Friday, November 11, 2016

A foundation for combating Flavivirus diseases?

       It is evident that human health worldwide is threatened by various established pathogens such as Zika, dengue, West Nile, yellow fever, and hepatitis C.  However, less apparent is the fact that the aforementioned pathogens are all members of the same family of viruses known as the Flaviviridae. The Flaviviridae are characteristic for their positive-sense, single-stranded RNA genome and their cytoplasmic replication (1).  These characteristics govern the life cycle of a flavivirus; how a virus produces new virions is largely dependent on the genome and its features and how it is utilized to produce new infectious particles.  And, as with many biological processes, regulatory mechanisms exist throughout a viral life cycle, governing tasks such as replication and particle assembly to create these new virions.
       Notably, modifications to RNA (post-transcriptionally or on the RNA genome itself), as observed with human mRNA transcripts, have been investigated and observed as a regulatory mechanism in viral life cycles.  For example, the N6-methyladenosine (m6A) RNA modification has recently been shown to serve as a positive regulator of HIV-1 by enhancing viral gene expression (2).  Cellular machinery exists that is capable of making this m6A modification via a methyltransferase complex, deleting the m6A modification via a demethylase, or reading the m6A modification via the proteins composing the cyotplasmic YTH-domain family (YTHDF).  Ultimately, m6A modifications can drive the translation of modified RNA (in transcript form, mRNA), or the degradation of modified RNA, all via interactions with the YTHD proteins.  Since viral progeny production depends on the production and presence of various viral proteins translated from the RNA, it is important to understand that RNA-based regulation through m6A modifications of RNA viral genomes therefore plays a fundamental role in their life cycles.  As a result, modifications such as m6A pose an interesting area for researchers to explore and understand the roots of infection from such viruses.
       The role that many members of the Flaviviridae family play as prevalent human pathogens generate much motivation to understanding aspects of flavivirus infection in order to propose modes of intervention to promote health and well-being around the world.  Thus, RNA modifications describe an essential area of research for gaining insight into flavivirus infections.  However, until recently, m6A has only been known to be present in the RNA transcripts of viruses carrying out replication in the nucleus, considering that the m6A cellular machinery is present there (3).  This has prevented research on m6A modification in cytoplasmic replicating viruses like the Flaviviridae.  
       Although, a team of researchers, primarily out of Duke University Medical Center, motivated by the fact that the flaviviruses present an RNA genome that serves directly as a transcript (positive-sense RNA), and by the fact that regulation of RNA transcripts can play an essential role in a viral life cycle, sought to understand if m6A modifications can also play a role in a cytoplasmic positive-sense RNA genome family of viruses: the Flaviviridae.  Through multiple experiments utilizing hepatitis C virus (HCV), which will be summarized subsequently, the team ultimately suggests a fundamental role of m6A in the regulation of the HCV life cycle and strongly predicts a role of m6A in other pathogenic flaviviruses... despite the fact that m6A activity was previously only defined in the nucleus.
       The first experiment carried out to determine broadly if m6A regulates the HCV life cycle, was done by depleting the m6A methyltransferases and by separately depleting the m6A demethylases. By observing the levels of an HCV protein utilized as a proxy for understanding viral replication, it was found that the methyltransferases negatively regulate the HCV life cycle (decrease progeny), while demethylases postiviely regulate the HCV life cycle (increase progeny).  In the broad sense, the researchers understood that moving forward with more specific experiments is reasonable considering that their findings ultimately suggest some role of m6A machinery in the HCV life cycle. Thus, subsequent experiments aimed to reveal the finer details of the role of the m6A machinery.  The next experiments reveal that the regulatory action of the machinery is not on HCV translation or genome replication, but rather on the production/release of newly formed infectious particles, ultimately defining the assembly stage of the HCV life cycle as the point of m6A regulation.  This was due to the observation that viral titers (concentration of virus present) were affected, while the level of viral replication was not (observed using an HCV reporter virus).
