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New research reveals why some patients may test positive for COVID-19 long after recovery
An image of lung cancer cells infected with the SARS-CoV-2 virus. Blue represents DNA, green shows the SARS-CoV-2 nucleocapsid protein, and red represents double-stranded RNA, which occurs when the virus replicates its genome. A new study from the Jaenisch lab suggests that some virus RNA can be reverse transcribed and inserted into the human genome, which may explain why some patients continue to test positive for COVID-19 even after recovery. Credit Alexsia Richards/Whitehead Institute
In the early months of the COVID-19 pandemic, healthcare workers analyzing test results began noticing something strange: patients who had already recovered from COVID-19 would sometimes inexplicably test positive on a PCR test weeks or even months later.
Although people can catch COVID-19 for a second time, this did not appear to be the case for these patients no live viruses were isolated from their samples, and some studies found these false positive results even while holding participants in quarantine. Also, RNAs generally have a short life—most only stick around for a few minutes—so it was unlikely for positive tests to be the result of residual RNAs.
Now, a new paper from the lab of Whitehead Institute Member and MIT professor of biology Rudolf Jaenisch may offer an answer to why some patients continue to test positive after recovery from COVID-19. In the paper, published online May 6 in the Proceedings of the National Academy of Sciences, Jaenisch and collaborators show that genetic sequences from the RNA virus SARS-CoV-2 can integrate into the genome of the host cell through a process called reverse transcription. These sections of the genome can then be "read" into RNAs, which could potentially be picked up by a PCR test.
SARS-CoV-2 is not the only virus that integrates into the human genome. Around eight percent of our DNA consists of the remnants of ancient viruses. Some viruses, called retroviruses, rely on integration into human DNA in order to replicate themselves. "SARS-CoV-2 is not a retrovirus, which means it doesn't need reverse transcription for its replication," says Whitehead Institute postdoc and first author Liguo Zhang. "However, non-retroviral RNA virus sequences have been detected in the genomes of many vertebrate species, including humans."
With this in mind, Zhang and Jaenisch began to design experiments to test whether this viral integration could be happening with the novel coronavirus. With the help of Jaenisch lab postdoc Alexsia Richards, the researchers infected human cells with coronavirus in the lab and then sequenced the DNA from infected cells two days later to see whether it contained traces of the virus' genetic material.
To ensure that their results could be confirmed with different methodology, they used three different DNA sequencing techniques. In all samples, they found fragments of viral genetic material (though the researchers emphasize that none of the inserted fragments was enough to recreate a live virus).
Zhang, Jaenisch and colleagues then examined the DNA flanking the small viral sequences for clues to the mechanism by which they got there. In these surrounding sequences, the researchers found the hallmark of a genetic feature called a retrotransposon.
Sometimes called "jumping genes," transposons are sections of DNA that can move from one region of the genome to another. They are often activated to "jump" in conditions of high stress or during cancer or aging, and are powerful agents of genetic change.
One common transposon in the human genome is called the LINE1 retrotransposon, which is made up of a powerhouse combination of DNA-cutting machinery and reverse transcriptase, an enzyme that creates DNA molecules from an RNA template (like the RNA of SARS-CoV-2).
"There's a very clear footprint for LINE1 integration," Jaenisch says. "At the junction of the viral sequence to the cellular DNA, it makes a 20 base pair duplication."
Besides the duplication, another feature as evidence for LINE1-mediated integration is a LINE1 endonuclease recognition sequence. The researchers identified these features in nearly 70 percent of the DNAs that contained viral sequences, but not all, suggesting that the viral RNA may be integrating into cellular DNA via multiple mechanisms.
To screen for viral integration outside of the lab, the researchers analyzed published datasets of RNA transcripts from different types of samples, including COVID-19 patient samples. With these datasets, Zhang and Jaenisch were able to calculate the fraction of genes that were transcribed in these patients' cells which contained viral sequences that could be derived from integrated viral copies. The percentage varied from sample to sample, but for some, a relatively large fraction of viral transcripts seem to have been transcribed from viral genetic material integrated into the genome.
A previous draft of the paper with this finding was published online on the preprint server bioRxiv. However, recent research revealed that at least some of the viral-cellular reads could be the product of misleading artifacts of the RNA sequencing method. In the present paper, the researchers were able to eliminate these artifacts that could have been obscuring the results.
Instead of simply tallying transcripts that contained viral material, the researchers looked at which direction the transcripts had been read. If the viral reads were the result of live viruses or existing viral RNAs in the cell, the researchers would expect that most of the viral transcripts would have been read in the correct orientation for the sequences in question in acutely infected cells in culture, more than 99 percent are in the correct orientation. If the transcripts were the product of random viral integration into the genome, however, there would be a near 50-50 split—half the transcripts would have been read forwards, the other half backwards, relative to the host genes. "This is what we saw in some patient samples," says Zhang. "It suggests that much of the viral RNA in some samples could be transcribed from integrated sequences."
Because the dataset they used was quite small, Jaenisch emphasizes that more information is needed to establish exactly how common this phenomenon is in real life and what it might mean for human health.
It is possible that only a very few human cells experience any kind of viral integration at all. In the case of another RNA virus that integrates into the host cell genome, only a fraction of a percent of infected cells (between .001 and .01) contained integrated viral DNA. For SARS-CoV-2, the frequency of integration in humans is still unknown. "The fraction of cells which have the integrating with could be very small," says Jaenisch. "But even if it's rare, there are more than 140 million people who have been infected already, right?"
In the future, Jaenisch and Zhang plan to investigate whether the fragments of SARS-CoV-2 genetic material could be made into proteins by the cell. "If they do, and trigger immune responses, it may provide continuous protection against the virus," Zhang says.
