Replication of Positive-Sense Single-Stranded RNA Viruses

Introduction to positive-sense RNA viruses

1.1 Baltimore classification

Positive-sense single-stranded RNA (ssRNA) viruses belong to Class IV of the Baltimore classification system.

Their genome consists of a single strand of positive-sense RNA. Because the RNA is positive-sense, it can function directly as messenger RNA (mRNA) and be translated by the host cell's ribosomes.

This means that, unlike double-stranded RNA viruses, positive-sense RNA viruses do not need to carry all the enzymes required for genome replication inside the viral particle. Once the genome enters the host cell, it can immediately be translated to produce viral proteins.

1.2 Double-stranded RNA intermediates

Although their genomes are single-stranded, positive-sense RNA viruses produce double-stranded RNA intermediates during replication.

These intermediates are formed when the virus synthesises a complementary negative-sense RNA strand using the positive-sense genome as a template.

The negative-sense strand can then act as a template to produce new positive-sense RNA molecules. These positive-sense RNAs can be used as:

• New viral genomes.

• mRNAs for producing viral proteins.

However, the double-stranded RNA intermediates are not necessarily stable or long-lived. This is important because dsRNA can trigger host-cell antiviral responses. Positive-sense RNA viruses therefore face a similar challenge to dsRNA viruses, but their genomes are not permanently present as dsRNA.

1.3 Two strategies for positive-sense RNA viruses

The lecture introduces two broad strategies used by positive-sense RNA viruses.

Strategy 1: Act like host mRNA

The viral genome resembles a normal cellular mRNA and uses the host cell's translation machinery.

Strategy 2: Use a different translation mechanism

The viral genome deliberately differs from normal host mRNA and uses alternative mechanisms to recruit the translation machinery. This can allow viral translation to continue even when the host cell attempts to shut down its own protein synthesis.

The lecture explores these strategies using two examples:

• Alphaviruses: Use a genome that resembles conventional mRNA.

• Poliovirus: Uses a cap-independent translation mechanism.

Coronaviruses are also mentioned as positive-sense RNA viruses that share similarities with the alphavirus strategy, although they are not covered in detail in this lecture.

2. Alphaviruses

2.1 General characteristics

Alphaviruses are enveloped, positive-sense RNA viruses.

Their structure includes:

• A positive-sense RNA genome.

• A capsid surrounding the genome.

• A lipid envelope surrounding the capsid.

• Viral proteins embedded in the envelope.

The lecturer identifies Semliki Forest virus as a commonly used model for studying positive-sense RNA virus replication because it is relatively simple to work with.

Chikungunya virus is another example. It is mosquito-borne and is associated with joint swelling.

2.2 Alphavirus genome organisation

The alphavirus genome resembles a conventional cellular mRNA.

It has:

• A 5′ cap.

• Viral genes.

• A poly(A) tail at the 3′ end.

The genome contains two main open reading frames (ORFs), which encode two groups of proteins:

1. Non-structural proteins: Involved in RNA replication and the regulation of the replication process.

2. Structural proteins: Used to assemble new viral particles.

The proteins are initially produced as polyproteins, which are subsequently cleaved into individual proteins.

A polyprotein is a long protein translated from a single RNA sequence and then processed into several functional proteins.

2.3 General replication cycle

The alphavirus replication cycle involves the following stages:

1. The virus enters the host cell through receptor-mediated endocytosis.

2. The viral envelope fuses with the endosomal membrane.

3. The viral capsid is released into the cytosol.

4. The positive-sense RNA genome is translated into viral proteins.

5. Viral proteins establish the machinery required for RNA replication.

6. New positive-sense RNA molecules and structural proteins are produced.

7. Viral proteins are processed and assembled into new particles.

8. New virions acquire their envelopes and leave the cell through budding.

The following sections explain the main molecular events in this process.

3. Alphavirus entry and membrane fusion

3.1 Endocytosis and endosomal acidification

Alphaviruses enter cells through receptor-mediated endocytosis.

After the virus has been taken into the cell, it enters an endosome. As the endosome progresses through the endocytic pathway, its internal pH decreases.

