IV. VIRUSES

F. Animal Virus Life Cycles

4. The Life Cycle of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2)

Fundamental Statements for this Learning Object:

1. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a helical nucleocapsid that belong to the viral family Coronaviridae.
2. Most human coronaviruses typically cause mild symptoms of the common cold in children and adults.
3. Three human coronaviruses, however, can cause potentially severe and possibly fatal infections: MERS-CoV, SARS-CoV, and SARS-CoV-2 (the virus causing COVID-19.
4. SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2), the virus responsible for COVID-19, is characterized by a rapid and virulent human-to-human spread.
5. SARS-CoV-2 is an enveloped virus with a positive-sense single-stranded RNA (+ssRNA) genome containing approximately 30,000 nucleotides. The RNA genome is located within a helical nucleocapsid which, in turn, is enclosed within the viral envelope.
6. There are 3 envelope proteins: S, M, and E.
7. The S or spike protein. The S1 subunit is responsible for virus binding to ACE-2 enzyme during viral adsorption; the S2 subunit is responsible for viral envelope-host cell membrane fusion during viral entry.
8. The M or Membrane protein. The M protein appears to be the central organizer for coronavirus assembly. It plays a role in determining the shape of the virus envelope, binds to all other viral structural proteins, helps to stabilize nucleocapsids or N proteins, and promotes viral assembly or maturation.
9. The E or envelope protein. The E protein plays a role in the production, maturation, and release of this virus. Abundant at the site of intracellular trafficking (ER/Golgi) where viral maturation occurs.
10. In addition, SARS-CoV-2 produces 16 non-structural proteins (nsps), 15 that compose the viral replication and transcription complex (RTC) responsible for viral RNA synthesis. They also play important roles in evading host immune responses and modulating host cell intracellular membranes into viral replication organelles.
11. During viral adsorption, the S or spike protein in the viral envelope binds to human ACE-2 enzyme found on cells of the respiratory tract, gastrointestinal tract, kidneys, bladder, and heart. 12. Following adsorption, the virus appears to enter the host cell two ways: Fusion of the viral envelope with the host cell plasma membrane, or by endocytosis where the virus is placed in an endosome.
13. Coronaviruses possess a (+) single-stranded RNA genome.
14. To replicate the viral genome, RNA-dependent RNA polymerase enzymes copy the sense (+) RNA genome producing antisense ss (-) RNA. RNA-dependent RNA polymerase enzymes then copy the antisense (-) RNA strands producing copies of the sense ss (+) RNA viral genomes.
15. To produce viral mRNA molecules, RNA-dependent RNA polymerase enzymes copy portions of the antisense (-) RNA strand into subgenomic mRNAs that code for viral proteins. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes.
16. Early in the SARS-CoV-2 replication cycle, interactions between various nsps and host cell factors also initiate the formation of double-membrane replication organelles derived from the endoplasmic reticulum of the host cell. Double-stranded RNA (dsRNA), often thought to be an intermediate in the replication of coronaviruses, segregates into these double-membrane organelles where RNA synthesis has been shown to occur.
17. During maturation, the small subgenomic mRNAs coding for the N protein are translated by cytoplasmic ribosomes where they then bind to the genomic (+) ss RNAs to form viral nucleocapsids.
18. During maturation, other small subgenomic mRNAs coding for viral structural protein such as the S, M, and E proteins are translated at the ribosomes of the rough endoplasmic reticulum (ER). The ER then packages these proteins and transports them to the Golgi by way of ER-Golgi intermediate compartments (ERGIC).
19. Viral nucleocapsids in the cytoplasm bind to the ERGICs and are pulled in forming the viral envelope embedded with S, M, and E proteins. The intact viruses are then packaged in smooth secretion vesicles by the Golgi where they are released from the host cell by exocytosis.
20. As viruses replicate in alveolar epithelial cells, the cells become damaged, stimulating an inflammatory response through the release of inflammatory cytokines, danger-associated molecular patterns (DAMPs), and interferons. Excessive cytokine release into the blood as a result of a severe infection is referred to as a cytokine storm and can lead to acute respiratory distress syndrome (ARDS) in some people with COVID-19.
21. SARS-CoV-2 proteins can interfere with both innate immunity and adaptive immunity through a variety of mechanisms.  