They also hope to investigate whether these integrated sections of DNA could be partly to blame for some of the long-term autoimmune consequences that some COVID-19 patients experience. "At this point, we can only speculate," says Jaenisch. "But one thing we do think we can explain is why some patients are long-term PCR positive."
Issues of Concern
A major concern is RNA mutations, which can disrupt the normal functioning of RNA and cause potentially life-threatening diseases. RNA errors can be the result of defects in the ribonucleoprotein complex, RNA itself, RNA binding proteins, or any RNA assembly factors. Myotonic dystrophy is a neuromuscular disease that is caused by a CTG nucleotide repeat on the DMPK gene resulting in a pathogenic RNA gain-of-function.ਊ mutation in splicing can result in a mutated SMN2 gene and lead to spinal muscular atrophy. Other concerning illnesses caused by RNA mutations include Prader Willi syndrome, prostate cancer, Fragile X syndrome, and amyotrophic lateral sclerosis (ALS).
The mutation rates of RNA viruses that cause various illnesses in humans are very high. It can be up to 1 million times higher than the mutation rate of their hosts . This increase accounts for their fast evolution and ability to produce newer variants with higher infectivity or increased resistance to antibiotics. Since such mutations are heritable, they provide a bottleneck for the development of drugs or vaccines to combat viral infections. HIV infections are an example of the emergence of many drug-resistant strains, where the viruses replicate and cause severe disease even in the presence of drugs.
Additionally, RNA viruses can also recombine and reassort with DNA and RNA from the host or other viral strains, potentially generating a newer strain. Influenza viruses have a very high ability to reassort for example, the H1N1 influenza strain recombined with the RNA segments from birds, humans, and pig viruses to generate the H1N1 strain that caused a pandemic in 2009.
Research catches double-stranded RNA virus in the act of transcription
Researchers led by Hong Zhou (top left) found that the dsRNA virus uses proteins on its surface to sense its environment and that when conditions are optimal, those proteins turn the switch on inside the virus.
In separate studies published in the peer-reviewed journals eLife and Nature, scientists at the California NanoSystems Institute at UCLA have revealed the three-dimensional atomic structure of a double-stranded RNA, or dsRNA, virus. The research demonstrates for the first time how viruses sense environmental conditions inside a host cell to trigger transcription, and presents key findings about how the dsRNA genome is organized inside the virus and RNA's mechanism for self-replication.
The researchers also discovered the biological nano-switch that turns on transcription—the process by which RNA self-replicates—and compared the switch's structure in the "off" and "on" states to determine why environmental conditions activate it.
They now know that the dsRNA virus uses proteins on its surface to sense its environment and that when conditions are optimal, those proteins turn the switch on inside the virus through a signal transduction pathway. This activates RNA transcription.
The research was led by Hong Zhou, a professor of microbiology, immunology and molecular genetics and faculty director of UCLA's Electron Imaging Center for Nanomachines. For both studies, researchers analyzed the cytoplasmic polyhedrosis virus, or CPV, which infects insects. Zhou said the team focused on CPV because it is the simplest dsRNA virus.
"As RNA came before DNA, studies like these can shed light on fundamental questions about the earliest stages of evolution, such as how and when RNA first replicated," Zhou said. "And because CPV can replicate and transcribe its RNA within the intact virus and in the absence of cells, it provides a great tool to probe RNA transcription in action and at the atomic level."
The studies are the culmination of research Zhou began with his colleagues in the 1990s, when electron microscopes were far less powerful than they are today.
Double-stranded RNA viruses are the largest family of viruses (others include retroviruses and papilloma viruses). One well-known dsRNA virus is rotavirus, which causes diarrhea in humans and each year kills half a million people around the world, mostly children in developing countries.
The UCLA scientists' explanation of the process through which dsRNA replicates—and the conditions in which it takes place—gives scientists targets for new drugs that could be developed to combat dsRNA viruses.
Nanoscale biological complexes such as viruses are difficult for scientists to see because of their vanishingly small sizes. To put their dimensions in context: The difference in size between a virus particle and a football is about the same as the difference between a football and the planet Earth.
Both studies were conducted using UCLA's Titan Krios cryo electron microscope, a highly sophisticated machine in which flash-frozen biological samples are preserved in their native state. Recent advances in cryo electron microscopy have enabled scientists to build—with unprecedented, atomic-level detail—3-D images that are capable of revealing the chemistry governing the assembly of the biological complexes.
The eLife study found that a small molecule called S-adenosyl-L-methionine, or SAM, can activate transcription by binding to a turret protein on the virus's shell.
The researchers discovered for the first time that when SAM binds to one region of the turret protein, it changes the virus shell, which enables another region of the turret protein to bind a small molecule called ATP and break it down. The energy released from breaking down ATP causes further changes to the shell that activate RNA transcription.
The study's lead author was Xuekui Yu, a UCLA project scientist in microbiology, immunology and molecular genetics.
Genome organization of dsRNA
In the Nature study, the researchers imaged the organization of the dsRNA genome and proteins inside the virus. They then captured images of the process of transcription by comparing the structure before and after.
Xing Zhang, the study's lead author and the scientific director of UCLA's Electron Imaging Center for Nanomachines, said that viruses are "smart"—they seem to know how to avoid replicating when it is not productive because they won't activate unless all key conditions are present outside the virus shell. When transcription begins, segments of nascent RNA are released into the cell's internal environment, where they package themselves in protein shells to make new viruses. These new viruses exit the cell, propagate into other cells and start the whole process again.
Xuekui Yu et al. A putative ATPase mediates RNA transcription and capping in a dsRNA virus, eLife (2015). DOI: 10.7554/eLife.07901
Evasion of RLR–MAVS-dependent immunity
All successful viral pathogens have effective strategies to evade or inhibit the activation of intracellular PRRs. In this section, we discuss the molecular strategies that are used by viruses to inhibit the activation of RLRs and MAVS. Many viruses also inhibit innate immune responses by targeting downstream molecules that are shared between RLRs and other PRRs, such as TBK1, IRF3, IRF7 and NF-κB, or they block IFNα/β receptor signalling or the function of specific antiviral effector proteins. However, these strategies are beyond the scope of this Review and have been discussed extensively elsewhere 51,52 .
