In today’s video, we’ll take a look at antiviral pathways. We will discuss how viruses are detected by the immune system, and how this generates an innate antiviral response.

The two major arms of an immune response are the innate and adaptive immune responses.

This applies to viral infections, with the initial response to a virus being the innate immune response that can react quickly by triggering the expression of genes that have intrinsic antiviral activity. The adaptive immune response takes longer to react, but will have memory of the virus once it has been exposed. Adaptive immunity can therefore protect the body from future infections in a quicker manner.

This video will focus on how a virus triggers the innate immune response and the expression of intrinsic antiviral genes after a viral infection has been detected.

Humans are constantly being exposed to microorganisms like bacteria, fungi, and viruses. Since many of these organisms are capable of causing severe disease in their hosts, our cells have evolved ways to recognize characteristics unique to the invaders. These are features that are not normally expressed by host cells, so their presence is a clear indication of infection.

Microorganisms that are able to cause disease are called pathogens, and the features that they commonly express that help our immune system recognize them are called pathogen-associated molecular patterns, or PAMPs. Viral PAMPs include structural components like their glycoproteins, which are expressed on the outside of the virus and used for attachment to host cells. They also have genomic PAMPs, which are hidden within the core of the virus particle and are not exposed until the virus enters the cell.

Viral genomes have important characteristics that distinguish them from host genetic material. For example, Reoviruses are a family of viruses that carry genomes composed of double stranded RNA. Double stranded RNA is not produced by eukaryotic cells, so the presence of these RNA molecules is a warning to the cell that there is an infection occurring. Other viruses like West Nile virus, which causes West Nile Fever, and the seasonal flu virus, Influenza A, possess single stranded RNA genomes. These can be detected directly if localized to certain compartments within the cell or when they form double stranded RNA intermediates during the replication process.

Viruses that carry DNA genomes, like Herpes Simplex viruses, can also be recognized by their genetic features. This is due to the fact that viral DNA is significantly less methylated than human DNA, which allows human cells to easily tell the difference between our own DNA and the DNA belonging to a virus.

To sense the presence of viral PAMPs, host cells use receptors called Pathogen Recognition Receptors, or PRRs. PRRs are designed to recognize conserved PAMPs to trigger an innate immune response. These receptors can be expressed on the plasma membrane of cells, or can be found on the inside of cells to detect viruses as they attempt to replicate.

Toll-like Receptors, or TLRs, are a major family of PRRs. TLRs are transmembrane proteins found on the cell surface and in endosomes. Many TLRs are expressed on the plasma membrane, including TLR2 and TLR4, which bind bacterial components like peptidoglycan and lipopolysaccharide, respectively. However, TLR4 is also known to bind proteins expressed by several viruses, such as Ebola and the virus that causes Dengue fever.

Intracellular PRRs can be categorized as endosomal or cytoplasmic. It is important for the host to have detection methods in both locations, since some viruses reveal their genetic material in endosomes and others reveal their genetic material in the cytoplasm.

Endosomal PRRs include TLR3 and TLR7, which detect double stranded and single stranded viral RNA, respectively. TLR9 detects viral DNA. For example, retroviruses like HIV carry single stranded RNA genomes, which are known to trigger TLR7 sensing, while Herpesviruses have DNA genomes sensed by TLR9.

In the cytoplasm, there are several different types of PRRs that sense viral nucleic acids. RIG-I-like receptors, or RLRs, have helicase domains that are able to detect double stranded RNA from viruses. Viral DNA in the cytoplasm can be sensed by a PRR called c-GAS, which recognizes the sugar-phosphate backbone of DNA molecules. cGAS does not appear to recognize features specific to viral DNA, but since host DNA should be contained in the nucleus, cGAS detection of DNA in the cytoplasm is an indicator that the DNA is of foreign origin.

Binding of PRRs by viral PAMPs triggers signaling pathways that lead to activation of genes involved in the innate immune response. PRRs often initiate the signal through activation of an adaptor protein. Almost all TLRs can activate signaling through the shared adaptor MyD88, and some TLRs can activate signaling through the adaptor TRIF. Both adaptors can recruit TRAF proteins that eventually cause activation of transcription factors like NF-Kappa B, a regulator of innate immune genes.