 

LEARNING OBJECTIVES FOR THIS SECTION

Viruses are infectious agents with both living and nonliving characteristics.

1. Living characteristics of viruses

a. They reproduce at a fantastic rate, but only in living host cells.

b. They can mutate.

2. Nonliving characteristics of viruses

a. They are acellular, that is, they contain no cytoplasm or cellular organelles.

b. They carry out no metabolism on their own and must replicate using the host cell's metabolic machinery. In other words, viruses don't grow and divide. Instead, new viral components are synthesized and assembled within the infected host cell.

c. The vast majority of viruses possess either DNA or RNA but not both.

A. Coronaviruses

Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a helical nucleocapsid that belong to the viral family Coronaviridae. Their genome size is one of the largest among RNA viruses, ranging in size from 26,000-32,000 nucleotides. In electron micrographs they have club-shaped spikes extending from their envelope resembling a solar corona from which their name is derived.

1. SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) was a responsible for a pandemic in 2003 but has not circulated since 2004. Bats appear to be its natural host, from where it spread to palm civets and racoon dogs, and then to humans.

2. MERS-CoV (Middle East Respiratory Syndrome Coronavirus) was first reported in Saudi Arabia in 2012 and continues to infect humans, but with limited human-to human transmission. Bats appear to be its natural host, from where it infected dromedary camels, and from there to humans.

3. SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2). was first reported in Wuhan China in Dec. 2019. This virus is characterized by a rapid and virulent human-to-human spread. Bats appear to be its natural host, from where it spread to intermediate hosts, possibly pangolins, to humans, and then human-to-human. This is the virus responsible for the current worldwide pandemic called COVID-19 (Coronavirus Disease 2019).

We will concentrate here on SARS-CoV-2, the virus responsible for COVID-19.

- CDC: Get the Facts About Coronavirus

- CDC: Coronavirus Symptoms and Self-Checker

 SARS-CoV-2 is an enveloped virus with a positive-sense single-stranded RNA (+ssRNA) genome (def) containing approximately 30,000 nucleotides. The RNA genome is located within a helical nucleocapsid which, in turn, is enclosed within the viral envelope. In electron micrographs the viruses have club-shaped spikes extending from their envelope resembling a solar corona from which their name is derived (see Fig. 1A and Fig. 1B).

The viral envelope is derived from endoplasmic reticulum-Golgi intermediate compartments (ERGIC) produced during the host cell's secretory pathway, as shown below under viral maturation. There are three proteins that are associated with the viral envelope (see Fig. 1B):

1. S or spike protein. This is cleaved by host cell protease into 2 subunits, S1 and S2. The S1 subunit is responsible for virus binding to ACE-2 enzyme during viral adsorption; the S2 subunit is responsible for viral envelope-host cell membrane fusion during viral entry into the host cell.

In addition, SARS-CoV-2 produces 16 non-structural proteins (nsps) (def), 15 that compose the viral replication and transcription complex (RTC) responsible for viral RNA synthesis, RNA proofreading, and RNA modification. These nsps also play important roles in evading host immune responses and modulating host cell intracellular membranes into viral replication organelles.

- For Electron Micrographs of SARS-CoV-2 courtesy of NAID

1. Viral Adsorption to Host Cell

The S or spike protein of SARS-CoV-2 binds to the human ACE-2 enzyme (angiotensin-converting enzyme 2) (def) that is highly expressed in cells of the upper respiratory tract, type II alveolar cells (AT2) of the lungs, as well as cells such as enterocytes from the ileum and colon, kidney proximal tubule cells, bladder urothelial cells, and myocardial cells. As a result, people infected with SARS-CoV-2 not only might experience respiratory problems such as pneumonia leading to Acute Respiratory Distress Syndrome (ARDS), but also experience disorders of the digestive tract, kidneys, and heart.

