Viral Replication Strategies

Question 1

Central dogma of molecular biology

Answer 1

1. Replication (DNA makes more DNA)
2. Transcription (DNA to RNA)
3. Translation (RNA to proteins)

Question 2

tRNA

Answer 2

Bring amino acids to the ribosomes that are charged onto the newly growing amino acid strand

Question 3

rRNA

Answer 3

Make up ribosomes

Question 4

Central dogma in viruses

Answer 4

The central dogma does not hold true in viruses, genetic information can flow in any direction

Question 5

+ sense RNA

Answer 5

Also called mRNA. Has potential to be translated into a functional protein

Question 6

• sense RNA

Answer 6

Has a reverse complementary sequence to mRNA, but can not be used to make a functional protein

Question 7

Reverse transcription

Answer 7

RNA becomes DNA. + sense RNA is converted into double stranded DNA that is integrated into the host cell genome

Question 8

DNA polymerase

Answer 8

Responsible for synthesizing DNA: they add nucleotides one by one to the growing DNA chain, incorporating only those that are complementary to the template. Only adds nucleotides to the 3’ end of the strand. They can’t start making a DNA chain from scratch, but require a pre-existing chain or short stretch of nucleotides called a primer. They also proofread, removing the vast majority of “wrong” nucleotides that are accidentally added to the chain

Question 9

Reverse transcriptase

Answer 9

Found in retroviruses- converts RNA to DNA

Question 10

RNA polymerase

Answer 10

Link nucleotides to form an RNA strand (using a DNA strand as a template). Each nucleotide is added to the 3’ end, so the new RNA strand is synthesized in the 5’ to 3’ direction

Question 11

RNA dependent RNA polymerase

Answer 11

Found in all viruses with RNA genomes. They are used to replicate RNA from an RNA template

Question 12

Replication strategies (3)

Answer 12

1. RNA directed RNA synthesis
2. Transcription and DNA replication
3. Reverse transcription and integration- retroviruses

Question 13

All viruses must

Answer 13

Produce mRNA, regardless of their specific nucleic acid. They need mRNA to make viral proteins

Question 14

Transcription of double stranded DNA viruses

Answer 14

They undergo transcription to make mRNA, like a normal cell

Question 15

Transcription of single stranded DNA viruses

Answer 15

Single stranded DNA is converted to double stranded DNA, then transcribe into mRNA using host cell machinery

Question 16

Transcription of double stranded RNA viruses

Answer 16

These viruses have + sense and - sense RNA. The - sense RNA is used as a template to make more + sense RNA (mRNA)

Question 17

Transcription of + sense single stranded RNA viruses

Answer 17

This + sense RNA can be directly used as mRNA

Question 18

Transcription of - sense single stranded RNA viruses

Answer 18

+ sense RNA (mRNA) is produced using the - sense RNA as a template

Question 19

Transcription of retrovirus single stranded RNA

Answer 19

Reverse transcriptase produces DNA and RNA, eventually producing double stranded DNA that can be transcribed into mRNA

Question 20

Transcription of retrovirus double stranded DNA

Answer 20

These nucleic acids have gaps between them. The gaps must be filled. Reverse transcriptase is used to make mRNA

Question 21

DNA dependent RNA polymerase

Answer 21

DNA is used as a template to make RNA. This is what happens during transcription

Question 22

Double stranded RNA dependent RNA polymerase

Answer 22

A virally encoded polymerase that uses double stranded RNA as a template to make mRNA

Question 23

RNA polymerases in the eukaryotic cell

Answer 23

There are 3: RNA polymerase 1, 2, and 3. RNA polymerase 2 specifically transcribes DNA to make mRNA. Polymerase 3 produces tRNA and some rRNAs, polymerase 2 produces a variety of rRNAs

Question 24

Identification of RNA polymerases

Answer 24

David Baltimore discovered RNA polymerase in + and - strand RNA. He then discovered reverse transcriptase which was a big milestone because it fueled the development of gene expression studies.

Question 25

Sequence similarity among polymerases

Answer 25

RNA polymerases are the most conserved genes among viruses, which can be used to predict an evolutionary relationship (like 16s rRNA)

Question 26

In a - strand RNA genome, what is packaged inside the virus particle?

