Lecture 4 + 5

Question 1

How do DNA viruses transcribe mRNA (what does it use as a template)?

Answer 1

DNA viruses transcribe mRNA using the (-) strand of the DNA genome as a template.

Question 2

What enzyme is typically responsible for transcribing mRNA in DNA viruses?

Answer 2

For most DNA viruses, the enzyme responsible for transcription is the host RNA polymerase II.

Question 3

Where does mRNA transcription take place in infected cells for most DNA viruses?

Answer 3

mRNA transcription takes place in the nucleus of infected cells, where RNA polymerase II is located.

Question 4

What is a notable exception regarding the location of replication and the enzyme responsible for transcription among DNA viruses?

Answer 4

Poxviruses replicate in the cytoplasm of infected cells and thus encode their own RNA polymerase, because they can not rely on host RNA polymerase II for transcription (because its not in the cytoplasm)

Question 5

How are the genomes of most (+) stranded RNA viruses translated? What is an exception?

Answer 5

The genomes of most (+) stranded RNA viruses can be translated directly by cellular ribosomes.

Retroviruses are an important exception, as their genomes require reverse transcription to form DNA before translation.

Question 6

How do (-) stranded RNA viruses and double-stranded RNA viruses express their genes?

Answer 6

mRNA must be transcribed from the genome in order to have gene expression.

This function is performed by RNA-dependent RNA polymerases

Question 7

Do (+) stranded RNA viruses need to incorporate an RNA pol inside the virus particle?

Answer 7

No, because the first thing they make is an RNA dependent RNA polymerase which can transcribe mRNA from the genome in order to have gene expression.

Question 8

Do (-) stranded RNA viruses need to incorporate an RNA pol inside the virus particle?

Answer 8

Yes

These viruses package a virus-encoded RNA-dependent RNA polymerase into the virus particle, ensuring its presence in the infected cell

Question 9

What type of viruses are retroviruses, such as HIV?

How do they replicate?

Answer 9

Retroviruses, such as HIV, are (+) RNA viruses.

Retroviruses replicate through a DNA intermediate.
- The RNA genome is “reverse transcribed” by the viral enzyme Reverse Transcriptase into double-stranded DNA
- The double-stranded DNA integrates into the genome of the host cell.
- The integrated DNA or provirus serves as a template for the synthesis of viral mRNAs using host RNA polymerase II.
- Host RNA polymerase II also synthesizes genome length (+)RNAs using the provirus as a template

Question 10

Why is reverse transcription unique to retroviruses?

Answer 10

Animal cells do not contain an enzyme that can perform reverse transcription.

Question 11

What are the steps involved in retrovirus gene expression?

Answer 11

1. Virus binds to a receptor on the surface of the host cell, leading to fusion.
2. RNA contained within the viral capsid is reverse transcribed into double-stranded DNA by the enzyme reverse transcriptase.
3. The double-stranded DNA is integrated into the host cell genome with the help of the enzyme integrase.
4. The integrated DNA, known as a provirus, serves as a template for the synthesis of viral mRNAs using host RNA polymerase II.

Question 12

What potential consequence can occur
due to the randomness of virus integration… i.e. retroviruses integrate into certain locations within the host cell genome?

Answer 12

If retroviruses integrate into critical regions of the host cell genome, such as tumor suppressor genes or regulatory regions, it can lead to disruptions in cellular functions and potentially contribute to the development of cancer.

Question 13

Why do retroviruses want to take place in a partially disassembled capsid during this process?

Answer 13

Retroviruses prefer to carry out their replication process within a partially disassembled capsid to evade detection by cellular sensors.

Naked DNA in the cytoplasm can trigger potent antiviral responses, such as the production of interferons. By maintaining intermediates within the capsid, retroviruses can evade detection and avoid triggering these responses.

Question 14

What are the two groups of double-stranded DNA viruses based on their replication location?

Answer 14

1. Replication exclusively occurs in the nucleus, relatively dependent on cellular factors.
2. Replication occurs in the cytoplasm for viruses like Poxviruses, which have evolved all necessary factors for replication and are largely independent of cellular machinery.

Question 15

Where does replication occur for single-stranded DNA viruses?

Answer 15

Replication occurs in the nucleus for single-stranded DNA viruses. This process involves the formation of a double-stranded intermediate that serves as a template for the synthesis of single-stranded genomic DNA.

Question 16

How can certain DNA viruses lead to cancer?

Answer 16

Certain DNA viruses, such as those causing cancer, have relatively small genomes and replicate in the nucleus, utilizing the host cell machinery. Most cells in the body are not actively dividing, so they typically do not maintain large pools of the machinery required for DNA replication. Additionally, deoxyribonucleotide triphosphates (dNTPs), essential for DNA synthesis, are expensive molecules for the cell to produce.