The S protein is cleaved by a host cell protease into 2 subunits, S1 and S2. The S1 subunit is responsible for virus binding to ACE-2 enzyme; the S2 subunit contains a fusion peptide responsible for viral envelope-host cell membrane fusion (see Fig. 2 and Fig. 3).

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2. Viral Penetration of the Host Cell

Following adsorption of the S or spike protein in the envelope of SARS-CoV-2 to an ACE-2 enzyme on the surface of susceptible human cell, the virus appears to enter two ways: envelope fusion or endocytosis.

a. Entering by fusion of the viral envelope with the host cell plasma membrane (see Fig. 4). Proteolytic cleavage of the receptor-attached S protein into its S1 and S2 subunits by a host cell protease called TMPRSS2, found on the surface of the host cell, creates a fusion peptide that triggers the fusion of the viral envelope with the host cell plasma membrane (Fig. 4,step 1A) and the viral nucleocapsid, consisting of the (+) single-stranded RNA genome and N or nucleocapsid proteins, enters the host cell's cytoplasm (Fig. 4, step 1B).

b. Entering the host cell by endocytosis (def). (See Fig. 4). The virus enters by endocytosis and is placed in an endosome (Fig 4, step 2A). Proteolytic cleavage of the receptor-attached S protein into its S1 and S2 subunits by a host cell protease triggers the fusion of the viral envelope with the membrane of the endosome (Fig. 4, step 2B) and the viral nucleocapsid enters the host cell's cytoplasm (Fig. 4, step 2C).

 

3. Viral Replication

Coronaviruses possess a (+) single-stranded RNA genome (def). (See Fig. 5). To replicate the viral genome, RNA-dependent RNA polymerase (def). enzymes copy the sense (+) RNA genome (def) producing antisense ss (-) RNA (def). RNA-dependent RNA polymerase enzymes then copy the antisense (-) RNA strands producing copies of the sense ss (+) RNA viral genomes. To produce viral mRNA molecules, RNA-dependent RNA polymerase enzymes copy the antisense (-) RNA strand into (+) viral mRNAs. The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes.

The sense (+)ssRNA that enters the host cell's cytoplasm is also the viral mRNA that binds to host cell ribosomes in the cytoplasm. Approximately two thirds of the genome is transcribed into 2 polyproteins that are cleaved into the various nsps (def) that form the viral replication and transcription complex (RTC) needed for replication of the viral genome and transcription of the various viral mRNAs.

The RNA-dependent RNA polymerase then makes complementary (-)ssRNA copies of the (+)ssRNA genome. The (-)ssRNA strands are then copied by RNA polymerase into many (+)ssRNA genomic strands as well as many small subgenomic RNA strands (sgRNAs) through a process called discontinuous transcription. Leader transcription regulatory sequences (TRS) located at the 5' end of the RNA strand and multiple body TRS located along the body of the RNA strand enable transcription of several subgenomic mRNAs of varying length coding for various structural viral proteins such as S, M, E, and N proteins, as well as non-structural proteins (nsps). Remember that RNA polymerase can only bind to the 3' (def) end of an RNA strand and copies it in the 5' (def) direction (See Fig. 6).

Early in the SARS-CoV-2 replication cycle, interactions between various nsps (def) and host cell factors also initiate the formation of double-membrane replication organelles (def) derived from the endoplasmic reticulum of the host cell. These include double-membrane vesicles (DMVs) and convoluted membranes (CMs); direction (See Fig. 7). While there are still questions regarding the specific location of viral RNA synthesis within the infected host cell, double-stranded RNA (dsRNA), often thought to be an intermediate in the replication of coronaviruses, segregates into these double-membrane organelles where RNA synthesis has been shown to occur. These double-membrane enclosures for the viral dsRNA, are thought to create a protective microenvironment for viral genomic RNA replication and transcription of subgenomic mRNAs (sg mRNAs) of SARS-CoV-2. Remember that dsRNA and other viral RNAs are common pathogen-associated molecular patterns (PAMPs) (def) that bind to pattern-recognition receptors (PRRs) (def) in the cytoplasm of the host cell to initiate innate immunity against viruses.

