Viral Virtuosos

New understanding of noncoding RNAs may solve a long-standing puzzle about how viruses orchestrate lifelong infections.  

By | February 1, 2015

© MEHAU KULYK/SCIENCE PHOTO LIBRARY/CORBIS

When it comes to viruses, those that transiently infect their hosts and cause the most damage get a lot of attention. Hollywood makes movies about Ebola, or about fictional viruses that resemble souped-up versions of the 1918 pandemic flu. Another viral world—one much closer to home—rarely enters the collective consciousness: the human virome.

A diverse, abundant, and underappreciated viral community exists on and within us, from our skin to our eyes, blood, brain, and other organs—even within our own genomes.1 Unlike marauding Ebola-like viruses, these viruses establish a balanced coexistence that can persist for a host’s entire lifetime. This coexistence involves careful control of the viral life cycle: whereas Ebola virus infection is flashy, persistent infection is elegant. (See illustration.)

Among other challenges, persistent viruses must effectively subvert the host immune response. To accomplish this, these viruses control both the timing and amount of viral replication. Such nuanced infectious cycles involve carefully choreographed viral gene expression that can foster completely different lifestyles depending on host cell type, cell-signaling events, or other factors. One important class of regulators that helps to mediate these lifestyle switches is the noncoding regulatory RNAs (ncRNAs), which today stand at the center of an ongoing mini-revolution in our understanding of gene-expression control.

Previously thought of as a simple decoder of genetic information—serving as an intermediate between DNA and protein—RNA is now known to engage in a bevy of other important biochemical activities. Despite differences in ncRNA sequence, size, and function, a theme is emerging: diverse biological processes rely on ncRNAs to balance the timing and magnitude of gene expression.2 In eukaryotes, microRNAs (miRNAs) represent the best-characterized ncRNAs. About 22 nucleotides in length, these small RNAs bind to and repress target messenger RNA (mRNA) transcripts, allowing for fine-tuning of gene expression. The human genome encodes hundreds of different miRNAs to regulate numerous biological functions, and recent research is revealing how the viruses of the human body use these miRNAs, as well as their own, to establish and maintain long-term persistent infections.

MicroRNAs can be thought of as tools that viruses use to manipulate key aspects of our biology and immune response in order to hitchhike with us throughout our evolutionary history.

Persistent viruses may play roles both in defending their hosts—they have been speculated to “tune” our immune response, priming it to better combat more harmful microbes—and in directly causing serious disease in humans. This double-edged sword of viral persistence makes the phenomenon all the more important to understand. Elucidating how miRNAs and other ncRNAs function could spawn new therapies, while yielding insights about the evolutionary forces that dictate parasitism, mutualism, and other multiorganismal relationships.

Stealth tools

MiRNAs function as components of a widely conserved set of eukaryotic gene-silencing machinery involved in the process of RNA interference (RNAi). RNAi occurs when RNA of a particular structure is processed into a smaller RNA molecule that directs sequence-specific silencing of gene expression. (See “Molecular Snipers,” The Scientist, September 2014.) While the interaction of any single miRNA with a target frequently results in only subtle modulation of gene expression, a single miRNA can regulate hundreds of different target mRNAs. Conversely, multiple miRNAs can regulate a single transcript. The sum of numerous miRNA-target interactions can have profound effects.

SHORT- VERSUS LONG-TERM INFECTION: While many viruses simply infect, replicate, lyse cells, and spread, others take up long-term residence in their host, sometimes living within the organism’s cells for a lifetime. This type of infection, known as persistent infection, requires a delicate balance of viral processes to avoid alerting the host immune system to the viruses’ presence.
See full infographic: JPG | PDF
© LUCY READING-IKKANDA
In most eukaryotes, RNAi defends the host against mobile genetic elements such as endogenous transposons, and in some organisms, such as plants and insects, against viruses. In these organisms, foreign viral RNA is recognized and destroyed by a series of host machineries. Whether or not RNAi also serves as a meaningful antiviral response in mammals remains a controversial and unresolved issue.3,4 What is clear, however, is that multicellular eukaryotes, including mammals, generate endogenous miRNAs that enter the RNAi machinery to silence specific mRNAs, thus providing an additional layer of control for gene expression.

