10. RNA replication. reverse transcription

RNA replication

synthesis of RNA using an RNA template

discovered in early 1960s - experimented that there is a virus - specific enzyme that could copy RNA without DNA template

main enzyme: RNA dependant RNA polymerase (RdRP) or RNA replicase

substrate for RNA replicase - nucleoside triphosphates (NTPs)

2 stages: initiation and elongation

initiation

RNA starts at 3’ end of RNA template (can be primer - dependant or independant)

primer dependant requires viral protein

elongation

some nucelotidyl transfer reaction is repeated to generate the complementary RNA product

genome of RNA virus can be positive sense (+) or negative sense (-)

positive stranded (5’ - 3’) viral RNA can be directly translated into viral protein

(+) RNA replication

(+) RNA viruses enter animal cells by endocytosis (occur in membranous invaginations)\

inside host cell, (+) RNA genome is released into the cytosol and translated by host ribosomes

resulting proteins recruit the (+) RNA compartments → functional viral (VRCs) are assembled

small amount of (-) RNA is synthesised to serve as a template for synthesis of large amount of (+) RNA

new (+) RNAs is released from the VRCs, they start a new cycle, become encapsidated and exit the cell

negative stranded (3’ - 5’) viral RNA is complementary to mRNA, cannot be transcribed into protein directly, instead must be first transcribed into a positive - sense RNA which acts as mRNA

Non-segmented genomes of (-) RNA viruses: These typically do have one RNA molecule and replication generally occurs in the cytoplasm (e.g., Rhabdoviruses like Rabies virus).

Segmented genomes of (-) RNA viruses: These have multiple RNA segments, and replication typically occurs in the nucleus for viruses like Orthomyxoviruses (e.g., Influenza virus). However, there are exceptions where segmented (-) RNA viruses can replicate in the cytoplasm, such as Arenaviruses and Bunyaviruses.

(-) RNA replication

When a virion containing (-) RNA enters the host cell, the host ribosome indeed cannot recognize or translate the (-) RNA directly because it is not in the correct orientation (not mRNA). The negative-sense RNA must first be converted into positive-sense RNA.

The (-) RNA must be transcribed into (+) RNA by the viral RNA-dependent RNA polymerase, not through reverse transcription.

The RNA-dependent RNA polymerase binds to the (-) RNA strand and synthesizes several copies of (+) RNA.

The resulting (+) RNA can be used as a template by the host ribosome to synthesize viral proteins or as a template to produce more (-) RNA genomes for packaging into new virions.

reverse transcription

Reverse transcription is the process of transcribing RNA templates into complementary DNA (cDNA).

The enzyme responsible is RNA-dependent DNA polymerase, commonly known as reverse transcriptase.

Reverse transcriptase is primarily identified in retroviruses and also found in certain retroelements in bacteria, animals, and plants.

• Functions of Reverse Transcriptase:

  1. It builds a DNA strand based on an RNA template, which occurs at the enzyme’s polymerase active site.

  2. It removes the original RNA template using its RNase H activity (not a "nuclear active site").

  3. It then builds a second DNA strand to form a final double-stranded DNA, also performed at the polymerase site.

reproductive steps of retrovirus

1. Binding to the cell membrane and entering the cell: The retrovirus binds to specific receptors on the host cell membrane and enters the cell via membrane fusion or endocytosis.

2. Reverse transcription and transport of the proviral genome into the nucleus: The viral RNA is reverse-transcribed into double-stranded DNA by reverse transcriptase, and this proviral DNA is transported into the nucleus.

3. Integration of the proviral genome into cellular DNA: The proviral DNA integrates into the host cell’s DNA with the help of the viral enzyme integrase.

4. Transcription of the proviral genome: The integrated proviral DNA is transcribed into viral mRNA by the host's RNA polymerase.

5. Translation of viral mRNA into new viral proteins and virion assembly: The viral mRNA is translated into proteins, which are assembled with new viral RNA genomes into immature virions.

6. Maturation of immature virion into an infectious particle: Immature virions undergo maturation, facilitated by the viral protease enzyme, to become infectious particles as they bud from the host cell.