteins in the host cell plasma membrane, dock to them, and "bud" out of the cell, taking a portion of the plasma membrane with them

How do naked viruses exit host cells

They almost always exit the cell by lysis of the host cell

How does influenza exit the host cell

The viral enzyme neuraminidase is required for the exit of the flu virus from the cell

- Receptors on the host cell contain sialic acid

- The viral receptor proteins on viral cell are called hemagglutinin which bind to the sialic acid receptors

- Neuraminidase present on the flu virus membrane cleaves the receptor interactions, releasing the viral particle

How does tamiflu fight against influenza

Tamiflu inhibits neuraminidase and prevents the influenza virus from leaving the cell

- So tamiflu, neuraminidase inhibitors, bind to/coat all of the neuraminidase on the viral cell and prevent it from disrupting the interaction between the receptors containing sialic acid and the hemagglutinin receptor on flu virus, preventing the spread of infection because the virus is stuck to host cell

Why do we have limited amounts of antiviral drugs

We have limited, effective antiviral drugs for several reasons.

1. Viruses use many components of the host thus there are fewer targets that antivirals can target without hurting the host (ex. RNA polymerase ribosomes, etc)

2. Viruses have higher mutation rates and can quickly become resistant to anti-virals

What is the best way to fight viruses

For many viral infections, the best offense is a good defense meaning use vaccination because the host immune system will not get sick if it is already immune, but its much harder to fight the infection once it is established