Over the last decade, my lab and several others have uncovered virus-encoded miRNAs from diverse viruses.5,6 This provides an interesting twist—host machinery that serves as an antiviral response in many organisms has been usurped by viruses for their own benefit. By binding to and silencing viral transcripts, for example, or by targeting host genes important for viral gene expression, viral miRNAs can optimize the location and timing of virus replication to fly under the radar of the host immune response. Some viruses also utilize miRNAs to directly regulate host defenses. Herpesviruses use viral miRNAs to block cell suicide,7 for example, while retroviruses and anelloviruses, a family of small single-stranded DNA viruses, rely on viral miRNAs to block the signaling activity of secreted antiviral cytokines.8,9 Meanwhile, members of the herpesvirus and adenovirus families use viral miRNAs and other types of ncRNAs, respectively, to block components of the innate immune response.5,10

One advantage of miRNAs as mediators of gene expression is their tiny genomic footprint. Since virus genome size is constrained by the requirement to fit within the protein shell, or capsid, miRNAs afford the virus the ability to regulate numerous genes with minimal genomic burden. Additionally, the immune response of jawed vertebrates is especially adept at detecting foreign proteinaceous cues of pathogen infection but not foreign miRNAs, so viral miRNA is largely invisible to the adaptive immune system. This allows the virus to regulate viral and host gene expression without alerting the host immune response to its presence. Thus, miRNAs and other ncRNAs can be thought of as tools that viruses use to manipulate key aspects of our biology and immune response in order to hitchhike with us throughout our evolutionary history.

The enemy within

The list of viruses that encode miRNAs and other ncRNAs includes members of the herpesvirus, polyomavirus, anellovirus, adenovirus, and retrovirus families. (See "Striking a Balance.") These viruses possess diverse physical structures and genome chemistries. Despite those differences, the viruses invariably share the ability to take up long-term residence in a host.

The coexistence of virus and host involves careful control of the viral life cycle: whereas Ebola virus infection is flashy, persistent infection is elegant.

Although the majority of such persistent viral infections are subclinical, serious disease can arise. For example, in patients who are immunosuppressed due to HIV-AIDS or on a regimen of immunosuppressive drug therapies, persistent viruses can result in life-threatening disease, including cancer, neurological defects, organ-transplant rejection, birth defects, and encephalitis. Additionally, more benign conditions such as cold sores, genital lesions, respiratory illness, and eye infections are associated with persistent viral infections in immunocompetent hosts.

In addition to the immune status of the host, regulation of viral gene expression can influence whether persistent viruses cause illness. These disease states can be viewed as conditions resulting from a disturbance to viral homeostasis, the maintenance of balanced gene expression for long-term virus production and fitness. Elucidating the role of ncRNAs, both of viral and host origin, has created considerable excitement, in part for their likely importance in understanding persistent infection and associated pathologies. For example, recent work in the herpesvirus family implicates both host and viral miRNAs in controlling the cues and signaling pathways involved in induction of lytic infection (high virus productive state resulting in cell death), and therefore, also in the diseases associated with lytic infection, such as cold sores or Kaposi’s sarcoma.

Avian influenza virus, type A strain H5N1© NIBSC/SCIENCE PHOTO LIBRARY/CORBISEbola virusCDC/FREDERICK A. MURPHYIn their latent, or “cryptic,” state, some herpesviruses express only viral ncRNAs, while they can express hundreds of proteins when they switch to a productive state. During latency, miRNAs filter transcriptional noise, likely dampening the frequency of virus production and preventing expression of immunostimulatory antigens. When the host cell is no longer a safe haven—as a result of coinfection with other pathogens or physical stressors, for example—viruses will often initiate reproduction. Robust production of pro-lytic transcription factors by both the virus and its host overrides the inhibitory miRNA activity, resulting in a cascade of gene expression culminating in new virus production and eventual cell death. In addition, the fact that host and viral miRNA activity can be globally decreased by stress and other signaling events such as pathogen coinfection may allow viruses to utilize ncRNAs as a “barometer” of the optimal conditions for the switch from persistent to productive infection.11 Although best documented in the herpesviruses, such phenomena likely occur in other types of persistent viruses, especially the polyomaviruses, whose miRNAs have been shown to directly inhibit viral lytic gene expression.

