Gliogenic LTP is a new form of paracrine synaptic plasticity in the central nervous system and
may lead to pain amplification close to and remote from an injury or an inflammation. This is
in line with the concept of chronic pain as a glio-pathy involving neurogenic neuroinflammation
(7, 24). These new insights may pave the way for
novel pain therapies (25, 26). P2X7Rs play a key
role in chronic inflammatory and neuropathic
pain (27) and in other neurodegenerative and
neuropsychiatric disorders (28). Glial cells display considerable diversity between and within
distinct regions of the central nervous system
(29). If the presently identified gliogenic LTP also
existed at some brain areas, it could be of relevance
not only for pain but also for other disorders, such
as cognitive deficits, fear and stress disorders, and
chronic immune-mediated diseases (24, 29, 30).
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This work was supported by grants P 29206-B27 and W1205 from the
Austrian Science Fund (FWF) to J.S. We thank L. Czarnecki for
laboratory support and B. Heinke and G. Janeselli for technical support.
All of the data are archived on servers of the Center for Brain Research,
Medical University of Vienna. The authors declare no conflicts of
interest. M. T.K., R.D.-S., and J.S. designed the research. M. T.K., R.D.-S.,
M.G., S. D. H., and H.L. T. generated and analyzed the data. M. T.K.,
R.D.-S., and J.S. wrote the paper, with input from the other authors.
Materials and Methods
Figs. S1 to S6
15 July 2016; accepted 1 November 2016
Published online 10 November 2016
Zika virus produces noncoding RNAs
using a multi-pseudoknot structure
that confounds a cellular exonuclease
Benjamin M. Akiyama,1 Hannah M. Laurence,1,2,3 Aaron R. Massey,4*
David A. Costantino,1 Xuping Xie,5 Yujiao Yang,5 Pei-Yong Shi,5 Jay C. Nix,6
J. David Beckham,4 Jeffrey S. Kieft1,7†
The outbreak of Zika virus (ZIKV) and associated fetal microcephaly mandates efforts
to understand the molecular processes of infection. Related flaviviruses produce
noncoding subgenomic flaviviral RNAs (sfRNAs) that are linked to pathogenicity in
fetal mice. These viruses make sfRNAs by co-opting a cellular exonuclease via
structured RNAs called xrRNAs. We found that ZIKV-infected monkey and human
epithelial cells, mouse neurons, and mosquito cells produce sfRNAs. The RNA structure
that is responsible for ZIKV sfRNA production forms a complex fold that is likely found
in many pathogenic flaviviruses. Mutations that disrupt the structure affect
exonuclease resistance in vitro and sfRNA formation during infection. The complete
ZIKV xrRNA structure clarifies the mechanism of exonuclease resistance and identifies
features that may modulate function in diverse flaviviruses.
Globalization, urbanization, and climate change contribute to the spread of patho- genic mosquito-borne viruses, typified by the outbreak of Zika virus (ZIKV) (1). ZIKV infection can cause fetal microcephaly and
Guillain-Barré syndrome (2), motivating efforts
to understand the molecular drivers of pathology. ZIKV is a (+)-sense single-stranded RNA
mosquito-borne flavivirus (MbFV) related to
dengue virus (DENV), yellow fever virus (YFV),
and West Nile virus (WNV) (3). The structured 3′
untranslated regions (UTRs) of many MbFVs
are the source of noncoding subgenomic flaviviral RNAs (sfRNAs) that accumulate during
infection when RNA elements resist degradation
by the host 5′ → 3′ exonuclease Xrn1 (fig. S1A) (4).
These sfRNAs are directly linked to cytopathic
and pathologic effects (4); they dysregulate
RNA decay pathways and bind cellular proteins
important for antiviral responses (5–14). Preventing
sfRNA production could be a strategy for targeted therapeutics or for generating attenuated
virus for vaccines (15–17).
Because sfRNA formation during ZIKV infec-
tion has not been reported, we infected multiple
cell lines with ZIKV strain PRVABC59, isolated in
2015 from an infected U.S. mainland–Puerto Rico
traveler. Northern blot analysis of total RNA iso-
lated from infected cells showed discrete bands
containing parts of the ZIKV 3′UTR, consistent
with sfRNAs (Fig. 1A). Mouse primary neuron in-
fection resulted in very little infectious virus and
produced three weak sfRNA bands. Infection
of C6/36 (Aedes albopictus mosquito) cells pro-
duced two predominant sfRNAs, whereas Vero
(monkey) and A549 (human) epithelial cell in-
fection produced additional bands. Different cell
types produced different sfRNA patterns, but the
largest sfRNA was present in all. The importance
of this cell type–dependent variation in the sfRNA
patterns is unknown, although studies with DENV
suggest that sfRNA production is modulated to
enable host adaptation (16, 18).
The production of ZIKV sfRNAs suggests the
existence of Xrn1-resistant structures (xrRNAs)
in the viral 3′UTR. Two areas of the UTR match
the sequence pattern and potential secondary
structure of known MbFV xrRNAs (Fig. 1, B and
C, and fig. S1B) (4, 18). The putative xrRNAs are
in series near the 5′ end of the UTR—a location
and pattern similar to that of other MbFVs (Fig.
1D). Xrn1 halting at putative ZIKV xrRNA1 and
xrRNA2 would result in sfRNAs of sizes matching
the two produced in all cell types tested (Fig. 1A
and fig. S1C). To test whether these putative elements are indeed Xrn1-resistant, we challenged
in vitro transcribed full-length ZIKV 3′UTR RNA
with recombinant Xrn1 (19). Although multiple
sfRNAs were observed during ZIKV infection,
in vitro the upstream xrRNA1 quantitatively halted
the enzyme (Fig. 1E). However, a U TR lacking the
upstream xrRNA (DxrRNA1) allowed the enzyme
to stop at the downstream xrRNA2. The size of
the Xrn1-resistant RNAs matched those of the
infection-produced sfRNAs (fig. S1D). Thus, ZIKV
1Department of Biochemistry and Molecular Genetics,
University of Colorado Denver School of Medicine, Aurora,
CO 80045, USA. 2Howard Hughes Medical Institute (HHMI),
University of Colorado Denver School of Medicine, Aurora,
CO 80045, USA. 3School of Veterinary Medicine, University
of California, Davis, CA 95616, USA. 4Department of
Medicine, Division of Infectious Diseases, University of
Colorado Denver School of Medicine, Aurora, CO 80045,
USA. 5Department of Biochemistry and Molecular Biology,
University of Texas Medical Branch, Galveston, TX 77555,
USA. 6Molecular Biology Consortium, Advanced Light Source,
Lawrence Berkeley National Laboratory, Berkeley, CA 94720,
USA. 7RNA BioScience Initiative, University of Colorado
Denver School of Medicine, Aurora, CO 80045, USA.
*These authors contributed equally to this work. †Corresponding
author. Email: email@example.com