Answer 26

RNA dependent RNA polymerase (RdRp), RNA is coated with a protein (nucleocapsid). Examples- VSV and influenza A. RdRp is necessary because host cells do not have similar enzymes that the virus can use

Question 27

In a + strand RNA genome, what is packaged inside the virus particle?

Answer 27

There is no RdRp and naked DNA. Exceptions are retroviruses and coronavirus (long coated genome). Examples of this type of virus are flavivirus and poliovirus

Question 28

What is packaged in a double stranded RNA genome?

Answer 28

RdRp, naked RNA. Example- reovirus

Question 29

Rules for successful viral RNA synthesis (2)

Answer 29

1. Genomic RNA must be copied end to end without losing any information or
   sequence
2. Viral mRNA must be produced that can be recognized and efficiently translated
   by host cell machinery- viral enzymes can’t proofread and may have errors, but they must be able to be read by host machinery

Question 30

Universal rules of RNA dependent RNA synthesis (4)

Answer 30

1. Initiation and termination of RNA synthesis occurs at specific sites- this refers to mRNA
2. RdRp may initiate RNA synthesis de novo (primer independent) or require a primer
3. Additional viral or cellular proteins may be required
4. Chain elongation occurs in 5’-3’ direction with stepwise incorporation of nucleotides

Question 31

2 modes of initiation of RNA synthesis

Answer 31

1. De novo initiation
2. Primer-dependent initiation

Question 32

De novo initiation

Answer 32

When RNA-dependent RNA synthesis does not require a primer. This is generally what happens inside the cell. RNA dependent RNA polymerases add nucleotides using the template RNA molecule

Question 33

Primer-dependent initiation

Answer 33

When a primer is required for initiation- can be a terminal protein or a capped primer. With the protein primer, a virally encoded protein provides a hydroxyl end (3’) for the nucleotide to be added to the template strand. Nucleotides continue to be added. With a capped primer, a 5’ cap on is cleaved from newly synthesized viral mRNAs. The 5’ cap provides the hydroxyl end for nucleotides to be added- influenza is one example of when this occurs

Question 34

Examples of + strand RNA viruses

Answer 34

1. Picornaviruses (poliovirus)
2. Flaviruses (Zika, Dengue, West Nile)
3. Alphaviruses (VEEV, rubella)

Question 35

Poliovirus synthesis of mRNA

Answer 35

Poliovirus has a + strand genome (mRNA), so RNA can be translated immediately. They make a - strand complement. From the negative strand complement, more mRNA can be made and be used to make proteins. The ribosomes produce the full length protein chain, which will undergo proteolytic cleavage to make smaller protein fragments

Question 36

Classification of viruses

Answer 36

Inside the Baltimore classes, there are multiple families of viruses. The families are divided into genera, which are subdivided again into species, and then to different genotypes

Question 37

Poliovirus genome

Answer 37

Has 7,5000 nucleotides. Has a virally encoded protein called VPg linked to the 5’ end. It is used as a primer for RNA synthesis. The genome is translated to produce long polyproteins that are cut by viral proteases

Question 38

Viral polymerase structure

Answer 38

Cloverleaf structure on 5’ end, then the cis-acting replication element, then a pseudo knot on the 3’ end. The structural signatures determine the specificity for viral polymerase, so it can find the viral genome even with everything else going on in the cell. The clover lead structure is involved in the initiation of strand synthesis, which is primer dependent

Question 39

How does RNA synthesis occur with poliovirus RNA? (6)

Answer 39

1. Viral protein 3AB is anchored in the cell membrane
2. It binds to host protein PCBP, which is bound to the clover leaf structure of the polioviral genome
3. Viral 3CD is also bound, and interacts with poly-A binding protein
4. Poly-a binding protein binds to the 3’ end of the viral genome. These interactions make the viral genome circular, which is important for RNA synthesis
5. 3CD cleaves 3AB. It also tags Vpg, which binds to the adenine on the poly-A tail
6. This initiates RNA synthesis

Question 40

What if the Cre element is mutated in poliovirus?

Answer 40

Then the 3CD protein can’t bind to the Cre element and initiate RNA synthesis

Question 41

Coronavirus replication cycle

Answer 41

+ sense genomic RNA. Only partial mRNA is used to synthesize proteins- polypeptides 1a and 1b, which code for polymerases. The polymerases are used to replicate the genome and synthesize subgenomic mRNA to make structural proteins