Some viruses do not encode their own ncRNAs, but rather take advantage of those produced by their host. Papillomaviruses, causative agents of several different human cancers, take up long-term residence and have atypical life cycles that require differentiation of host cells for viral reproduction. Specifically, papillomaviruses express less viral protein in the inner skin cells of the dermal layer, where adaptive immune surveillance is greater, than they express in the more differentiated outer cells of the epidermis. Such a life cycle presumably requires tight regulation of viral gene expression, yet to date, no papillomavirus miRNAs have been identified. At least one type of papillomavirus, the cervical cancer–associated HPV31, solves this problem by taking advantage of a host miRNA whose expression levels inversely correlate with skin differentiation status.12 By maintaining a docking site for this host miRNA, HPV31 derives all the advantages of a viral-encoded miRNA without the burden of encoding an miRNA gene in its own genome.

Viral microRNAs can optimize the location and timing of virus replication to fly under the radar of the host immune response.

Although there are only a few reported examples of this sort, undoubtedly other papillomaviruses and other types of persistent viruses use a similar strategy. Even some herpesviruses that encode their own miRNAs have evolved to also take advantage of host miRNAs to optimize viral gene expression.13,14 These findings illustrate the ability of viruses to repurpose host machinery and underscore the utility of miRNAs for balancing gene expression during persistent infections.

Making use of ncRNAs

In addition to increasing our understanding of virus biology and associated pathogenesis, studying ncRNAs offers other insights. Because many viruses use ncRNAs to evade host defenses, these small molecules represent a valuable set of probes for uncovering key components of the antiviral response. Additionally, by engineering unnatural miRNA docking sites into viruses, researchers are able to generate viruses and viral vectors that better target specific tissues or species. This has the potential to improve the safety of gene therapies, vaccines, and cancer-fighting oncolytic viruses, and has even been proposed as a way to reduce the chances of harmful viruses escaping from laboratories.15 Because some ncRNAs have unique properties that make them useful biomarkers, viral ncRNAs may also be valuable for early detection of virus-associated disease.

As our appreciation of bacterial microbiota grows, a logical next step is to determine how the human virome contributes to health and disease. In the future, holistic examination of the full spectrum of viral diversity, not just of the viruses that make us ill, may be factored into medical decisions. For example, an increase in the levels of a benign persistent virus was recently suggested as a useful indicator for monitoring therapeutic immune suppression.16 Additionally, beneficially altering host viral communities, so-called provirotics, could conceivably contribute to improved human health. Advancing this exciting notion will require enormous effort to catalog and exploit the human virome and to decipher the contributions of the humble but elegant persistent viruses—and the ncRNAs that regulate them. 

Christopher S. Sullivan is an associate professor in the Department of Molecular Biosciences at the University of Texas at Austin.

STRIKING A BALANCE
An important part of maintaining a persistent infection is the regulation of viral gene expression. A number of common mammalian viruses have been found to use noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), to keep gene expression at an appropriate level so as not to be recognized and cleared by the host immune system, while still maintaining a viral genome able to produce new infectious virus.

Herpesviruses: Causative agents of cold sores, genital lesions, and cancers, these large viruses have the best-defined alternative gene-expression programs associated with cryptic latent infection or productive lytic infection. These viruses use miRNAs to regulate viral gene expression and to block cell suicide and components of the innate immune response. In addition, some herpesviruses encode other types of ncRNAs, which are generally less well understood than the miRNAs, but appear to play an important role in regulating gene expression.
 
Polyomaviruses: Like the herpesviruses, these viruses can establish long-term infections, and phylogenetically distant polyomaviruses have been identified that encode miRNAs, all with the ability to regulate viral gene expression. Polyomaviruses are associated with serious diseases such as cancer and rare forms of dementia, partly as a result of misregulated viral gene expression.

Anelloviruses: One of the most abundant viruses infecting humans, with some 104 genome copies per milliliter of blood, anelloviruses are masters of living peacefully in the body, causing no known diseases. Many of these viruses encode their own miRNA, which can help them evade a host cytokine-mediated antiviral defense.