The low pH triggers a conformational change in the viral envelope protein.

3.2 Conformational changes in the envelope protein

The envelope protein changes from its initial structure into a more open configuration.

This altered structure interacts with the host-cell membrane in a cholesterol-dependent manner.

The viral protein then undergoes further conformational changes that bring the viral and host membranes together, ultimately causing them to fuse.

Fusion allows the viral capsid to be released into the cytosol.

3.3 The point of no return

The lecturer distinguishes between two stages of the fusion process:

• Initial stage: Depends on the low pH found inside the endosome.

• Later stage: Does not depend on pH in the same way.

Once the protein has reached the later stage, the fusion process will proceed even if the pH conditions change.

The lecturer describes this as a point of no return.

This mechanism helps the virus avoid triggering membrane fusion prematurely outside the host cell. The initial conformational change occurs under the appropriate acidic conditions, allowing the virus to proceed with entry at the correct location.

The lecturer notes that acid-dependent membrane fusion is not unique to alphaviruses; it is used by other viruses as well.

4. Translation of the alphavirus genome

4.1 Immediate translation

Once the positive-sense RNA genome reaches the cytosol, it can be translated directly.

This gives positive-sense RNA viruses an advantage over viruses whose genomes cannot be translated immediately.

The first proteins produced include the non-structural proteins required to establish the replication machinery.

The virus therefore does not need to carry all its replication enzymes inside the incoming particle.

4.2 Non-structural polyprotein

The non-structural region of the alphavirus genome is translated into a polyprotein.

The lecture describes this as a precursor containing proteins NSP1–NSP4.

These proteins have different functions in the replication cycle.

Initially, the virus produces the non-structural proteins because it needs them to replicate its genome. The structural proteins are not required immediately, so their production is regulated to occur later.

4.3 Regulation of NSP4 production through a leaky stop codon

The virus controls how much of NSP4 is produced using a stop codon between the regions encoding NSP3 and NSP4.

The lecture identifies the stop codon as UGA and describes it as relatively leaky.

This means that ribosomes do not always stop translation at this position.

According to the lecture:

• Approximately 90% of the time, translation stops before NSP4, producing NSP1–NSP3.

• Approximately 10% of the time, the ribosome reads through the stop codon and produces the longer NSP1–NSP4 polyprotein.

This allows the virus to produce more of NSP1–NSP3 than NSP4.

The lecturer explains that this is useful because the virus does not require equal amounts of every non-structural protein.

4.4 Cleavage of the polyprotein

The polyprotein is processed by viral proteases.

The lecture identifies NSP2 as the protein responsible for cleaving NSP4 away from the initial polyprotein.

This releases NSP4 as an independent protein.

The cleavage process is important because the different forms of the non-structural proteins participate in different stages of RNA replication.

The lecturer emphasises that the processing of polyproteins is a common feature of RNA virus replication.

5. Alphavirus genome replication

5.1 Production of negative-sense RNA

The first stage of genome replication involves producing a negative-sense RNA copy of the positive-sense genome.

The lecture describes the NSP1–NSP3 polyprotein, together with the separately cleaved NSP4, as forming the functional RNA-dependent RNA polymerase (RdRp) complex.

This complex copies the positive-sense genome to produce a complementary negative-sense RNA strand.

The negative-sense RNA then provides a template for producing new positive-sense RNA molecules.

5.2 Changes in protein processing during infection

As infection progresses, the concentration of NSP2 increases.

The lecturer explains that higher levels of NSP2 lead to further cleavage of the non-structural polyprotein.

This produces NSP1, NSP2, NSP3 and NSP4 as independent proteins.

The change in processing alters the activity of the replication machinery.

5.3 Production of positive-sense RNA

When the four non-structural proteins are present as independent proteins, they can come together in a complex that supports the production of positive-sense RNA from the negative-sense template.

This allows the virus to regulate RNA synthesis at different stages of infection.

The lecturer presents the difference between the initial polyprotein form and the later, fully cleaved proteins as an established mechanism for controlling the timing of replication.

5.4 Temporal regulation of viral gene expression

The virus does not produce all its proteins at the same time.