Sequestration or modification of viral RNA ligands. Most RNA viruses replicate in the cytoplasm where RLRs are also present and well positioned to detect foreign RNA. Several viruses have evolved ways to sequester their genomes to escape surveillance by RLRs (Fig. 3). A major strategy that is used by viruses to prevent RLRs from accessing viral RNA is to induce the formation of specific replication compartments that are confined by cellular membranes, or to replicate on organelles, such as the endoplasmic reticulum, the Golgi apparatus and mitochondria. For example, DENV, a mosquito-borne flavivirus that causes dengue fever and the more severe dengue haemorrhagic fever, replicates in convoluted membranes of the endoplasmic reticulum, which efficiently conceal dsRNA from the cytosol, thus preventing the activation of RLRs. By contrast, JEV, a related flavivirus, fails to conceal dsRNA and markedly induces the production of type I IFNs 53 . Unlike members of the Flaviviridae family and most other RNA viruses, influenza A viruses (IAVs), which are responsible for seasonal outbreaks of flu, have atypical life cycles and replicate in the nucleus to avoid the sensing of viral RNA by RLRs in the cytoplasm. In fact, IAV contains eight RNA genome segments that would potently stimulate the activation of RIG-I if found in large quantities in the cytoplasm. In addition, IAV has developed a strategy to avoid the recognition of viral RNA during the short transit of the virus through the cytoplasm after viral entry. It has been shown that in some strains of IAV the viral polymerase subunit PB2 prevents the RIG-I-mediated recognition of incoming genomic RNAs, which are encapsidated by nucleoproteins (NPs) 31 . PB2 in mammalian-adapted strains of IAV harbours a lysine residue at position 627 and has an increased affinity for NP, and this tight packing of the viral genome hinders the binding of RIG-I. Similarly, other viruses use viral or host-encoded proteins that 'shield' viral RNA from RLRs. For example, viral protein 35 (VP35) from EBOV and Marburg virus, non-structural protein 1 (NS1) from IAV and the E3 protein from vaccinia virus bind to viral dsRNA to avoid detection by RIG-I 54,55,56,57,58,59 , whereas respiratory syncytial virus (RSV), a member of the Paramyxoviridae family that can cause severe infection of the respiratory tract especially in children, uses the host cellular RNA-binding protein La to prevent RIG-I from binding to viral leader RNA 60 . Finally, the C protein of human parainfluenza virus type 1 (HPIV1) has been shown to limit the accumulation of cytoplasmic dsRNA to prevent the activation of MDA5 and the expression of type I IFN genes 61 .
Most successful viral pathogens are equipped with effective strategies to evade or inhibit the activation of intracellular pattern recognition receptors (PRRs), such as retinoic acid-inducible gene-I protein (RIG-I) or melanoma differentiation-associated protein 5 (MDA5), or the activation of their adaptor mitochondrial antiviral signalling protein (MAVS). To prevent the activation of RIG-I, viral phosphatases can process the 5′-triphosphate moiety in the viral RNA, or viral nucleases, such as the nucleoprotein (NP) of Lassa virus, can digest free double-stranded RNA (dsRNA). Furthermore, viral proteins, such as viral protein 35 (VP35) from EBOV, non-structural protein 1 (NS1) or PB2 from influenza A virus (IAV) and the E3 protein from vaccinia virus, or host proteins (such as La) bind to viral RNA to inhibit the recognition of pathogen-associated molecular patterns (PAMPs) by RIG-I. Several viruses manipulate specific post-translational modifications of RIG-I and/or MDA5, thereby blocking their signalling abilities. For example, viruses prevent the Lys63-linked ubiquitylation of RIG-I by encoding viral deubiquitylating enzymes (DUBs). NS1 from IAV and the NS3–NS4A protease complex from hepatitis C virus (HCV) antagonize the cellular E3 ubiquitin ligases, tripartite motif protein 25 (TRIM25) and/or Riplet, thereby also inhibiting RIG-I ubiquitylation and thus its activation. Furthermore, subgenomic flavivirus RNA (sfRNA) from dengue virus (DENV) binds to TRIM25 to block sustained RIG-I signalling. To suppress the activation of MDA5, the V proteins from measles virus (MeV) and Nipah virus (NiV) prevent the PP1α-mediated or PP1γ-mediated dephosphorylation of MDA5, keeping it in its phosphorylated inactivate state, whereas the V protein of parainfluenza virus 5 (PIV5) blocks the ATPase activity of MDA5. Furthermore, VP35 from EBOV, NS1 from IAV and the 4a protein from Middle East respiratory syndrome coronavirus (MERS-CoV) target protein kinase R activator (PACT) to antagonize RIG-I. The NS3 protein from DENV targets the trafficking factor 14-3-3ɛ to prevent the translocation of RIG-I to MAVS at the mitochondria. Numerous viruses encode proteases ( Pro ) to cleave RIG-I, MDA5 and/or MAVS. PB1-F2 from IAV translocates into the mitochondrial inner membrane space to accelerate mitochondrial fragmentation. Other viruses subvert cellular degradation pathways to inhibit RLR–MAVS-dependent signalling. Specifically, the X protein from hepatitis B virus (HBV) and the 9b protein from severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) promote the ubiquitylation and degradation of MAVS. BDV, Borna disease virus CCHFV, Crimean–Congo haemorrhagic fever virus CVB3, coxsackievirus B3 EV71, enterovirus 71 HAV, hepatitis A virus K63-Ub, Lys63-linked ubiquitylation P, phosphate RSV, respiratory syncytial virus Ub, ubiquitin.