Adenoviruses: Common respiratory pathogens, adenoviruses can, in some contexts, also establish persistent infection. Adenoviruses encode ncRNAs called virus-associated (VA) RNAs that directly bind to and block host antiviral defense proteins. VA RNAs can also be processed further into the smaller miRNAs, although the functional relevance of these viral miRNAs remains unknown.

Retroviruses: Retroviruses are atypical in that they integrate into the host genome as part of their infectious cycle, enabling them to persist for long periods of time. Although no human retroviruses are widely accepted to encode their own miRNAs, it is likely that human retroviruses use host miRNAs to regulate viral gene expression. In addition, some nonhuman primate and bovine retroviruses encode abundant miRNAs that regulate host genes that control cell growth and immune functions.

Papillomaviruses: Papillomaviruses are persistent viruses associated with warts and cancer. To date, no papillomaviral miRNAs have been described. However, the cancer-associated human papillomavirus 31 uses a host miRNA to regulate viral gene expression.

© LUCY READING-IKKANDA

References

  1. H. W. Virgin, “The virome in mammalian physiology and disease,” Cell, 157: 142-50, 2014.
  2. M. S. Ebert, P. A. Sharp, “Roles for microRNAs in conferring robustness to biological processes,” Cell, 149:515-24, 2012.
  3. B. R. Cullen et al., “Is RNA interference a physiologically relevant innate antiviral immune response in mammals?” Cell Host Microbe, 14:374-78, 2013.
  4. J. M. Pare, C. S. Sullivan, “Distinct antiviral responses in pluripotent versus differentiated cells,” PLOS Pathog, 10:e1003865, 2014.
  5. A. Grundhoff, C. S. Sullivan, “Virus-encoded microRNAs,” Virology, 411:325-43, 2011.
  6. S. Pfeffer et al., “Identification of virus-encoded microRNAs,” Science, 304:734-36, 2004.
  7. E. Y. Choy et al., “An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival,” J Exp Med, 205:2551-60, 2008.
  8. R. P. Kincaid et al., “A human torque teno virus encodes a microRNA that inhibits interferon signaling,” PLOS Pathog, 9:e1003818, 2013.
  9. R. P. Kincaid et al., “Noncanonical microRNA (miRNA) biogenesis gives rise to retroviral mimics of lymphoproliferative and immunosuppressive host miRNAs,” MBio, 5:e00074-14, 2014.
  10. R. P. Kincaid, C. S. Sullivan, “Virus-encoded microRNAs: An overview and a look to the future,” PLOS Pathog, 8:e1003018, 2012.
  11. J. E. Cox, C. S. Sullivan, “Balance and stealth: The role of noncoding RNAs in the regulation of virus gene expression,” Annu Rev Virol, 1:89-109, 2014.
  12. V. Gunasekharan, L. A. Laimins, “Human papillomaviruses modulate microRNA 145 expression to directly control genome amplification,” J Virol, 87:6037-43, 2013.
  13. C. M. O’Connor et al., “Host microRNA regulation of human cytomegalovirus immediate early protein translation promotes viral latency,” J Virol, 88:5524-32, 2014.
  14. D. Pan et al., “A neuron-specific host microRNA targets herpes simplex virus-1 ICP0 expression and promotes latency,” Cell Host Microbe, 15:446-56, 2014.
  15. R. A. Langlois et al., “MicroRNA-based strategy to mitigate the risk of gain-of-function influenza studies,” Nat Biotechnol, 31:844-47, 2013.
  16. I. De Vlaminck et al., “Temporal response of the human virome to immunosuppression and antiviral therapy,” Cell, 155:1178-87, 2013.

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Avatar of: jln

jln

Posts: 2

February 2, 2015

Has anyone looked at a connection between these types of miRNAs and Alzheimer's? Years ago we published an abstract suggesting a correlation to inflammation due to herpes- related viruses. Could this be the missing piece of the puzzle?

Avatar of: James V. Kohl

James V. Kohl

Posts: 435

February 2, 2015

Thank you Dr. Sullivan for this extremely timely update. I had not considered that viral microRNAs might facilitate genomic stablility to benefit the virus. 

