Viral Replication Strategies Flashcards
Central dogma of molecular biology
- Replication (DNA makes more DNA)
- Transcription (DNA to RNA)
- Translation (RNA to proteins)
tRNA
Bring amino acids to the ribosomes that are charged onto the newly growing amino acid strand
rRNA
Make up ribosomes
Central dogma in viruses
The central dogma does not hold true in viruses, genetic information can flow in any direction
+ sense RNA
Also called mRNA. Has potential to be translated into a functional protein
- sense RNA
Has a reverse complementary sequence to mRNA, but can not be used to make a functional protein
Reverse transcription
RNA becomes DNA. + sense RNA is converted into double stranded DNA that is integrated into the host cell genome
DNA polymerase
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
Reverse transcriptase
Found in retroviruses- converts RNA to DNA
RNA polymerase
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
RNA dependent RNA polymerase
Found in all viruses with RNA genomes. They are used to replicate RNA from an RNA template
Replication strategies (3)
- RNA directed RNA synthesis
- Transcription and DNA replication
- Reverse transcription and integration- retroviruses
All viruses must
Produce mRNA, regardless of their specific nucleic acid. They need mRNA to make viral proteins
Transcription of double stranded DNA viruses
They undergo transcription to make mRNA, like a normal cell
Transcription of single stranded DNA viruses
Single stranded DNA is converted to double stranded DNA, then transcribe into mRNA using host cell machinery
Transcription of double stranded RNA viruses
These viruses have + sense and - sense RNA. The - sense RNA is used as a template to make more + sense RNA (mRNA)
Transcription of + sense single stranded RNA viruses
This + sense RNA can be directly used as mRNA
Transcription of - sense single stranded RNA viruses
+ sense RNA (mRNA) is produced using the - sense RNA as a template
Transcription of retrovirus single stranded RNA
Reverse transcriptase produces DNA and RNA, eventually producing double stranded DNA that can be transcribed into mRNA
Transcription of retrovirus double stranded DNA
These nucleic acids have gaps between them. The gaps must be filled. Reverse transcriptase is used to make mRNA
DNA dependent RNA polymerase
DNA is used as a template to make RNA. This is what happens during transcription
Double stranded RNA dependent RNA polymerase
A virally encoded polymerase that uses double stranded RNA as a template to make mRNA
RNA polymerases in the eukaryotic cell
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 1 produces a variety of rRNAs
Identification of RNA polymerases
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.
Sequence similarity among polymerases
RNA polymerases are the most conserved genes among viruses, which can be used to predict an evolutionary relationship (like 16s rRNA)
In a - strand RNA genome, what is packaged inside the virus particle?
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
In a + strand RNA genome, what is packaged inside the virus particle?
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
What is packaged in a double stranded RNA genome?
RdRp, naked RNA. Example- reovirus
Rules for successful viral RNA synthesis (2)
- Genomic RNA must be copied end to end without losing any information or
sequence - 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
Universal rules of RNA dependent RNA synthesis (4)
- Initiation and termination of RNA synthesis occurs at specific sites- this refers to mRNA
- RdRp may initiate RNA synthesis de novo (primer independent) or require a primer
- Additional viral or cellular proteins may be required
- Chain elongation occurs in 5’-3’ direction with stepwise incorporation of nucleotides
2 modes of initiation of RNA synthesis
- De novo initiation
- Primer-dependent initiation
De novo initiation
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
Primer-dependent initiation
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
Examples of + strand RNA viruses
- Picornaviruses (poliovirus)
- Flaviruses (Zika, Dengue, West Nile)
- Alphaviruses (VEEV, rubella)
Poliovirus synthesis of mRNA
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
Classification of viruses
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
Poliovirus genome
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
Viral polymerase structure
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
How does RNA synthesis occur with poliovirus RNA? (6)
- Viral protein 3AB is anchored in the cell membrane
- It binds to host protein PCBP, which is bound to the clover leaf structure of the polioviral genome
- Viral 3CD is also bound, and interacts with poly-A binding protein
- 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
- 3CD cleaves 3AB. It also tags Vpg, which binds to the adenine on the poly-A tail
- This initiates RNA synthesis
What if the Cre element is mutated in poliovirus?
Then the 3CD protein can’t bind to the Cre element and initiate RNA synthesis
Coronavirus replication cycle
+ 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
