Fluorometric Estimation of Viral Thermal Stability

Rayaprolu, Vamseedhar; Kruse, Shannon; Kant, Ravi; Movahed, Navid; Brooke, Dewey; Bothner, Brian

doi:10.21769/bioprotoc.1199

Cited by 11 publications

(12 citation statements)

References 3 publications

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“… 19 , 20 , 21 Sample purity and capsid integrity were confirmed by SDS-PAGE and negative-stain electron microscopy (EM), respectively ( Figure 1 A). The T m for the purified full and empty capsids, diluted into PBS buffer, was determined by DSF, 22 as described in Materials and Methods . The T m is the temperature at which 50% of the sample is unfolded and bound with dye.…”

Section: Resultsmentioning

confidence: 99%

Thermal Stability as a Determinant of AAV Serotype Identity

Bennett

Patel

Mietzsch

et al. 2017

Molecular Therapy - Methods & Clinical Development

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102

103

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Currently, there are over 150 ongoing clinical trials utilizing adeno-associated viruses (AAVs) to target various genetic diseases, including hemophilia (AAV2 and AAV8), congenital heart failure (AAV1 and AAV6), cystic fibrosis (AAV2), rheumatoid arthritis (AAV2), and Batten disease (AAVrh.10). Prior to patient administration, AAV vectors must have their serotype, concentration, purity, and stability confirmed. Here, we report the application of differential scanning fluorimetry (DSF) as a good manufacturing practice (GMP) capable of determining the melting temperature (Tm) for AAV serotype identification. This is a simple, rapid, cost effective, and robust method utilizing small amounts of purified AAV capsids (∼25 μL of ∼1011 particles). AAV1-9 and AAVrh.10 exhibit specific Tms in buffer formulations commonly used in clinical trials. Notably, AAV2 and AAV3, which are the least stable, have varied Tms, whereas AAV5, the most stable, has a narrow Tm range in the different buffers, respectively. Vector stability was dictated by VP3 only, specifically, the ratio of basic/acidic amino acids, and was independent of VP1 and VP2 content or the genome packaged. Furthermore, stability of recombinant AAVs differing by a single basic or acidic amino acid residue are distinguishable. Hence, AAV DSF profiles can serve as a robust method for serotype identification of clinical vectors.

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Section: Resultsmentioning

confidence: 99%

Thermal Stability as a Determinant of AAV Serotype Identity

Bennett

Patel

Mietzsch

et al. 2017

Molecular Therapy - Methods & Clinical Development

Self Cite

102

103

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“…The differences in particle diameter and resistance to ionic strength suggested that 4D CP in CLPs interacted differently than WT CP in CLPs assembled with oligo. To explore this possibility, we used differential scanning fluorometry to compare the thermal stability of cargofree 4D CLPs to CLPs assembled with WT CP and 27mer and 48hp cargos (figure 3(D)) [43,44]. Based on the stability of the 4D CLPs under increased ionic strength, we expected 4D CLPs to be more stable to thermal denaturation than WT CLPs assembled in the presence of cargo: that the CP-CP interactions in 4D CLPs would be stronger than the cargo-CP scaffolding interactions in WT, cargo-containing CLPs.…”

Section: Mutant Cp Self-assembles Into Clps Independent Of Cargomentioning

confidence: 99%

Length of encapsidated cargo impacts stability and structure ofin vitroassembled alphavirus core-like particles

Rayaprolu¹,

Moore²,

Wang³

et al. 2017

J. Phys.: Condens. Matter

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In vitro assembly of alphavirus nucleocapsid cores, called core-like particles (CLPs), requires a polyanionic cargo. There are no sequence or structure requirements to encapsidate single-stranded nucleic acid cargo. In this work, we wanted to determine how the length of the cargo impacts the stability and structure of the assembled CLPs. We hypothesized that cargo neutralizes the basic region of the alphavirus capsid protein and if the cargo is long enough, it will also act to scaffold the CP monomers together. Experimentally we found that CLPs encapsidating short 27mer oligonucleotides were less stable than CLPs encapsidating 48mer or 90mer oligonucleotides under different chemical and thermal conditions. Furthermore, cryo-EM studies showed there were structural differences between CLPs assembled with 27mer and 48mer cargo. To mimic the role of the cargo in CLP assembly we made a mutant (4D) where we substituted a cluster of four Lys residues in the CP with four Asp residues. We found that these few amino acid substitutions were enough to initiate CLP assembly in the absence of cargo. The cargo-free 4D CLPs show higher resistance to ionic strength and increased temperature compared to wild-type cargo containing CLPs suggesting their CLP assembly mechanism might also be different.

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“…Differential scanning fluorimetry. DSF was performed essentially as described previously (23). Desired virus and control (lysozyme) samples were resuspended either in Nanopure sterile water (pH 7.1), in virus suspension buffer (50 mM sodium acetate, 8 mM magnesium acetate, pH 4.5), or in 100 mM phosphate buffer (pH 7.2).…”

Section: Methodsmentioning

confidence: 99%

“…To test the relative thermal stability of B1 V , B2 V , and B3ϩ4 V , a differential scanning fluorimetry (DSF) assay was performed as described in Materials and Methods. The hydrophobic dye used in this assay increases the intensity of the fluorescence following its binding to the accessible hydrophobic regions of the viral CP during thermal denaturation (23). Results of the DSF analysis of the temperature-dependent melting of virions of B1 V , B2 V , and B3ϩ4 V and control samples (wt BMV and lysozyme) under three buffer and pH conditions are summarized in Fig.…”

Section: Fig 3 Legend (Continued)mentioning

confidence: 99%

Unravelling the Stability and Capsid Dynamics of the Three Virions of Brome Mosaic Virus Assembled Autonomously In Vivo

Chakravarty

Reddy

Rao

2020

J Virol

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Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of Brome mosaic virus (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasisymmetry. Type 1 (B1V) and type 2 (B2V) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4V) virions copackage genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta. Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K65, R103, K111, and K165 on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1V and B2V released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4V capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids. IMPORTANCE The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that, in solution, virions are highly dynamic assemblies. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

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Fluorometric Estimation of Viral Thermal Stability

Cited by 11 publications

References 3 publications

Thermal Stability as a Determinant of AAV Serotype Identity

Thermal Stability as a Determinant of AAV Serotype Identity

Length of encapsidated cargo impacts stability and structure ofin vitroassembled alphavirus core-like particles

Unravelling the Stability and Capsid Dynamics of the Three Virions of Brome Mosaic Virus Assembled Autonomously In Vivo

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