As RLRs specifically recognize certain features of viral nucleic acids (Box 2), several viruses modify their genomes to prevent detection or hinder the activation of RLRs. Members of the Bunyaviridae family, such as Hantaan virus, and Crimean–Congo haemorrhagic fever virus (CCHFV), and also Borna disease virus (BDV) in the Bornaviridae family, encode phosphatases to process the 5′-triphosphate group on their genomes to 5′-monophosphate to escape surveillance by RIG-I 62,63 . Arenaviruses, such as Junin virus, which can cause haemorrhagic fever in humans, have a 5′-triphosphate group on their genomes, but with an atypical 5′ unpaired nucleotide overhang, which does not trigger the production of type I IFNs 64 . Mechanistically, 5′-triphosphate dsRNA with a nucleotide overhang binds to RIG-I without causing its activation, thereby acting as a decoy RNA 65 . Lassa virus has evolved another unique strategy in which the C-terminal half of its nucleoprotein (NP) adopts a 3D fold similar to the DEDD superfamily of exonucleases and has authentic 3′–5′ exonuclease activity. This activity enables the NP from Lassa virus to digest free dsRNA, which prevents the activation of RIG-I 66,67 .
Manipulation of the post-translational modification of RLRs and MAVS. In recent years, it has become clear that the RLR signalling pathway is intricately regulated by post-translational modifications — ubiquitylation and serine/threonine phosphorylation in particular — of RLRs and downstream signalling molecules. As the Lys63-linked ubiquitylation of RIG-I is a crucial step for its activation, it is not surprising that several viruses target this type of ubiquitylation to inhibit RIG-I signalling (Fig. 3). Some viruses directly target the cellular E3 ubiquitin ligases that are responsible for the ubiquitylation of RIG-I. For example, the NS1 proteins of many strains of IAV interact directly with TRIM25 through its coiled-coil domain this inhibits the homo-oligomerization of TRIM25, which is crucial for its enzymatic activity to attach Lys63-linked polyubiquitin to Lys172 in the CARDs of RIG-I 68 . The NS1 proteins of some IAV strains can also bind to Riplet, which inhibits the Lys63-linked polyubiquitylation of RIG-I at its C-terminal domain 69 . Similarly, the NS3–NS4A protease complex of HCV cleaves Riplet to prevent the Lys63-ubiquitylation of RIG-I 23 . Other viruses — both RNA viruses and DNA viruses — directly remove the Lys63-linked ubiquitylation of RIG-I through virus-encoded DUBs, such as ORF64 from KSHV, papain-like protease (PLP) from severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), leader proteinase (L pro ) from foot-and-mouth disease virus (FMDV) and the ovarian tumour (OTU)-type DUBs of arteriviruses and nairoviruses 70,71,72,73 . Furthermore, viruses can inhibit the Lys63-linked ubiquitylation of RIG-I independent of protein–protein interactions, by modulating the abundance of cellular microRNAs or through RNA–protein interactions. For example, the 3C protein of enterovirus 71 (EV71), a member of the Picornaviridae family that causes hand, foot and mouth disease, and occasionally severe central nervous system diseases, downregulates the host microRNA miR-526a to increase the expression of the cellular DUB enzyme CYLD, thus inhibiting the activation of RIG-I 74 . More recently, the subgenomic flavivirus RNA (sfRNA) of an epidemic strain of DENV that was isolated in Puerto Rico has been shown to bind to TRIM25 in a sequence-specific manner and prevent its deubiquitylation 75 , which has been shown to be crucial for sustained RIG-I signalling 76 .
The phosphorylation of serine or threonine residues keeps RIG-I and MDA5 inactive in the absence of an infection, whereas the recruitment of PP1α or PP1γ and the dephosphorylation of specific phosphorylation marks in RLRs are crucial for innate immune activation following viral infection. Measles virus (MeV), a morbillivirus in the Paramyxoviridae family, uses its non-structural V protein to bind to and sequester PP1α and PP1γ from MDA5, a mechanism that relies on the presence of a specific PP1-binding motif in the C-terminal 'tail' region of the viral protein 77 . Nipah virus (NiV), a related virus that has been observed to cause encephalitis and respiratory illness during outbreaks in South Asia and Southeast Asia, is also able to target PP1α and PP1γ and it is likely that other paramyxoviruses may have evolved a similar PP1-antagonistic strategy. The V proteins of MeV and NiV are, in turn, dephosphorylated by PP1α or PP1γ, which suggests that these viral proteins may act as decoy substrates alternatively, it is possible that the dephosphorylation of the V proteins by PP1α or PP1γ may promote other functions of the V proteins that could be beneficial for viral replication and/or virus-induced pathogenesis. In addition, MeV has evolved a V protein-independent mechanism to inhibit the dephosphorylation of both RIG-I and MDA5 by PP1α or PP1γ in dendritic cells. Specifically, MeV binds to the CLR protein dendritic-cell-specific ICAM3-grabbing non-integrin (DC-SIGN also known as CD209), and triggers its activation. DC-SIGN signalling then activates the kinase RAF1, which negatively regulates the activity of PP1α and PP1γ and inhibits the dephosphorylation of both RIG-I and MDA5 (Ref. 78).
