SARS-CoV-2
carries the largest single-stranded RNA genome and is the causal
pathogen of the ongoing COVID-19 pandemic. How the SARS-CoV-2 RNA genome
is folded in the virion remains unknown. To fill the knowledge gap and
facilitate structure-based drug development, we develop a virion RNA in
situ conformation sequencing technology, named vRIC-seq, for probing
viral RNA genome structure unbiasedly. Using vRIC-seq data, we
reconstruct the tertiary structure of the SARS-CoV-2 genome and reveal a
surprisingly “unentangled globule” conformation. We uncover many
long-range duplexes and higher-order junctions, both of which are under
purifying selections and contribute to the sequential package of the
SARS-CoV-2 genome. Unexpectedly, the D614G and the other two
accompanying mutations may remodel duplexes into more stable forms.
Lastly, the structure-guided design of potent small interfering RNAs can
obliterate the SARS-CoV-2 in Vero cells. Overall, our work provides a
framework for studying the genome structure, function, and dynamics of
emerging deadly RNA viruses.
Duplexit , quadruplexit ja stem loop- muodostumat ihmisen genomsisa:
https://academic.oup.com/nar/article/48/18/10567/5909921
Duplex formation in a G-quadruplex bulge
Published:
22 September 2020
Article history
Previously, it has been shown that increasing the bulge size led to decreasing stability of the G-quadruplex (44). In this study, as observed in the case of B4-dx2, B4-dx3 and B4-dx4,
increasing the size of a duplex bulge did not lead to a decrease in the
stability of the G-quadruplex. On the contrary, the melting temperature
slightly increased from 46.2°C to 50.5°C, as the duplex bulge size was
increased from 15 to 33 nt (Supplementary Figure S6). A similar trend was also observed when a duplex of increasing size was incorporated within a G-quadruplex loop (49). The melting temperature of a G-quadruplex was observed to increase by ∼13°C (Supplementary Figure S6) when a non-structured bulge (B4–15T; Table 1) is replaced by a duplex bulge with the same length (B4-dx2),
indicating that a G-quadruplex with a duplex bulge exhibited
significantly higher thermal stability than a G-quadruplex containing a
non-structured bulge of the same length.
CONCLUSION
In this study, we showed that a duplex stem can
be incorporated into a bulge of a G-quadruplex. The NMR solution
structure of a G-quadruplex containing a duplex bulge was presented,
showing a unique quadruplex–duplex junction with the duplex bulge
stacking below the 3′-end G-tetrad. Breaking up of the immediate base
pair step at the quadruplex–duplex junction, coupled with a narrowing of
the duplex groove within the context of the bulge, led to a progressive
transition between the quadruplex and duplex segments. Duplex bulges
can occur at various positions of a G-quadruplex scaffold and increasing
the length of a duplex bulge does not lead to a decrease in the
stability of the overall G-quadruplex core. The formation of a long
duplex bulge within G-quadruplexes would expand the current knowledge on
predicting G-quadruplex-forming sequences. Potential existence of such
structures in the human genome may serve as unique targets for designing
ligands for specific G-quadruplex targeting.
VIRUSPARTIKKELI ILMAN genomia:
VLP, viruksen kaltainen partikkeli joka on ilman genomisisältöään, tuhoutuu nopeammin,
PMCID: PMC7699159
PMID:
33272571 Structural stability of SARS-CoV-2 virus like particles degrades with temperature
,a ,a ,a ,b ,b ,b ,a,c,d,∗ and a,c,d,∗∗
This article has been
cited by other articles in PMC.
Abstract SARS-CoV-2
is a novel coronavirus which has caused the COVID-19 pandemic. Other
known coronaviruses show a strong pattern of seasonality, with the
infection cases in humans being more prominent in winter. Although
several plausible origins of such seasonal variability have been
proposed, its mechanism is unclear. SARS-CoV-2 is transmitted via
airborne droplets ejected from the upper respiratory tract of the
infected individuals. It has been reported that SARS-CoV-2 can remain
infectious for hours on surfaces. As such, the stability of viral
particles both in liquid droplets as well as dried on surfaces is
essential for infectivity. Here we have used atomic force microscopy to
examine the structural stability of individual SARS-CoV-2 virus like
particles at different temperatures. We demonstrate that even a mild
temperature increase, commensurate with what is common for summer
warming, leads to dramatic disruption of viral structural stability,
especially when the heat is applied in the dry state. This is consistent
with other existing non-mechanistic studies of viral infectivity,
provides a single particle perspective on viral seasonality, and
strengthens the case for a resurgence of COVID-19 in winter.
Keywords: SARS-CoV-2, Virus-like particle, Stability, Temperature
Go to: 1. Introduction SARS-CoV-2 is a virus of zoonotic origin which was first identified in humans in late 2019 [
1]. Similar to other coronaviridae [
2], the viral particles are enveloped and polymorphic decorated by a variable number of S protein spikes on their membrane [
3].
One of the most confusing and yet urgently pressing questions at the
time of this writing is whether the COVID-19 pandemic caused by
SARS-CoV-2 will show seasonal character. Climate and seasonal dependence
was expected early in the pandemic [
4] due to similarity with other human coronavirus diseases [
5],
however the rates of infections have failed to strongly decline in the
summer of 2020, leading to widespread doubts about COVID-19 seasonality.
At the same time, a mounting body of evidence, from theoretical studies
[
6] to experimental research on viral populations and their infectivity [
7,
8]
suggest that seasonality is indeed to be expected. However an
understanding of how SARS-CoV-2 survives different environmental
conditions is still incomplete and mechanisms of virus particle
degradation are poorly mapped out. This then creates uncertainty for
public health policy and its forward projection.A key
challenge in studying SARS-CoV-2 is the extreme level of threat
associated with the live virus and the resultant need for high safety
standards for such work. Aside from the envelope and S proteins,
SARS-CoV-2 also packages the positive sense RNA genome encapsidated with
thousands of copies of nucleocapsid, N proteins. SARS-CoV-2 also
packages thousands of copies of matrix protein (M) which consists of
three membrane spanning helixes with small intraluminal and extra
luminal domains. In addition, an unknown number of envelope (E)
proteins, which contain a single membrane spanning helix, are also
packaged in each virion. We have previously shown that similar to SARS-CoV [
9],
the
expression of SARS-CoV-2 M, E, and S proteins in transfected human
cells is sufficient for the
formation and release of virus like
particles (VLPs) through the same biological pathway as used by the
fully infectious virus [10].
These VLPs faithfully mimic the external structure of the SARS-CoV-2
virus. The VLPs however,
possess no genome and thus present no
infectious threat which enables rapid studies with reduced safety
requirements. The ability to produce non-infectious VLPs further enabled
us to devise and rapidly validate novel strategies for manipulation of
these particles, most notably via the addition of protein tags to the S
and M proteins (these findings are detailed in a separate manuscript). Here,
we report studies of VLPs subjected to variable
temperature conditions
before or after being immobilized and dried out on a functionalized
glass surface. We show that exposure of VLPs to a mildly elevated
temperature (34C) for as little as 30 min is sufficient to induce
structural degradation. The effect is weaker for particles exposed to
elevated temperatures in solution and stronger for exposure in the dry
state. Overall, these results provide insight into the viral seasonality
of SARS-CoV-2.
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