structural stability of sars cov 2 virus like particles degrades with temperature CORD-Papers-2022-06-02 (Version 1)

Title: Structural stability of SARS-CoV-2 virus like particles degrades with temperature
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.
Published: 2020-11-28
Journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2020.11.080
DOI_URL: http://doi.org/10.1016/j.bbrc.2020.11.080
Author Name: Sharma A
Author link: https://covid19-data.nist.gov/pid/rest/local/author/sharma_a
Author Name: Preece B
Author link: https://covid19-data.nist.gov/pid/rest/local/author/preece_b
Author Name: Swann H
Author link: https://covid19-data.nist.gov/pid/rest/local/author/swann_h
Author Name: Fan X
Author link: https://covid19-data.nist.gov/pid/rest/local/author/fan_x
Author Name: McKenney R J
Author link: https://covid19-data.nist.gov/pid/rest/local/author/mckenney_r_j
Author Name: Ori McKenney K M
Author link: https://covid19-data.nist.gov/pid/rest/local/author/ori_mckenney_k_m
Author Name: Saffarian S
Author link: https://covid19-data.nist.gov/pid/rest/local/author/saffarian_s
Author Name: Vershinin M D
Author link: https://covid19-data.nist.gov/pid/rest/local/author/vershinin_m_d
sha: 3fc1061863d1567d88a51b485566a00a6d38cbfb
license: no-cc
license_url: [no creative commons license associated]
source_x: Elsevier; Medline; PMC
source_x_url: https://www.elsevier.com/https://www.medline.com/https://www.ncbi.nlm.nih.gov/pubmed/
pubmed_id: 33272571
pubmed_id_url: https://www.ncbi.nlm.nih.gov/pubmed/33272571
pmcid: PMC7699159
pmcid_url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699159
url: https://www.sciencedirect.com/science/article/pii/S0006291X20321239?v=s5 https://api.elsevier.com/content/article/pii/S0006291X20321239 https://www.ncbi.nlm.nih.gov/pubmed/33272571/ https://doi.org/10.1016/j.bbrc.2020.11.080
has_full_text: TRUE
Keywords Extracted from Text Content: liquid droplets humans SARS-CoV-2 virus coronaviruses c t SARS-CoV-2 b s t r a SARS-CoV-2 COVID-19 droplets upper respiratory tract coronavirus GSGS SpyCatcher human K560 coronaviridae [2 GE PIPES-KOH human kinesin-1 membrane [3 matrix MTs DNA poly-L-lysine 180C Santa Barbara MT K560-mScarlet-Strep-SpyCatcher NIST-traceable NP-40 AFM surface glycerol AAA HEPES nucleocapsid single membrane SARS-CoV-2 M PMSF SARS-CoV-2 locales K560-T92 N microtubules Strep tag FL late 2019 [1 biotin Mono Q column SARS-CoV-2 virus GF150 buffer human coronavirus SARS-CoV-2 pathogen spycatcher-Kinesin SARS viruses LE human cells ATP COVID-19 surface e SARS-CoV [9 cells mScarlet Spytag-S VLPs body Cells HB buffer humans virus particles tip VLP VLPs e VLPs isopropyl-b-D-thiogalactopyranoside potassium acetate MgSO4 E pET28a-based cell lysates IPTG particle BL21(DE3) cells EGTA membrane NIGMS RJM NSF RAPID 2026657 SS, NIGMS grant R35GM133688
Extracted Text Content in Record: First 5000 Characters:a b s t r a c t 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. 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. During initial refinement of VLP purification strategies and associated VLP characterization [10] , we have found that such particles remain stable for at least a week if stored in liquid buffer at near 0 C conditions (on ice). We therefore examined whether they would remain stable at room temperature under dry conditions on a model surface (Fig. 1) . Spytag-S VLPs were adhered to microtubules (MTs) via spycatcher-Kinesin and these complexes were abundantly retained on poly-L-lysinated glass surfaces. The shapes observed via AFM imaging at 22 C ( Fig. 1A) have nearly monodisperse sizes and only slight shape variability consistent with individual VLPs. Features indicati
Keywords Extracted from PMC Text: LE AAA GSGS locales surface PA lateral AFM mScarlet GF150 buffer cell lysates single membrane COVID-19 membrane [3 late 2019 [1 spycatcher-Kinesin MT SARS-CoV [9 coronaviridae [2 isopropyl-β-D-thiogalactopyranoside X.F. Porcine microtubules R.J.M. S-protein PIPES-KOH EGTA SARS-CoV-2 M poly-L-lysine glycerol SARS-CoV-2 virus KIF5B-spycatcher 180C membrane SARS viruses MA Mono Q column Ethanol FL BL21(DE3) cells nucleocapsid Spy Cells human kinesin-1 K560-T92 N tubulin Cytoskeleton DNA matrix body potassium acetate SARS-CoV-2 pathogen microtubules NP-40 HEPES GE ∼140 biotin pET28a-based human coronavirus humans human K560 MgSO4 cells IPTG VLP KIF5B-spycatcher + VLP virus particles MT-VLP Santa Barbara Spytag-S VLPs GTP SARS-CoV-2 Taxol tip ATP Catcher Strep tag 's particle K560-mScarlet-Strep-SpyCatcher HB buffer B.P. SpyCatcher human cells MgSo4 PMSF MTs Poly-L-Lysine VLPs HsKIF5B(1–560 NIST-traceable E
Extracted PMC Text Content in Record: First 5000 Characters: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. During initial refinement of VLP purification strategies and associated VLP characterization [10], we have found that such particles remain stable for at least a week if stored in liquid buffer at near 0 °C conditions (on ice). We therefore examined whether they would remain stable at room temperature under dry conditions on a model surface (Fig. 1 ). Spytag-S VLPs were adhered to microtubules (MTs) via spycatcher-Kinesin and these complexes were abundantly retained on poly-L-lysinated glass surfaces. The shapes observed via AFM imaging at 22 °C (Fig. 1A) have nearly monodisperse sizes and only slight shape variability consistent with individual VLPs. Features indicative of envelope disruption and VLP aggregation are rare. Note that individual MTs are usually washed out during sample preparation leaving behind characteristic depressions in the poly-L-lysinated surface. In addition, surface clumping of poly-L-lysine is discernible in the AFM data as background height inhomogeneity. VLP imaged at high humidity are similarly stable (Fig. S2). However, identically prepared samples imaged via AFM at 34 °C under dry conditions (Fig. 1B) are harder to image stably due to high amount of background noise which obscures poly-L-lysine inhomogeneity and MT washout sites (likely due to loose debris on the surface – plausibly a by-product of particle degradations). Features consistent with intact VLPs are prominent relative to noise levels and hence easy to resolve (Fig. 2 ), but they are so exceedingly rare at 34 °C that they are considered outliers (Fig. 3 ). They were seen in large area surveys in which each particle is mechanically probed only a few times. Such particles do not survive intact during even a single detailed zoomed-in scan (Fig. 2). It takes 20–30 min to install the sample into the AFM, approach the surface and validate tip condition. Therefore mildly elevated temperature has a rapid ef
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