Cleavage or degradation of RLRs and MAVS. One of the most effective ways to inhibit PRR signalling is to eliminate the sensor or a key component of its signalling pathway. As such, many viruses encode viral proteases that directly cleave RLRs (Fig. 3). The 3C pro proteases of both poliovirus and EV71 cleave RIG-I, whereas the 2A pro protease of EV71 cleaves MDA5 (Refs 79,80). As MAVS is crucial for both RIG-I-mediated and MDA5-mediated signalling, it is not surprising that numerous viral proteases target and cleave MAVS, such as 3C pro from hepatitis A virus (HAV), 2A pro from EV71, NS3–NS4A from HCV and GB virus B, 2A pro and 3C pro from rhinovirus, and 3C pro from coxsackievirus B3 (CVB3) 80,81,82,83,84,85 . By contrast, IAV uses a non-proteolytic mechanism to degrade MAVS. Specifically, PB1-F2 from IAV, a small accessory protein that contributes to viral pathogenicity, translocates into the mitochondrial inner membrane space to reduce the inner membrane potential, which accelerates the fragmentation of mitochondria and thereby inhibits MAVS signalling 86 . Furthermore, PB1-F2 binds to the transmembrane region of MAVS to block the induction of IFN production 87 .
Another strategy that is used by viruses to decrease the abundance of RLRs and MAVS is to subvert cellular degradation pathways. The X protein of hepatitis B virus (HBV), a DNA virus in the Hepadnaviridae family that also produces RNA species during its life cycle, binds to MAVS and promotes its degradation through the ubiquitylation of Lys136 however, the identity of the cellular ubiquitylating enzyme that is involved is unknown 88 . Infection with poliovirus leads to MDA5 being cleaved independent of the viral proteases 2A pro and 3C pro . Instead, the cleavage of MDA5 occurs in a proteasome-dependent and caspase-dependent manner 89 . Moreover, infection with MeV triggers selective autophagy to degrade mitochondria, a process termed mitophagy, which decreases the abundance of MAVS 90 . SARS-CoV, which causes severe acute respiratory syndrome, has evolved a strategy in which its 9b protein localizes to mitochondria and subverts the cellular E3 ubiquitin ligase atrophin-1-interacting protein 4 (AIP4) to degrade MAVS 91 . Another study showed that the NS1 and NS2 proteins of RSV trigger the proteasome-dependent degradation of RIG-I and numerous other immune molecules, but not MAVS, by assembling a large degradative complex on mitochondria 92 . More recently, a novel mechanism has been proposed for DENV and WNV in which their capsid proteins bind to peroxisomal biogenesis factor 19 (PEX19), which leads to a decrease in the number of peroxisomes in the cell and thereby impedes the MAVS-dependent production of type III IFNs 93 . However, further studies to elucidate the mechanistic details are still required.
Sequestration or relocalization of RLRs. To keep MDA5 sequestered and in an inactive state, the V proteins of several paramyxoviruses, including parainfluenza virus 5 (PIV5), directly bind to the helicase domain of MDA5 to block its ATPase activity 6,94,95,96 (Fig. 3). VP35 from EBOV, NS1 from IAV, and the 4a protein from Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) target PACT, which inhibits the activation of RIG-I (and possibly MDA5) and thus prevents the production of IFNs 97,98,99 . In addition, the membrane protein (M) from SARS-CoV, the Z protein from arenaviruses and the glycoprotein (G) from human metapneumovirus (hMPV) bind to RIG-I to sequester it from downstream signalling molecules 100,101,102,103 .
Following the recognition of RNA ligands in the cytosol, RIG-I and MDA5 need to translocate to signalling-competent organelles, such as mitochondria, MAMs and peroxisomes, on which MAVS is localized. Recently, it was shown that the NS3 protein of DENV binds to the mitochondrial-targeting chaperone protein 14-3-3ɛ to prevent the translocation of RIG-I to mitochondria that contain MAVS 104 . Intriguingly, NS3 uses a highly conserved phosphomimetic 64 RXEP 67 motif, which resembles a canonical phosphorylated serine-containing or threonine-containing motif of cellular 14-3-3-binding proteins, to interact with 14-3-3ɛ and impair RIG-I signalling and immune activation. A recombinant DENV encoding a mutant NS3 protein that is deficient in 14-3-3ɛ binding is impaired in RIG-I antagonism and elicited an augmented production of IFNβ, pro-inflammatory cytokines and ISGs, compared with wild-type DENV. The mutant DENV also induced a stronger activation of T cells. Interestingly, in contrast to the related NS3–NS4A protease complex of HCV, which cleaves MAVS to block RIG-I signalling, NS2B–NS3 of DENV antagonizes RIG-I-dependent signalling in a proteolysis-independent manner.
Another strategy used by viruses to sequester RLRs is to relocalize them to other subcellular sites, often virus-induced structures. The nucleoprotein (N) of RSV binds to MDA5 and relocalizes it (and also MAVS) to large viral inclusion bodies 105 , whereas the non-structural protein in small segment (NSs) of severe fever with thrombocytopenia syndrome virus (SFTSV), a recently described phlebovirus in the Bunyaviridae family, relocalizes RIG-I and its upstream activator TRIM25 into cytoplasmic endosome-like structures, the formation of which is induced by infection with SFTSV 106 . Furthermore, stress granules are cytoplasmic bodies that have been proposed to act as antiviral platforms for RLR signalling, and the 3C pro of poliovirus and encephalomyocarditis virus (EMCV) cleave the cellular protein RAS GTPase-activating protein-binding protein 1 (G3BP1) to prevent the formation of RLR-containing stress granules 107,108 .
Finally, as NLR proteins also have a major role in virus sensing, several viruses have, in turn, evolved mechanisms to counteract NLR-mediated signalling pathways (Box 3).