The early stage is focused on producing non-structural proteins that establish the replication machinery.

The later stage allows the production of structural proteins needed to assemble new viral particles.

This is an example of temporal regulation of viral gene expression.

The advantage is that the virus does not waste resources producing large amounts of structural proteins before they are needed.

The lecturer compares this with the timing of gene expression in DNA viruses and double-stranded RNA viruses, although the molecular mechanism described here is particularly well established.

6. Subgenomic RNA and structural protein production

6.1 What is subgenomic RNA?

Once the alphavirus replication machinery has reached the appropriate stage, it can produce RNA corresponding only to the structural genes.

This RNA is called subgenomic RNA because it contains only part of the viral genome rather than the complete genome.

It is positive-sense RNA and can therefore be translated into proteins.

The subgenomic RNA contains the information needed to produce the structural proteins required for new viral particles.

6.2 Why produce a separate RNA?

Producing a separate subgenomic RNA allows the virus to regulate structural protein production independently of non-structural protein production.

The virus can produce the non-structural proteins early, when they are needed to establish replication, and produce structural proteins later, when new viral particles are being assembled.

This also means that structural protein production does not have to occur in the same amounts as non-structural protein production.

6.3 Processing the structural polyprotein

The structural proteins are initially translated as a polyprotein containing several components.

This polyprotein is then cleaved to produce the individual proteins required for assembly.

The lecture explains that cleavage can be carried out by different enzymes:

• Viral proteases.

• Host-cell enzymes, including signal peptidase and furin.

The virus therefore makes use of host-cell enzymes to process some of its proteins.

The lecturer notes that this can affect which cells the virus is able to infect. If a cell lacks a host enzyme needed for a particular processing step, the virus may be unable to complete that step successfully.

6.4 Downstream hairpin loop (DLP)

The structural-protein subgenomic RNA contains an RNA structure called the downstream hairpin loop (DLP).

This RNA structure is important for translation of the structural proteins.

During infection, the host cell can respond to viral infection by reducing protein synthesis. This limits the production of proteins by both the host and the virus.

The DLP helps the virus maintain translation of its structural proteins under these conditions.

The lecturer explains that the DLP interacts directly with methionyl-tRNA and helps recruit it to the viral RNA, allowing translation to proceed even when the normal host translation process is being inhibited.

The ribosome is still required to translate the RNA into protein. The DLP helps the viral RNA bypass part of the normal translation initiation process rather than eliminating the need for a ribosome altogether.

This gives the virus a way to continue producing structural proteins when the host cell is attempting to shut down protein synthesis.

7. Alphavirus assembly and release

Once the viral proteins have been produced and processed, new viral particles can be assembled.

The main stages are:

1. The capsid proteins assemble around the viral genome.

2. The assembled capsid moves towards the host cell's plasma membrane.

3. Viral envelope proteins are present in the membrane, having been produced earlier.

4. The capsid interacts with the membrane-associated viral proteins.

5. The virus buds through the membrane and acquires its lipid envelope.

The newly released virion therefore contains its RNA genome, capsid and surrounding lipid envelope.

This completes the alphavirus replication cycle.

8. Poliovirus

8.1 General characteristics

Poliovirus is a positive-sense single-stranded RNA virus.

Unlike alphaviruses, poliovirus is non-enveloped. It consists of an RNA genome surrounded by a protein capsid.

The capsid has an icosahedral structure and is formed from four capsid proteins.

The lecturer notes that the capsid proteins can spontaneously assemble into capsid structures when expressed in cells, even without the viral genome being present. This makes capsid assembly relatively straightforward to study experimentally.

8.2 General replication cycle

The poliovirus replication cycle involves:

1. Attachment to the host cell.

2. Entry into the cell.

3. Release of the viral genome into the cytosol.

4. Translation of the positive-sense RNA genome.

5. Production of viral replication proteins.

6. Replication of the RNA genome.

7. Assembly of capsids and packaging of the genome.

8. Release of new viral particles.

The key difference from alphaviruses is how poliovirus releases its genome and how it translates its RNA.