Consequently, cells often have limited availability of dNTPs. To facilitate their replication, these cancer-causing viruses induce host cells to enter the S phase of the cell cycle, where DNA synthesis occurs. By inducing cell division, these viruses ensure a supply of cellular machinery and dNTPs for their replication.

If the viral infection is incomplete or the virus is defective, some infected cells may survive and continue to proliferate, leading to transformation into cancer cells. These cancer cells can evade cell cycle checkpoints and continue growing uncontrollably, contributing to tumorigenesis.

Question 17

Where do most RNA viruses (viruses with RNA genomes) replicate? What is an exception?

Answer 17

In the cytoplasm of infected cells.

Orthomyxoviruses, such as influenza viruses, are a notable exception as they replicate in the nucleus of infected cells.

Question 18

What is the general process of gene expression and replication for (+) RNA viruses?

Answer 18

The genome of (+) RNA viruses can be directly translated, producing a polyprotein. One of the proteins synthesized is RNA-dependent RNA polymerase, which copies the (+) strand into a (-) strand, serving as a template for the production of more (+) strands.

Question 19

How do Flaviviruses and Picornaviruses regulate protein production from their (+) RNA genomes?

Answer 19

Flaviviruses and Picornaviruses produce a single polyprotein from their (+) RNA genomes.

The entire genome gets translated and cleaved into smaller parts by viral proteases.

However, this process results in a 1:1:1:1… ratio of proteins, which can be inefficient as some proteins are produced in excess while others are produced in insufficient quantities.

Question 20

How do other (+) RNA viruses regulate protein production differently than flavi- and picornaviruses?

Answer 20

Other (+) RNA viruses, such as Alphaviruses, initially translate only the first open reading frame of their genome, producing RNA-dependent RNA polymerase.

This enzyme initiates replication, producing a full-length (-) strand, which serves as a template for more (+) strands. Additionally, the (-) strand can serve as a template for subgenomic mRNAs, which are smaller molecules initiating internally and translating into different proteins.

Alphaviruses can efficiently regulate protein production by generating more of one subgenomic mRNA, thus ensuring a more balanced production of proteins. This regulation is controlled by the efficiency with which the RNA-dependent RNA polymerase sits in front of a particular reading frame.

Question 21

What is the consequence of the translation process for Flaviviruses and Picornaviruses in terms of protein ratios, in contrast to alpha viruses?

Answer 21

Flaviviruses and Picornaviruses produce proteins in a 1:1:1:1… ratio, which can be inefficient as some proteins are produced in excess while others are produced in insufficient quantities. This imbalance results in a surplus of capsid proteins relative to other essential proteins.

In contrast, Alphaviruses can adjust protein production by generating more of one subgenomic mRNA, ensuring a more efficient utilization of cellular resources. Additionally, (+) strand viruses can synthesize RNA-dependent RNA polymerase without needing to bring it into the cell, which enhances their replication efficiency.

Question 22

How do segmented (-) RNA viruses regulate gene expression and replication?

Answer 22

Segmented (-) RNA viruses, such as influenza virus, have their genomes divided into multiple segments.

Each segment generally encodes a single protein.

The viral RNA polymerase copies the (-) strand into a complementary (+) strand, which can serve as mRNA for translation.

Additionally, the RNA polymerase synthesizes another (+) strand that does not get translated but serves as a template for the production of more (-) strands, which ultimately get packaged into new virus particles.

Question 23

How do non-segmented (-) RNA viruses regulate gene expression and replication?

Answer 23

Non-segmented (-) RNA viruses, such as Ebola virus, have a single piece of RNA for their genome. These viruses synthesize sub-genomic mRNAs for gene expression.

The viral RNA polymerase transcribes mRNAs from the (-) strand for different genes.

Additionally, the anti-genome complement of the genome is made to serve as a template for the production of more genomes.

Question 24

What are ambisense RNA viruses?

Answer 24

Ambisense RNA viruses have a genomic RNA that contains both (-) and (+) polarities.

Ambisense RNA viruses are often very deadly and have segmented genomes, with one segment referred to as L (large) and the other as S (small). Both segments encode two open reading frames: one for L and one for Z.

Question 25

What is the significance of the L and Z proteins in ambisense RNA viruses?

Answer 25

The L protein is the largest protein synthesized by these viruses and is typically associated with the polymerase function. L is (-) polarity

The Z protein is of positive polarity.

Question 26

How do ambisense RNA viruses regulate gene expression?

Answer 26

The RNA segments of ambisense RNA viruses lack a cap, so they are not directly translated. Thus, the viruses must bring RNA-dependent RNA polymerase into the cell. The polymerase synthesizes mRNA for the nucleocapsid, using the (-) polarity as a template. It also produces a full-length anti-genome copy, with the nucleocapsid segment in the (+) polarity and the glycoprotein segment in the (-) polarity.

