Acceptor Fluorophores Research Articles

Engineering of weak helper interactions for high-efficiency FRET probes

Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.

Microsecond dynamics of an unfolded protein by a line confocal tracking of single molecule fluorescence

We present a new method for high speed tracking of fluorescence time series from single proteins. The method uses a fast sample flow and a modified confocal microscopy, line confocal microscopy, and achieves the time resolution of less than 20 μs. The obtained time series from the B domain of protein A labeled with donor and acceptor fluorophores suggest conformational heterogeneity and dynamic fluctuations in the unfolded state.

Squeezing at Entrance of Proton Transport Pathway in Proton-translocating Pyrophosphatase upon Substrate Binding

Homodimeric proton-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H(+)-PPase consists of 14-16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H(+)-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H(+)-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H(+)-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H(+)-PPase upon substrate binding.

Pivotal role of P450–P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone

We investigated the relationship between oligomerization of CYP3A4 (cytochrome P450 3A4) and its response to ANF (α-naphthoflavone), a prototypical heterotropic activator. The addition of ANF resulted in over a 2-fold increase in the rate of CYP3A4-dependent debenzylation of 7-BFC [7-benzyloxy-4-(trifluoromethyl)coumarin] in HLM (human liver microsomes), but failed to produce activation in BD Supersomes or Baculosomes containing recombinant CYP3A4 and NADPH-CPR (cytochrome P450 reductase). However, incorporation of purified CYP3A4 into Supersomes containing only recombinant CPR reproduced the behaviour observed with HLM. The activation in this system was dependent on the surface density of the enzyme. Although no activation was detectable at an L/P (lipid/P450) ratio ≥750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization, we probed P450-P450 interactions with a new technique that employs LRET (luminescence resonance energy transfer). The amplitude of LRET in mixed oligomers of the haem protein labelled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes™. The addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450-P450 interactions.

Estimating the distance separating fluorescent protein FRET pairs

Förster resonance energy transfer (FRET) describes a physical phenomenon widely applied in biomedical research to estimate separations between biological molecules. Routinely, genetic engineering is used to incorporate spectral variants of the green fluorescent protein (GFPs), into cellular expressed proteins. The transfer efficiency or rate of energy transfer between donor and acceptor FPs is then assayed. As appreciable FRET occurs only when donors and acceptors are in close proximity (1–10nm), the presence of FRET may indicate that the engineered proteins associate as interacting species. For a homogeneous population of FRET pairs the separations between FRET donors and acceptors can be estimated from a measured FRET efficiency if it is assumed that donors and acceptors are randomly oriented and rotate extensively during their excited state (dynamic regime). Unlike typical organic fluorophores, the rotational correlation-times of FPs are typically much longer than their fluorescence lifetime; accordingly FPs are virtually static during their excited state. Thus, estimating separations between FP FRET pairs is problematic. To overcome this obstacle, we present here a simple method for estimating separations between FPs using the experimentally measured average FRET efficiency. This approach assumes that donor and acceptor fluorophores are randomly oriented, but do not rotate during their excited state (static regime). This approach utilizes a Monte-Carlo simulation generated look-up table that allows one to estimate the separation, normalized to the Förster distance, from the average FRET efficiency. Assuming a dynamic regime overestimates the separation significantly (by 10% near 0.5 and 30% near 0.75 efficiencies) compared to assuming a static regime, which is more appropriate for estimates of separations between FPs.