8.3 Poliovirus receptor

The lecturer discusses the poliovirus receptor as an example of how viruses have helped researchers identify previously poorly understood host-cell proteins.

For a long time, the receptor was known primarily because it bound poliovirus. Researchers subsequently identified other roles for the protein, including roles in immune signalling.

This illustrates how studying virus–host interactions can reveal previously unknown functions of host-cell proteins.

8.4 Genome release into the cytosol

Poliovirus enters the cell and undergoes uncoating within the endosome.

During this process, capsid proteins undergo conformational changes that allow the viral genome to pass through the membrane.

The lecture identifies VP4 as a capsid protein involved in forming a pore in the membrane.

The viral RNA is injected through the pore into the cytosol, while the capsid remains within the endosome.

This is different from the membrane-fusion mechanism described for alphaviruses. Poliovirus does not need to fuse a viral envelope with the endosomal membrane because it does not have an envelope.

Instead, it uses changes in its capsid proteins to allow the genome to cross the membrane.

9. Poliovirus genome organisation

9.1 One open reading frame

The poliovirus genome contains a single open reading frame.

This means that the entire protein-coding region is translated as one large polyprotein, which is subsequently cleaved into individual proteins.

The resulting proteins include:

• Structural proteins, which form the capsid.

• Non-structural proteins, which are involved in RNA replication and other stages of the viral life cycle.

Unlike the alphavirus example, where the lecture describes regulation through separate non-structural and structural regions and subgenomic RNA, poliovirus translates its protein-coding region as one large polyprotein.

The lecturer explains that this arrangement limits the virus's ability to independently control the initial production of each protein. The proteins are initially produced together and subsequently separated through cleavage.

9.2 VPg and RNA synthesis

Poliovirus has a protein called VPg that is covalently attached to the 5′ end of its RNA genome.

VPg plays a role in initiating RNA synthesis and acts as a primer for the production of new viral RNA.

The lecturer compares this with the use of primers in DNA synthesis.

A particular tyrosine residue in VPg is important for this function. The hydroxyl group on tyrosine participates in initiating RNA synthesis, allowing nucleotides to be added during the production of RNA.

The lecture discusses research showing that this tyrosine is conserved in related viruses and is essential for VPg's priming function.

If the relevant tyrosine is altered so that it can no longer perform this role, VPg cannot function properly as a primer.

9.3 The 5′ end and poly(A) tail

Unlike conventional cellular mRNA and the alphavirus genome described earlier, poliovirus RNA does not have a conventional 5′ methylguanosine cap.

However, the genome does have a poly(A) tail.

The absence of a 5′ cap means poliovirus needs an alternative mechanism to recruit the host translation machinery.

This is the basis of its cap-independent translation strategy.

10. Cap-independent translation in poliovirus

10.1 Internal ribosome entry site (IRES)

Poliovirus uses an internal ribosome entry site (IRES) to initiate translation without a conventional 5′ cap.

The IRES is a complex RNA structure formed by multiple stem-loops.

Rather than relying on the normal cap-dependent initiation process, this structure recruits the components needed for translation to an internal site on the viral RNA.

The ribosome can then initiate translation of the viral polyprotein.

The lecturer describes the IRES as a complex structure with multiple components, each contributing to its function.

10.2 Why is cap-independent translation advantageous?

Host-cell mRNAs normally rely on cap-dependent translation.

Poliovirus, however, does not need the conventional cap-dependent process because it uses the IRES.

This gives poliovirus an advantage when it interferes with the host's normal translation machinery: even if host mRNA translation is inhibited, poliovirus can continue translating its own RNA through its alternative mechanism.

10.3 The IRES and poliovirus vaccine mutations

The lecturer discusses three mutations associated with the attenuated poliovirus vaccine strain.

These mutations occur within the IRES region.

The lecture explains that changes to this RNA structure can substantially reduce the virus's ability to cause disease, producing an attenuated strain.

The lecturer refers to these as the Sabin mutations, associated with the oral poliovirus vaccine.

The importance of these mutations demonstrates how sensitive poliovirus function can be to changes in its RNA structure.