 

4. Viral Maturation and Release (See Fig. 8).

The small subgenomic mRNAs coding for the N protein are translated by cytoplasmic ribosomes where they then bind to the genomic (+) ss RNAs to form viral nucleocapsids (Fig. 8, steps 1 & 2). Other small subgenomic mRNAs coding for viral structural protein such as the S, M, and E proteins, as well as a few other non-structural viral proteins, are translated at the ribosomes of the rough endoplasmic reticulum (ER) where they enter the ER (Fig. 8, step 3). The ER then packages these proteins and transports them to the Golgi by way of ER-to-Golgi intermediate compartments (ERGIC) containing the S, M, and E proteins. Viral nucleocapsids in the cytoplasm bind to the ERGICs and are pulled in forming the viral envelope embedded with S, M, and E proteins (Fig. 8, step 4). The intact viruses are then packaged in smooth secretion vesicles by the Golgi (Fig. 8, step 5) where they are released from the host cell by (def) exocytosis (Fig 8 step 6 and Fig. 9).

 

5. Cytokine Storm

As was learned in Unit 3 on Bacterial Pathogenicity, an excessive inflammatory response as a result of overproduction of proinflammatory cytokines can be harmful to the body, and in some cases fatal. The same applies to COVID-19.

Review from Unit 3: The Ability of PAMPs to Trigger the Production of an Excessive Inflammatory Response

For example, as viruses replicate in alveolar epithelial cells, the cells become damaged, potentially stimulating a strong, dysregulated pro-inflammatory response through the release of inflammatory cytokines (def) and danger-associated molecular patterns (DAMPs) (def). These, in turn, attract alveolar macrophages which produce inflammatory cytokines such as tumor necrosis-alpha (TNF-alpha), interleukin-1 (Il-1), Interleukin-6 (Il-6), and chemokines (def) such as Interleukin-8 (Il-8). An excessive production of inflammatory cytokines by the body is referred to as a cytokine storm (def). TNF-alpha and Il-1 increase capillary permeability and the expression of adhesion molecules on leukocytes and the inner wall of the capillaries for diapedesis (def), while chemokines attract neutrophils (def), macrophages (def) and cytotoxic T-lymphocytes (CTLs) (def) into the alveoli (def). Increased capillary permeability also enables plasma to flow out of the capillaries into the alveoli causing pulmonary edema (def) and loss of gas exchange. This results in dyspnea (def) and hypoxemia (def). Neutrophil and macrophage extracellular killing of alveolar cells, as well as the killing of infected cells by CTLs, causes further damage to alveolar cells and lung cells leading to more release of DAMPs and cytokines, as well as less production of surfactant which contributes to alveolar collapse. Damaged alveolar cells release leukotrienes (def) and prostaglandins (def) - along with TNF-alpha, IL-1 and Il-6 cause fever and further inflammation and infiltration of fluids, neutrophils, macrophages, and CTLs. Hypoxemia leads to tachypnea (def) and tachycardia (def). Collectively, this referred to as acute respiratory distress syndrome or ARDS (def).

6. Resisting Innate Immunity (def)

Some of the nsps (def) of SARS-CoV-2 dampen the production of type-I interferons (def) as well as other host proteins involved in innate immunity. Interferons are cytokines that activate hundreds of genes that collectively can produce a variety of antiviral protein interfering with every step in the viral life cycle and blocking viral replication. Interferons are ultimately able to:

1. Strengthen the cells outermost defenses to interfere with viral entry into the host cell.
2. Degrade mRNA to block transcription and curb protein synthesis within the host cell.
3. Block maturation of the viruses.
4. Block the release of mature viruses from infected cells.
5. Activate macrophages (def) and NK-cells (def), as well as recruit B-lymphocytes (def) and T-lymphocytes (def) to the area for adaptive immune responses against the virus and the viral-infected cells.