Box 3: Viral evasion of NLR-dependent immunity
Several viruses target the NOD-like receptor (NLR)-mediated activation of the inflammasome. Non-structural protein 1 (NS1) from influenza A virus (IAV) inhibits the production of interleukin-1β (IL-1β) and IL-18 during infection, although the molecular details remain elusive 159 . An IAV mutant with a partial deletion of the amino-terminal RNA-binding domain of NS1 induces the production of IL-1β and IL-18, which suggests that this domain is responsible for antagonizing the activation of caspase 1. Furthermore, PB1-F2 from IAV translocates into the mitochondrial inner membrane space to decrease mitochondrial membrane potential and to accelerate the fragmentation of mitochondria, thus blocking both the activation of the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome and retinoic acid-inducible gene-I protein (RIG-I)–mitochondrial antiviral signalling protein (MAVS) signalling 86 . The V protein of measles virus (MeV) interacts with NLRP3 and relocalizes it to the perinuclear region following activation of the inflammasome, thereby inhibiting the secretion of IL-1β 160 . The M13L protein of myxoma virus and the vPOP protein of Shope fibroma virus bind to and target the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) to prevent the activation of caspase 1 (Refs 161,162). Finally, the F1L protein of vaccinia virus and the ORF63 protein of Kaposi sarcoma-associated herpesvirus (KSHV) interact with NLRP1 to inhibit the activation of the inflammasome 163,164 , although it is unclear whether NLRP1 is directly involved in nucleic acid sensing. Interestingly, the ORF63 protein of KSHV is a viral homologue of NLRP1 and is able to interact with NLRP1, NLRP3 and nucleotide-binding oligomerization domain-containing protein 2 (NOD2), which suggests a broad antagonism of cellular NLRs.
The most well-known retrovirus that infects humans is HIV. However, there are several other human retroviruses. These include the human T-cell lymphotropic virus 1 (HTLV-1). HTLV-1 is associated with certain T-cell leukemias and lymphomas. There are many additional retroviruses which have been identified as infecting other species.
HIV treatment is one of the reasons that people have become more familiar with the concept of retroviruses. Reverse transcriptase inhibitors make up some of the well-known classes of HIV drugs.
Reverse transcriptase inhibitors prevent HIV from becoming integrated into the genome of the host cell. This, in turn, keeps the cell from making copies of the virus and slows the progression of the infection. However, there are growing problems with resistance to many drugs in these classes.
Retroviruses are also sometimes used as gene delivery methods during gene therapy. This is because these viruses are both easy to modify and easily integrated into the host genome.
This means that, in theory, they can be used to cause the cellular machinery to make proteins in an ongoing way. For example, scientists have used retroviruses to help diabetic rats make their own insulin.
IL-10 Is Induced in Response to dsRNA𠅊 number of viruses have been found to be potent inducers of IL-10 (20). To explore this phenomenon, spleen-derived macrophage cells (SMs) were treated with pIC to simulate dsRNA intermediates of viral replication. RNA from pIC-treated cells was used for real-time quantitative PCR. IL-10 mRNA was found to be highly induced ( Fig. 1A ). Appropriately, IL-10 protein was also induced in cells, with levels in the culture medium peaking at 8 h ( Fig. 1B ). As LPS has previously been shown to induce IL-10, this treatment was used to confirm that the SM cell line responds appropriately ( Fig. 1C ). The data show the reported induction of IL-10 by viral infection is, at least in part, via a dsRNA response.
IL-10 is induced by dsRNA. Levels of IL-10 were measured in SM cells treated with the dsRNA mimic pIC (50 μg/ml) for the indicated time periods. A, total RNA was extracted and analyzed by real-time quantitative PCR to measure the induction of the IL-10 transcript. B, the cell supernatants were analyzed by ELISA to measure induction of IL-10 protein. C, SM cells were treated with LPS (10 ng/ml) for the indicated time periods, and the cell supernatants were analyzed by ELISA for IL-10 levels.
JNK and NF-κB Regulate IL-10 Induction—To identify the essential factors that mediate IL-10 induction, signaling pathways regulated by dsRNA were explored. Members of the MAP kinase family with NF-㮫 have been reported to regulate IL-10 induction in macrophages in response to LPS (8, 26, 34). To assess a role for these proteins in the dsRNA-mediated induction of IL-10, SM cells were treated with the chemical inhibitors, SP600125, PD98059, SB203580, or BAY-11-7085, to gauge the involvement of JNK, Erk1/2, p38, and NF-㮫 (respectively). Induction of both IL-10 mRNA and protein was significantly blocked in response to pIC only in the presence of the JNK and IKK inhibitor ( Fig. 2, A and B ).
dsRNA-mediated IL-10 induction requires JNK and NF-㮫. A, identity of signal transduction proteins that regulate the induction of IL-10 was investigated by a 1-h pretreatment of SM cells with the inhibitors SP600125 (SP 10 μ m ), PD98059 (PD 20 μ m ), SB203580 (SB 4 μ m ), or BAY-11-7085 (BAY-11 5 μ m ), or as a control the solvent (DMSO), or untreated (NT), prior to stimulation with pIC (50 μg/ml) for 16 h. IL-10 levels were then measured by ELISA. B, IL-10 mRNA levels were measured by real-time quantitative PCR after 1 h of pretreatment of SM cells with SP600125 (10 μ m ) and BAY-11-7085 (5 μ m ) followed by stimulation with pIC (50 μg/ml) for 3 and 6 h. C, dsRNA-mediated induction of IL-10 was assessed using a firefly luciferase reporter construct directed by the murine IL-10 promoter fragment to +65 (pGL3B-IL-10-LUC). The reporter construct was transiently cotransfected into RAW264.7 cells with constructs expressing GFP (eGFP), dominant negative (dn) JNK1, c-Jun, and ATF2, or the I㮫α super-repressor (dn JNK, I㮫αSR, dn cJUN, dn ATF2, respectively). Following transfection, untreated controls (NT) were compared with cells treated with pIC (50 μg/ml) for 16 h. IL-10 promoter activity was measured by luciferase assay. The experiment was done in triplicate.
