This is second part on the Life cycle of SARS-CoV-2. In the previous post, we wrote about the entry, penetration and the genome of the virus. In this post, we shall see about the RNA replication, transcription, protein synthesis, assembly of the viral particle and finally the release of the progeny virions.
• RNA synthesis, Transcription and Translation:
As mentioned in a previous post, genome of the SARS-CoV-2 is a single stranded positive sense RNA (fig 1). It has a 5′ cap and a 3′ polyA tail, allowing it to mimic the mRNA.
(Just for info: Read our post on the general information on SARS-CoV-2 & COVID-19)
Hence, following the release of the viral RNA genome into the cell, the host translation components recognises it and the 80S ribosomal complex is assembled at the translation initiation codon. The host translational machinery then synthesizes the pp1a.
A -1 ribosomal frameshifting takes place in the overlapping region between ORFs 1a and 1b, and the pp1ab is synthesised.
The two polyproteins, pp1 (∼500 kDa) and pp1ab (∼800 kDa), are then processed by viral proteinases (nsp 3), generating sixteen non-structural proteins (nsps), which are essential for the viral genome replication and transcription.
The 16 nsps and their functions are given in the table no. 1:
The first non-structural protein (nsp) to be synthesized is Papain-like proteinase (PL proteinase, nps3), the largest component of the replication-transcription complex. The PL proteinase in nsp3 cleaves nsps 1-3. The nsp1 helps in evasion of host innate immune response and promotes host mRNA degradation.
Non-structural proteins like nsp3, nsp4, and nsp6 cause rearrangement of the cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes (CMs) (fig 2). The viral replication-transcription complex are anchored into these structures and become the site of replication and transcription of viral RNA.
(Just for info: Read this paper by Knoop et al. (2008) to know about the organelle like replicative structure seen in cells infected by the Coronavirus -SARS CoV).
These viral genome encodes for two different proteins with RNA dependent RNA polymerse activity; nsp8 (with primase activity) and nsp12 (without primase activity). Initially the negative sense RNA are produced, then the RdRp synthesize positive sense mRNAs (fig 3).
The two viral RNA polymerases (nsp8 and nsp12) produce genome-length RNA (gRNA) and set of shorter RNAs called as subgenomic (sg) RNAs. The full-length positive sense RNAs are used as progeny viral genome, while the (positive sense) sg mRNAs are used for the expression of the structural and accessory proteins. Also, the sg mRNAs lack encapsidation signals, hence cannot be packaged into progeny viral particles.
The genome mRNA synthesis is a continuous process, using a full-length complementary negative-strand RNA as the template. Subgenomic RNA synthesis is a discontinuous step and involves discontinuous extension of the 3′-end of the negative strand been synthesized.
Most of the (positive sense) sg mRNA, obtained from negative sense RNA, lacked few of the 5′ genes, but all had 5′ cap, forming a “nested set” of mRNAs of different length (fig 3), with identical 5′ and 3′ ends.
It was observed that all also had an identical leader sequence (around 70-100 nt) at the 5’-end. This leader sequence is present only once in the genomic RNA, near the 5’-end, and nowhere in the middle of the genome.
However, there exists another sequence of around 8-9 nucleotides, downstream to the leader sequence, called the transcription regulating sequence (TRS-L). TRS is also found within the genome, just at the upstream of the ORFs-encoding structural proteins. The TRS found at the upstream of the structural gene ORFs are called TRS body (TRS-B).
Now, The RNA-dependent RNA polymerase (RdRp) copies the genomic positive-sense RNA into a negative-sense template until it reaches a TRS-B. Then the RdRp may jump to the TRS-L (5′ end) of the genome and complete the short negative-sense sgRNA. These negative-sense sgRNAs serve as templates for the synthesis of the corresponding positive-sense sg mRNAs (fig 4).
The positive sense mRNAs are produced in almost 50- to 100-fold more in number than the minus-strand RNA.
Only the 5′-terminal ORF of each of these subgenomic mRNAs is translated into the respective structural or accessory proteins. The protein synthesis takes place on the rough endoplasmic reticulum (RER).
• Assembly and Release:
Virion assembly takes place on membranes of RER. Genomic RNA is bound by N protein, associates with M protein and buds into endoplasmic reticulum-Golgi intermediate compartment (ERGIC).
M proteins packs tightly into membranes. S and E are also membrane proteins and are taken up during the budding process, which takes place in the Golgi body, leading to vesicle formation with viral particles.
[Just for info: Read this paper to know more about the endoplasmic reticulum-Golgi intermediate compartment (ERGIC)]
The E protein acts as ion channel, increasing the pH of the transport vesicles and thereby promoting virus release by exocytosis. Once released the progeny virus can infect other neighbouring cells by attaching to the hACE2.
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High Pressure Liquid Chromatgraphy (HPLC).
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References:
Angeletti et al (2020) COVID‐2019: The role of the nsp2 and nsp3 in its pathogenesis Journal of Medical Virology. https://doi.org/10.1002/jmv.25719
Cornillez-Ty et al. (2009) Severe Acute Respiratory Syndrome Coronavirus non-structural protein 2 interacts with a host protein complex involved in mitochondrial biogenesis and intracellular signalling. Journal of Virology: 10314-10318.
Fehr and Perlman. (2015) Coronaviruses: An Overview of Their Replication and Pathogenesis. Methods Mol Biol. 1282: 1–23. doi:10.1007/978-1-4939-2438-7_1.
Graham et al. (2008) SARS coronavirus replicase proteins in pathogenesis. Virus Res. 133(1): 88–100.
Hagemeijer et al.( 2012) Biogenesis and Dynamics of the Coronavirus Replicative Structures. Viruses 4:3245-3269. doi:10.3390/v4113245.
Hoffman et al. (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Hoffmann et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor, Cell 181:1–10, https://doi.org/10.1016/j.cell.2020.02.052
Knoops et al. (2008) SARS-Coronavirus Replication Is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum. PLoS Biol 6(9): e226. doi:10.1371/journal.pbio.0060226
Masters (2019) Virology, Coronavirus genomic RNA packaging. Virology 537: 198-207.
Maxwell (2014) Structural determinants of Coronavirus nsp5 function and inhibition. Thesis submitted to Vanderbilt University.
Nakagawa et al (2016) Viral and Cellular mRNA Translation in Coronavirus-Infected Cells. Adv Virus Res. 96:165–192. doi:10.1016/bs.aivir.2016.08.0
Sztuba-Solińska et al. (2011) Subgenomic messenger RNAs: Mastering regulation of (+)-strand RNA virus life cycle. Virology 412 (2) :245-255.
Walls et al (2020) Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein [published online ahead of print]. Cell doi:10.1016/j.cell.2020.02.058
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