       Now that the stage of the HCV life cycle that is regulated has been defined, the researchers tested whether the YTHDF proteins, the known mediators of m6A regulation, had similar effects on the HCV life cycle.  The findings suggest that the YTHDF proteins do in fact bind to viral RNA, and negatively regulate infectious HCV production while having no effect on HCV replication.  This is consistent with the findings of m6A regulation, indicating that the m6A regulatory mechanism described in other biological processes is likely the same occurring in HCV.  Taking it one step further to suggest such, the researchers utilized microscropy to conclude that the localization of the YTHDF proteins is enriched around the liquid droplets in association with the endoplasmic reticulum membrane, which is the site of HCV particle assembly.  Similarly, the researchers used an antibody that specifically recognizes m6A in a pool of RNA harvested from HCV-infected cells to confirm that the m6A machinery is in fact present in the cytoplasm.  These findings all strongly support the idea that m6A regulatory functions are performed on the HCV RNA at the point of particle assembly, considering that they are all localized to the same region.
Figure 1. Liquid droplet depiction.
The YTHDF proteins were found to be localized at the site of HCV particle assembly.   
       The final step taken by the researchers of this study was to map the m6A sites in the HCV genome, and specifically those that overlap with the YTHDF binding regions to confirm the function of these regulatory sites.  Although 42 binding sites for YTHDF proteins were found in the genome, only two of high confidence overlapped with the m6A sites suggesting that the YTHDF proteins may interact with the HCV genome is other ways as well.  This therefore poses as a future direction for research interested in RNA modification in flaviviruses.  Although, mutations within these high-confidence regions resulted in virion production behaviour consistent with the previous findings; mutations in the m6A sites that YTHDF proteins also interact with led to an increase in viral titer but no alteration in viral replication, further suggesting the role of m6A modification in the HCV life cycle.
       In summary, all of this work has identified that HCV particle assembly is regulated by m6A machinery.  Although, the researchers have yet to identify the mechanism for such other than indicating that YTHDF proteins interact with the m6A on the RNA (ie. degradative processes, activation of a pathway, etcetera).  The researchers noted that the m6A sites that had YTHDF interactions were located in the E1 gene, and linked this to the prior understanding that the E1 gene in the HCV genome interacts with the core protein (associates with nucleic acid in new viral "particle").  The researchers utilized the same m6A site mutations from prior experiments to see if the binding of RNA to the core protein was altered by these mutations.  The observations suggest that the mutations increase the binding of RNA to the core protein, providing a foundation for understanding a potential mechanism that next research can investigate and fine-tune.  In other words, the researchers speculate some regulatory affect on the interaction between the E1 region of the HCV genome and the core protein as the mechanism for altering virion assembly.   
Figure 2.  Depiction of the major findings of m(6)A modification
 on HCV particle assembly. 
       However, one may be puzzled by some of the findings of this work, "Why would HCV want to negatively regulate its production of infectious particles?"  The answer to this may lie in viral fitness, and the fact that slower assembly/regulated assembly may be liked to persistent infection through mechanisms like evading the immune response of the host.  As a result, uncovering the mechanisms by which a virus may persist, or evade the immune response of the host is important when understanding pathogenesis.  For example, this work has given us insight towards the regulatory strategies of a pathogenic flavivirus.  And, since the researchers subsequently identified similar m6A sites in other pathogenic flaviviruses such as dengue, yellow fever, West Nile, and Zika, future research can identify clinical intervention points for disrupting such a process and hopefully aiding the host in managing or clearing the pathogenic microbe.  More specifically, this work may provide a new foundation for developing a vaccine for Hepatitis C, allowing the population to finally have vaccinations for now three prevalent hepatitis pathogens (A, B, C).  

Primary Article:
Gokhale, Nandan S. et al. (2016). N6-Methyladenosine in Flaviviridae viral RNA genomes regulates infection. Cell Host & Microbe, 20, 1-12.

Works Cited
1. http://web.stanford.edu/group/virus/flavi/2008/flavi.html
2. Kennedy et al. (2016) Posttranscriptional m(6)A editing of HIV-1 mRNAs enhances viral gene expression.  Cell Host & Microbe, 19, 675-685.
3.  Gokhale, Nandan S. et al. (2016). N6-Methyladenosine in Flaviviridae viral RNA genomes regulates infection. Cell Host & Microbe, 20, 1-12.

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