Question 27

How does the RNA-dependent RNA polymerase in ambisense RNA viruses facilitate gene expression?

Answer 27

The RNA-dependent RNA polymerase produces mRNA for the glycoprotein, using the full-length anti-genome copy as a template. This copy also serves as a template for making new S RNA, ultimately getting packaged into new virus particles.

Question 28

What is the role of the L RNA segment in ambisense RNA virus gene expression?

Answer 28

The L RNA segment, when inside the cytoplasm, can produce mRNA encoding the L protein. It is also copied into an antisense strand, serving as a template for the production of the Z RNA, ensuring the presence of both L and Z proteins in the virus replication process.

Question 29

How is the packaging of the genome into icosahedral capsids performed?

Answer 29

The capsid assembles around the virus genome in a process that is poorly understood.
or
The genome is “fed” into preformed capsids.

Question 30

How is the assembly process different for helical nucleocapsids than icosahedral capsids in RNA viruses?

Answer 30

For helical nucleocapsids, the viral genomic RNA is generally coated with nucleocapsid protein during its synthesis.

Question 31

Big viruses (large capsids) make a _________ first, then builds capsomeres that come together to form the ______________

Answer 31

Big viruses (large capsids) make a PRO-CAPSID (a structure built around the scaffold proteins) first, then builds capsomeres that come together to form the CAPSID

Question 32

How does herpes virus capsid assembly occur?

Answer 32

• Herpes virus capsid assembly involves the formation of a procapsid first, which is built around scaffold proteins associated with a scaffold protease.
• Capsomeres are then assembled to form the icosahedral structure. In herpes viruses, there are 28 faces and 12 vertices, with one special vertex called the portal, a dodecameric ring of protein through which viral genes are pumped into the capsid.
• Assembly starts with the portal to prevent incorporation of multiple portals, which could lead to issues.
• A motor called terminase binds to the genome end and pumps DNA into the capsid. The process continues until the terminase reaches the genome’s end, triggering an endonuclease to snip it off.
• This motor operates purely by pressure, exerting high pressure to facilitate DNA pumping.

Question 33

What does terminase (the motor in herpesvirus capsid assembly) rely on?

a) ATP
b) light
c) pressure
d) PMF

Answer 33

c)

• This motor operates purely by pressure, exerting high pressure to facilitate DNA pumping.

Question 34

What are inclusions in virus-infected cells, and how do they relate to assembly?

Answer 34

• Inclusions are cellular structures that form during virus assembly, often causing significant cytopathic effects (CPE).
• They can appear either in the cytoplasm or nucleus, depending on where the virus assembly occurs within the cell.
• The abundance of capsid proteins produced during assembly leads to the formation of these inclusions, which are visible under light microscopy.
• The size and location of these inclusions in infected cells often provide characteristic features specific to particular virus infections.
• e.g. Negri bodies

Question 35

How does egress differ between viruses with envelopes and those without?

Answer 35

Viruses with “naked” capsids are often released from infected cells by lysis, a process involving viroporins that disrupt the cell membrane, leading to cell death.

In contrast, viruses with envelopes can acquire their envelope through budding from various cellular membranes. Those that bud from the plasma membrane are released directly into the extracellular environment, while those budding from the Golgi apparatus or endoplasmic reticulum are secreted from the infected cell.

Question 36

How does replication take place in influenza A virus?

Answer 36

In influenza A virus, which has a segmented genome with negative polarity, replication occurs in the nucleus of infected cells. The nucleocapsid synthesis takes place in the cytoplasm and then returns to the nucleus.

Piece of RNA coded with nucleocapsid protein, has to find its 7 other partners somehow. This ssRNA wrapped in the capsid in some place is not wrapped, and a piece of RNA can leak out from inside the protein. The other 7 capsids do the same with a bit different sequences. These RNA sequences can base pair with each other (complimentary). Also explains the structure that’s formed (1 in the middle, 7 around it as they keep joining around it)

When all 8 pair, the virus buds from the cell. This process ensures that each virion contains one of the 8 specific segments, encoding different gene products.

Question 37

Describe the replication and egress process of herpes viruses.

Answer 37

Herpes viruses replicate their genomes in the nucleus and package them into capsids. These capsids move along microtubules to the nuclear core complex, where they inject their DNA into the nucleus.

After replication, the viruses exit the nucleus and acquire an envelope by budding into the inner nuclear membrane. This places them in the perinuclear space, which is contiguous with the endoplasmic reticulum (ER).

They then fuse their membrane with the outer nuclear membrane, releasing a capsid into the cytoplasm.

After budding into the Golgi membrane to acquire the final envelope, the now infectious viruses are trafficked through the Golgi, where glycoproteins in the envelope are modified by glycosylation.

The mature virus particles then move to the cell surface and fuse with the plasma membrane. At the cell surface, polymerization of actin behind the virus propels it towards adjacent cells, facilitating viral spread.