Most SARS-CoV-2 proteins ultimately play a role in blocking interferon function. For example:

1. Viral RNA is a major pathogen-associated molecular pattern (PAMP) (def) responsible for activating innate immunity and interferon production. Certain SARS-CoV-2 proteins interfere with the host cell's ability to recognize viral RNA.
2. Other SARS-CoV-2 proteins block the cellular signaling molecules used to activate the transcription of interferon genes in the host cell's nucleus.
3. Some SARS-CoV-2 proteins block the actual synthesis of interferons by the host cell.
4. Some SARS-CoV-2 proteins interfere with the interferon-induced activation of a wide variety of other host genes that confer antiviral activity.

As mentioned above, the double-membrane replication organelles (def) that enclose the viral dsRNA, are also thought to create a protective microenvironment for viral genomic RNA replication and transcription of sub-genomic mRNAs (sg mRNAs) of SARS-CoV-2. Remember that dsRNA and other viral RNAs are common pathogen-associated molecular patterns (PAMPs) (def) that bind to pattern-recognition receptors (PRRs) (def) in the cytoplasm of the host cell to initiate innate immunity against viruses. The early suppression of innate immunity by SARS-CoV-2 promotes viral replication.

In addition, Some proteins also block autophagy (def) . within infected host cells. Autophagy is the process by which a cell will breakdown its own proteins and, in the process, digest viruses and viral proteins within that cell.

7. Resisting Adaptive Immunity (def)

During humoral immunity (def), virus-neutralizing antibodies are made that recognize and bind to the S-protein of SARS-CoV-2 and block the adsorption (def) of the virus to the ACE-2 enzyme (angiotensin-converting enzyme 2) (def) receptors on the host cell. Mutation of the viral genome can alter the shape of the S-protein (def) of SARS-CoV-2 so that antibodies made against the S-protein of previous SARS-CoV-2 strains do not "fit" the newer mutant strain as well and thus decrease their neutralizing ability.

Mutations in the S-protein can also modify its shape in a way that enables the virus to more readily bind to the ACE-2 enzyme on infected cells, making the virus strain more contagious. For example, omicron strains of SARS-CoV-2 have stabilized the receptor binding domain of its S-protein so that it more effectively binds to Ace-2 receptors. Also, mutations have caused the receptor binding domain of the omicron's S-protein to become more positively charged. Since the ACE-2 receptors on the host cell are negatively charged, the S-protein of the virus is more readily attracted to the ACE-2 enzyme. (Since antibodies have a positive charge, the increased positive charge of the S-protein may also make it more difficult for antibodies to react with the viral S-protein.)

During cell-mediated immunity (def), a major antiviral body defense is the production of cytotoxic T-lymphocytes (CTLs) (def). CTLs use their T-cell receptors (def) and CD8 molecules (def) to bind to viral epitopes (def) bound to MHC-I molecules (def) on the surface of virus-infected host cell. They then kill the infected cell by inducing apoptosis (def). Some SARS-CoV-2 proteins suppress the production of MHC-I molecules by the infected host cell. MHC-I molecules are responsible for presenting epitopes of SARS-CoV-2 to the T-cell receptors and CD8 molecules on T8-lymphocytes (def) in order for these cells to proliferate and differentiate into cytotoxic T-lymphocytes (CTLs). During severe disease, there is also a decrease in the number of dendritic cells (def), NK cells (def), alveolar macrophages (def), and T-lymphocytes (def).

 

Concept map for the life cycle of SARS-CoV-2.

 

Medscape article on infections associated with organisms mentioned in this Learning Object. Registration to access this website is free. Cytomegalovirus