The role of NF-㮫 and JNK in pIC-regulated IL-10 induction was further examined by promoter reporter assay. Dominant negative JNK1 or the I㮫α super-repressor (I㮫α-SR) was transfected into the murine macrophage cell line, RAW 264.7, expressing the murine IL-10 promoter (/+65) as a luciferase reporter construct, and then treated with pIC. As was shown for the endogenous IL-10 mRNA and protein, the IL-10 promoter was induced more than 3-fold in response to pIC ( Fig. 2C ). The promoter induction was significantly reduced by dominant negative JNK1 and I㮫α-SR constructs. In contrast, the dominant negative c-Jun and ATF2 constructs did not alter the activation of the IL-10 promoter.
NF-κB Regulates the IL-10 Promoter via a Novel Distal Binding Site—The involvement of NF-㮫 in dsRNA-mediated IL-10 induction was verified using an electrophoretic mobility shift assay with lysates of SM cells expressing the I㮫α-SR. Treatment of SM cells with pIC showed significant NF-㮫 DNA binding activity ( Fig. 3A ). Control treatments compared TNFα and IL-1β treatments. Appropriately, NF-㮫 DNA binding activity was significantly reduced in cells expressing I㮫α-SR. Accordingly, the induction of IL-10 was reduced 3-fold in response to pIC in cells expressing I㮫α-SR compared with control GFP-expressing cells ( Fig. 3B ).
dsRNA-mediated NF-㮫-dependent regulation of IL-10 at a novel distal site on the gene promoter. A, direct activation of NF-㮫 was measured by EMSA in whole cell lysates (20 μg) from SM cells stably expressing I㮫α-SR or GFP. Cells were untreated (C), or stimulated with pIC (50 μg/ml), and as controls TNFα (25 ng/ml), or IL-1β (5 ng/ml) for 30 min. B, role of NF-㮫 in dsRNA induction of IL-10 was verified by comparing IL-10 protein levels in supernatants from SM cells either stably expressing the I㮫α-super-repressor (IκBα-SR) or, as a control, GFP (eGFP), either untreated or treated with pIC (50 μg/ml) for 16 h. C, binding of NF-㮫 to specific elements within the IL-10 promoter was measured by ChIP analysis. Protein complexes were immunoprecipitated with anti-p65, or anti-p50 antibodies from SM cell lysates either untreated or stimulated with pIC (50 μg/ml) for 6 h. Two regions of the IL-10 promoter, surrounding regions / and /, were amplified from DNA obtained from the ChIP. As a positive control amplification of regions surrounding the α-amylase promoter is shown using input DNA before ChIP (Input). The specificity of the ChIP assay was tested using rabbit IgG. D, the difference in NF-㮫 binding to the proximal (/) or distal (/) sites in the IL-10 promoter was measured by quantitative PCR. Measures were normalized to the zero time point for each site. E, the functionality of each NF-㮫 binding site within the murine IL-10 promoter was tested by transient transfection of RAW264.7 cells with pGL3B-IL-10-LUC that were either wild-type (IL-10), mutated at the distal site (㮫 ), or proximal site (㮫 ), and at both sites (㮫 /).
To verify the binding of NF-㮫 specifically to the IL-10 promoter, a ChIP assay was performed. SM cells were stimulated with pIC for 6 h and ChIP performed using anti-p65 and p50 antibodies. Analysis of the murine IL-10 promoter sequence (using TESS software) identified a putative NF-㮫 binding site between the nucleotides / (relative to the transcription start site). This site, with a second previously reported site at / within the IL-10 promoter (26), was interrogated. Fig. 3C confirms that NF-㮫 associates with the murine IL-10 promoter and identifies the distal, /, region as the pertinent regulatory motif mediating the response to pIC. Increased binding to the distal NF-㮫 site in the IL-10 promoter was confirmed by quantitative PCR ( Fig. 3D ). Although the relevance of either NF-㮫 site can only be accurately assessed in context of the cellular chromatin, we confirmed the functionality of the distal site in an exogenous promoter context using an IL-10 promoter (/+65) reporter assay in which the proximal and distal sites were mutated, separately and together, to prevent NF-㮫 binding ( Fig. 3E ). The data demonstrate that JNK and NF-㮫 are key regulators of the dsRNA-mediated induction of IL-10 in SM and RAW 264.7 cells.
dsRNA-mediated IL-10 Induction Is PKR-dependent𠅊s PKR is implicated in signaling pathways triggered by dsRNA, as well as LPS in macrophage cells, and is reported to activate both JNK and NF-㮫 (4, 35), a role in IL-10 induction was investigated. The response to pIC was compared between wt and pkr-ko SM cells (characterized in Fig. 4A and supplemental Fig. S2) by measuring IL-10 levels in the culture supernatant with an ELISA. Remarkably, pIC-mediated IL-10 induction was entirely lost in the pkr-ko cells ( Fig. 4B ). Treatment of wt SMs with a chemical inhibitor of PKR, 2-aminopurine, or RNAi targeting PKR and then treatment with LPS (10 ng/ml) verified that the difference observed between the wt and pkr-ko cell lines was entirely due to the genetic deletion of pkr (supplemental Fig. S3).
PKR dependence of the dsRNA-mediated IL-10 induction was also measured in primary BMM isolated from wt or pkr-null mice. Supporting the observation using SMs, the induction of IL-10 mRNA was found to be 4-fold higher in the wt BMM compared with the pkr-ko BMM after 4 h of pIC treatment ( Fig. 4C ). Interestingly, this defect was largely rectified in the BMM at later time points (data not shown).
To confirm the importance of PKR in the IL-10 response in a biologically relevant stress response, wt and pkr-ko SM cells were infected with Sendai virus, and the levels of IL-10 in cell supernatants were measured by ELISA. As shown for pIC, IL-10 was highly induced by Sendai virus only in the wt SM cells ( Fig. 4D ).