For comparison, last month, SICB (2015) presenters inadvertently may have linked  viral microRNAs to biodiversity across species from crustaceans to insects. If so, they did it via established links from enymes and hormones to metabolic networks and genetic networks. Unfortunately, they did not clarify precisely how ecological variation leads to ecological adaptations in the context of what is known about biologically-based cause and effect. See for example: All in the (bigger) family.

Last week, Gunter Wagner's group added experimental evidence and thoughts on the role of transposons, which Victor Lynch reportedly referred to elsewhere as"genomic parasites." That representation may be confusing to SICB-ers and others who still think in terms of mutations and evolution.

It implies that the parasites might still be linked to mutations and rapid evolution of the mammalian placenta. See for comparision this article from The Scientist: How Transposons Shaped Pregnancy. The problem for SICB-ers may be the Wagner group's focus on rapid emergence of an organ sans mutations. For comparison, the placenta in some species of fish appear to be an ecological adapation that facilitates their rapid divergence via sexual selection. See: Biologists link sexual selection and placenta formation (sans mutations?)

Works from Christopher Sullivan's group now include Pan-viral-microRNA screening identifies interferon inhibition as a common function of diverse viruses. This suggests the need to tell more researchers about links from nutritional epigenetics to metabolic networks and genetic networks via changes in the microRNA/messenger RNA balance that may lead to abnormal cell type proliferation or cell type differentiation that is controlled across species via nutrient-dependent sexual selection.

 

Nutrient energy appears to cause base pair changes, which also may be caused by other environmental effects. However, the conserved molecular mechanisms of nutrient-dependent organizing base pair changes are attributed to the epigenetic effects of food odors and the pheromone-controlled physiology of reproduction. This excludes explanations in the context of genomic parasites and mutations. See for review Human pheromones and food odors: epigenetic influences on the socioaffective nature of evolved behaviors and Nutrient-dependent/pheromone-controlled adaptive evolution: a model.

 

Indeed, nutrient-dependent RNA-directed DNA methylation of the carbon-5 position of cytosine, which results in differences in 5hmCs (see Unmasking Secret Identities), may be the most commonly studied type of nutrient-dependent pheromone-controlled structural and functional eukaryotic modification that results from organizing base pair changes and amino acid substitutions.

The amino acid substitutions appear to differentiate all cell types in all individuals of all species via additional genomic stability in the context of viral microRNAs. Without the amino acid substitutions organism-level thermoregulation might be perturbed by viral microRNAs -- if they destablize the chemistry of protein folding during thermodynamic cycles of protein biosynthesis and degradation.

It seems likely that nutrient uptake supports the immunne system and prevents damage by viral microRNAs. If so, the nutrient-dependent pheromone-controlled physiology of reproduction probably links the benefits of nutrient uptake to the immune system and to biodiverity in species from microbes to man via the need to avoid damage from nutrient stress and social stress. Stress reduction probably helps to maintain the most functional metabolic networks and genetic networks, which are linked by RNA-mediated events to enzymes, hormones, and species diversity in the presentations at SICB (2015).

Avatar of: James V. Kohl

James V. Kohl

Posts: 435

Replied to a comment from jln made on February 2, 2015

February 2, 2015

Micro spies from the brain to the periphery: new clues from studies on microRNAs in neuropsychiatric disorders

MicroRNA-132 Is Enriched in Developing Axons, Locally Regulates Rasa1 mRNA, and Promotes Axon Extension

Please provide a link to your published work. The missing piece of the puzzle appears to be de novo creation of olfactory receptor genes in neurodegenerative disorders and other physiopathology.

My model links food odors and pheromones via olfaction to GnRH and luteinizing hormone. The context of this patent may be of particular interest to others.

Pheromones and the luteinizing hormone for inducing proliferation of neural stem cells and neurogenesis

Species-specificity is addressed in other works, like this one:

MicroRNA Expression and Regulation in Human, Chimpanzee, and Macaque Brains

Sex differences in cell types are addresses in the molecular epigenetics section of our 1996 Hormones and Behavior review. From Fertilization to Adult Sexual Behavior

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