The lecturer also explains that attenuated live poliovirus strains can potentially acquire mutations that restore characteristics of wild-type virus. This is one reason the use of live attenuated vaccines requires careful consideration and monitoring.

The lecture mentions monitoring wastewater and rivers for poliovirus as part of surveillance, because virus shed from infected or vaccinated individuals can enter the environment.

11. Poliovirus takes over host-cell translation

11.1 Experimental evidence

The lecturer describes an experiment similar to the one discussed for rotavirus, in which proteins produced by infected cells were labelled and analysed using a blot.

Following poliovirus infection, the protein pattern changed rapidly.

Within approximately three hours of infection, the detected proteins were dominated by poliovirus proteins rather than the broad range of proteins normally produced by the host cell.

Because poliovirus produces a relatively small number of proteins, the viral protein bands could be identified individually.

This demonstrates how rapidly poliovirus can suppress host-cell protein production and redirect the cell towards producing viral proteins.

11.2 Poliovirus proteases

Poliovirus encodes proteases, including 2A and 3C, which contribute to the inhibition of host-cell cap-dependent translation.

These proteases interfere with components of the host translation machinery.

The lecturer particularly highlights the disruption of cap-dependent translation initiation, including the degradation of a cap-binding protein involved in the host process.

As these components are disrupted, normal host-cell translation becomes less effective.

Poliovirus, however, can continue translating its RNA using the IRES.

The result is that the host cell's normal protein production is suppressed while viral protein production continues.

11.3 How quickly does this happen?

The lecturer describes experimental evidence showing degradation of a host translation protein within approximately an hour of infection.

By around an hour and a half, the protein was substantially depleted or present as a degraded fragment in the experiment described.

This demonstrates that the inhibition of host translation begins early in infection.

The lecturer also notes that other virus families use different proteins to interfere with cap-dependent translation. Poliovirus is one example of a broader strategy used by viruses to prioritise the translation of their own RNA.

12. Poliovirus assembly and genome packaging

12.1 Capsid assembly

After the viral polyprotein has been processed, the individual structural proteins assemble into capsid structures.

The lecturer describes capsid assembly as progressing through intermediate structures, including monomers and trimers, before the complete capsid forms.

The capsid proteins can assemble even in the absence of a viral genome, making this process relatively easy to study.

However, a capsid without a genome is not a complete infectious virion.

12.2 Genome incorporation

The viral RNA genome must be incorporated into the capsid during assembly.

The lecturer explains that researchers have observed the addition of RNA during the assembly process, followed by further structural changes that produce the complete particle.

However, how poliovirus specifically recognises its own genome and ensures that it is packaged correctly remains unclear, according to the lecture.

This is an unresolved question in the assembly process described.

13. Poliovirus release from the host cell

13.1 Traditional model: cell lysis

Poliovirus is traditionally described as a non-enveloped, lytic virus.

In the classical model, infected cells eventually break apart, releasing the newly assembled virions.

Because poliovirus does not require an envelope for its normal infectious particle, it can be released as a capsid containing its RNA genome.

13.2 Evidence for membrane-associated release

The lecturer describes more recent evidence suggesting that poliovirus can also leave cells inside membrane-bound structures.

In this process:

• Groups of poliovirus particles become enclosed within lipid membranes.

• These membrane-associated structures leave the cell through exocytosis.

• The particles can subsequently become exposed as infectious capsids.

These structures are not considered conventional viral envelopes in the same sense as those of enveloped viruses.

Instead, the virus is enclosed within host-derived membrane material.

13.3 Potential advantage of membrane-associated release

The lecturer suggests that membrane enclosure may help protect poliovirus particles from the immune system.

Because the surrounding membrane is derived from the host cell, the enclosed viral particles may be less readily recognised as foreign.

The membrane can subsequently be lost or degraded, leaving infectious capsids able to infect another cell.

The lecturer emphasises that this is a relatively recent development in the understanding of poliovirus release. The traditional lytic mechanism remains relevant, and the virus is still classified as non-enveloped.

14. Comparison of the two viral strategies

The lecture uses alphaviruses and poliovirus to demonstrate two different ways of replicating positive-sense RNA viruses.