The preceding data predict that PKR mediates the induction of IL-10 via activation of JNK and NF-㮫. To confirm this, we examined the activation status of JNK and NF-㮫inwtand pkr-ko SM cells. Consistent with the previous observations, JNK activation was decreased in pkr-ko SM cells, as measured by the levels of the phosphorylated protein detected by Western analysis ( Fig. 4E ). Similarly, NF-㮫 activity was severely impaired in the pkr-ko compared with the wt cells treated with pIC, measured by EMSA ( Fig. 4F ). The data show that PKR is critical in the dsRNA signaling pathway leading to IL-10 production in SM cells, and is upstream to JNK and NF-㮫.
Activation of STAT3 in Response to dsRNA Is PKR-dependent𠅊 consequence of IL-10 induction is the subsequent activation of STAT3 (36). To measure the downstream effect of IL-10 induction following pIC treatment, and to corroborate a role for PKR in this pathway, we examined the activation of STAT3 in response to pIC in wt and pkr-ko SM cells. Remarkably, STAT3 activation, measured by phosphorylation of the tyrosine residue at position 705, was exclusively observed in the wt and not in the pkr-ko SM cells on pIC treatment ( Fig. 5A ). Appropriately, a neutralizing antibody to IL-10 ablated STAT3 activation ( Fig. 5B ). As predicted from our previous results, JNK and NF-㮫 inhibitors (SP600125 and BAY-11-7085) significantly decreased pIC-mediated STAT3 activation ( Fig. 5C ). Furthermore, STAT3 phosphorylation was unaffected by inhibitors of Erk1/2 and p38 MAP kinases.
PKR activates STAT3 via production of IL-10 independent of IFN. A, STAT3 activation in wt and pkr-ko SM cells treated with pIC was measured by Western blot analysis using a phospho-Y705-STAT3 antibody. Total STAT3 level was measured to verify equal loading. B, STAT3 activation was attenuated in wt SM incubated with an IL-10 neutralizing antibody (IL-10 Ab, 20 μg/ml) for 1 h prior to stimulation with pIC for 6 h. C, role for JNK and NF-㮫 in dsRNA-mediated, IL-10 induction and ensuing STAT3 activation was corroborated by pretreating wt SM cells with DMSO, or the inhibitors SP600125 (SP 10 μ m ), PD98059 (PD 20 μ m ), SB203580 (SB 4 μ m ), and BAY-11-7085 (BAY 5 μ m ) for 1 h prior to stimulation with pIC (50 μg/ml) for 6 h, followed by Western blot analysis using a phospho-Y705-STAT3 and total STAT3 antibodies. D, induction of the IFN-inducible protein p56 was measured by Western blot of lysates from wt and pkr-ko SM cells treated with pIC (50 μg/ml) for indicated times. β-Actin levels were measured to verify equal loading. E, the role of the IFN response in IL-10 signaling was assessed by incubating wt SM cells with control IgG or an IFNβ neutralizing antibody (12.5 μg) for 1 h before stimulation with pIC for 6 and 8 h. Cell lysates were probed by Western blot to measure STAT3 activation using the phospho-Y705 STAT3 and total STAT3 antibodies. Suppression of the IFN response was gauged by probing for levels of p56.
LPS-mediated induction of IL-10 has been reported to be dependent upon induction of IFNβ (37). PKR has been shown to regulate induction of IFNβ, via NF-㮫 and IRF1 in mouse embryonic fibroblasts (38). More recent reports, however, have emphasized the role of TLR3 and RIG-I in production of type I IFNs in response to viral infection (39, 40). We sought to estimate the contribution of PKR by monitoring induction of p56, a well-established IFN-inducible gene product, using wt and pkr-ko SM cells. Induction of p56 was markedly reduced in the absence of PKR ( Fig. 5D ), implying PKR is required for full induction of IFN. To gauge the requirement of IFNs in the dsRNA-mediated induction of IL-10, SM cells were treated with a neutralizing antibody to IFNβ or, as a control, IgG antibody, prior to pIC. This treatment did not markedly reduce the production of IL-10 (data not shown) and did not affect STAT3 phosphorylation ( Fig. 5E ). Appropriately, the IFN-dependent amplification of p56 was blocked. Thus PKR-mediated IL-10 induction and STAT3 activation are largely independent of IFNβ production in SM cells.
We would like to thank Molly Gale Hammell, Magdalena Götz, Sten Linnarsson, Chris Douse, Florence Cammas and Bryan Cullen for providing valuable reagents and input on the manuscript. We also thank, M. Persson Vejgården, U. Jarl, and A. Hammarberg for technical assistance. We are grateful to all members of the Jakobsson laboratory. The work was supported by grants from the Swedish Research Council (2018-02694, JJ & 2018-03017, PJ), the Swedish Brain Foundation (FO2019-0098, JJ), Cancerfonden (190326, JJ), Barncancerfonden (PR2017-0053, JJ), Formas (2018-01008, PJ) and the Swedish Government Initiative for Strategic Research Areas (MultiPark & StemTherapy).
MicroRNA-like fragment encoded by Ebola virus (EBOV)
EBOV is a negative-strand RNA virus which duplicates in the cytoplasm and leads to a severe hemorrhagic fever . It is reported that EBOV can encode miRNA-like fragment to destroy host immune defenses [56, 57]. Chen et al.  speculates three pre-miRNAs by the EBOV/Yambuku-Mayinga sequence and keeps one pre-miRNA after alignment with 125 EBOV genomes, then this pre-miRNA creates one mature miRNA sequence, miR-VP-3p. Further research discovers the miRNA-like fragment exists in serum of Ebola virus disease (EVD) patients by Northern blotting, qRT-PCR and TA-cloning/sequencing. Interesting, subsequent consequences discover that this miRNA-like fragment exists during the acute phase but not during recovery phase in the serum of EBOV-positive patients. With great clinical importance, this miRNA-like fragment is detectable before the detection of Ebola genomic RNA, which may improve the diagnosis of EVD.
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