Upon binding of RLRs, like RIG-I and MDA5, by viral RNA in the cytoplasm, a mitochondria-associated adaptor protein called MAVS is activated. MAVS then acts as a signaling scaffold to induce activation of NF-Kappa B and Interferon Regulatory Factors, or IRFs. IRFs are transcription factors that regulate innate immune signals known as interferons.

cGAS, the PRR that detects viral DNA, utilizes the secondary messenger, cyclic GAMP, to activate an adaptor protein called STING, which then induces signaling to activate interferon production.

PRR signaling leads to the production of cytokines that are important for regulating immune responses. One class of cytokines that is critical for the innate antiviral response is Interferons. Interferons are divided into three types: type I – which includes interferon alpha and beta, primarily produced by innate immune cells such as Macrophages and plasmacytoid dendritic cells; type II – which refers to interferon gamma, produced by a number of cell types including lymphocytes like Natural Killer cells and cytotoxic CD8+ T cells; and type III – which consists of interferon lambda subtypes that act primarily on epithelial cells on mucosal surfaces, like the gut.

All three types of interferons activate the expression of genes called interferon stimulated genes, or ISGs. Hundreds of ISGs have been discovered, and many of them produce proteins that directly antagonize viral replication by inhibiting various steps of the virus life cycle, from virus entry, to genome replication and viral release.

These steps are displayed in the figure on the right, and the proteins in red text are ISGs that inhibit the designated replication steps. As you can see, the majority of the steps in a virus’ life cycle have an associated ISG that has evolved to restrict the activity of the virus at a specific stage.

A few examples of ISGs include eye-fit-em (IFITM), which inhibits viral membrane fusion and initial entry into the cell, Ribonuclease L, which directly cleaves viral RNA, and Tetherin, a trasmembrane protein that tethers new virus particles to the cell membrane to stop their release.

Though ISGs are generally considered to be part of the innate immune response, PRR sensing can also lead to processes that aid in adaptive immunity. For example, PAMP detection induces activation and maturation of antigen presenting cells, characterized by upregulation of co-stimulatory molecules such as CD80 and CD86 and production of cytokines. Other properties may be induced depending on the cell type, and it includes increased antigen presentation capacity, migratory patterns, or increased pathogen killing.

Thus, PRR stimulation does not only activate an intrinsic antiviral response, but it is also needed for lasting adaptive immunity to viral infections.

The coexistence between viruses and their hosts have led viruses to evolve their own weapons against their hosts’ immune systems. Indeed, many successful viral pathogens are known to use various mechanisms to evade PRR detection.

A common evasion method is hiding viral genomic material from host sensors. Dengue virus leverages the complex membrane system of the endoplasmic reticulum to hide its own genome during replication, while Influenza A and the Ebola virus encode their own specialized proteins that bind their RNA genomes and shield them from detection by RIG-I-like receptors.

Another method that viruses utilize is the post-translational modification of PRRs to inhibit their function. For example, ubiquitination of RLR’s is needed for their proper activation, so several viruses, including influenza A and the original SARS-coronavirus, encode proteins that block this process from happening.

Instead of indirectly blocking the activation of PRRs, some viruses have methods to directly destroy these sensors. Poliovirus encodes a protease that cleaves the RIG-I-like receptor, MDA5, while hepatitis A and C viruses carry proteases to degrade MAVS – the adaptor protein needed for RLR signaling.

Finally, viruses can also target the end product of PRR sensing, which is the ISG-encoded antiviral proteins themselves. A prime example of this is HIV, which possesses an array of viral antagonists for ISG proteins, including its VPU protein that targets the ISG Tetherin for degradation.

The effectiveness of a virus’ methods for evading host detection is a major contributor of its replicative success within a host species.

In summary, viruses have biological features that are not found in humans, and are therefore easily detectable by receptors called PRRs.

Stimulation of these receptors induce the activation of pathways for intrinsic antiviral responses, including the activation of interferon stimulated genes.

Successful pathogenic viruses have evolved mechanisms to evade detection by their hosts, and understanding how viruses interact with antiviral pathways at a molecular level will help scientists develop therapies that directly block viral replication, or boost immunological responses to viral infections.

You can utilize reliable and highly specific antibodies from BioLegend to characterize activation of antiviral signaling factors like IRFs, recombinant proteins like interferons to stimulate expression of ISGs, or ELISAs and LEGENDplex assays to measure cytokine responses to viral infection.

Curious about what happens when immune responses to viral infections are uncontrolled? View our short video lecture on Cytokine Storm to learn about how our own immune systems cause inflammatory disease during infections. And, be sure to send any comments to us here.