A Toolbox of Fluorescence Microscopic Approaches Reveals Dynamics and Assembly of a Membrane-Associated Protein

A Toolbox of Fluorescence Microscopic Approaches Reveals Dynamics and Assembly of a Membrane-Associated Protein

Columnar Mesomorphism of Fluorescent Board-Shaped Quinoxalinophenanthrophenazine Derivatives with Donor–Acceptor Structure

Quinoxalino[2',3':9,10]phenanthro[4,5-abc]phenazine (QPP) dyes have been studied as electron acceptor materials, fluorophores, and building blocks for self-organizing organic semiconductors. Condensation of tetraketopyrene with electron-rich diamino-terphenylene and -triphenylene derivatives generates new donor-acceptor QPP derivatives that display columnar mesomorphism over wide ranges of temperature; are fluorescent in solution, liquid crystal, and solid phases; and have electron acceptor properties. Also reported are the synthesis and properties of the first diamino-(tetraalkoxy)triphenylene as a valuable new synthon.

Development of a Robust Model System of FRET using Base Surrogates Tethering Fluorophores for Strict Control of Their Position and Orientation within DNA Duplex

Although distance dependence of Förster resonance energy transfer (FRET) is well-studied and FRET has been extensively applied as "molecular ruler", only limited examples of orientation-dependent FRET have been reported. To create a robust FRET system that precisely reflects the orientation between donor and acceptor, donor and acceptor fluorophores were introduced into a DNA via a D-threoninol scaffold. Strong stacking interactions among intercalated dyes and natural base-pairs suppress free movement of the dyes, clamping them in the duplex in a fixed orientation. Pyrene and perylene were used as donor and acceptor, respectively, and both the distance and orientation between these dyes were systematically controlled by varying the number of intervening AT pairs from 1 to 21 (corresponding to two turns of helix). FRET efficiency determined from static fluorescence measurement did not decrease linearly with the number (n) of inserted AT pairs but dropped significantly every 5 base pairs (i.e., n = 8, 13, and 18), corresponding to a half-turn of the B-type helix. This clearly demonstrates that FRET efficiency reflects the orientation between pyrene and perylene. We also measured time-resolved fluorescence spectroscopy with a streak camera and successfully observed the time-course of the energy transfer directly. As expected, the FRET efficiencies determined from the lifetime of pyrene emission were in good agreement with static measurements. Theoretical calculation of FRET efficiency assuming that the DNA duplex is a rigid cylinder with B-type geometry coincided with the experimental results. We believe that our method of using d-threoninol will contribute to further development of FRET-based measurement techniques.

RAG and HMGB1 create a large bend in the 23RSS in the V(D)J recombination synaptic complexes

During V(D)J recombination, recombination activating gene proteins RAG1 and RAG2 generate DNA double strand breaks within a paired complex (PC) containing two complementary recombination signal sequences (RSSs), the 12RSS and 23RSS, which differ in the length of the spacer separating heptamer and nonamer elements. Despite the central role of the PC in V(D)J recombination, little is understood about its structure. Here, we use fluorescence resonance energy transfer to investigate the architecture of the 23RSS in the PC. Energy transfer was detected in 23RSS substrates in which the donor and acceptor fluorophores flanked the entire RSS, and was optimal under conditions that yield a cleavage-competent PC. The data are most easily explained by a dramatic bend in the 23RSS that reduces the distance between these flanking regions from >160 Å in the linear substrate to <80 Å in the PC. Analysis of multiple fluorescent substrates together with molecular dynamics modeling yielded a model in which the 23RSS adopts a U shape in the PC, with the spacer located centrally within the bend. We propose that this large bend facilitates simultaneous recognition of the heptamer and nonamer, is critical for proper positioning of the active site and contributes to the 12/23 rule.

Diffusion-Enhanced FRET between Membrane Proteins

Forster resonance energy transfer (FRET) is often used to study interactions between membrane proteins. The use of long-lived (∼10−3 sec) lanthanide donors allows for a delay between excitation and detection of emission, greatly enhancing the signal-to-noise ratio. However, this long excited state lifetime also allows donor- and acceptor-labeled membrane proteins to diffuse significant distances towards each other while the donor is excited, potentially enhancing energy transfer. Here we show that diffusion enhances lanthanide-based FRET (LRET) between membrane proteins, and that this phenomenon provides information concerning membrane protein organization. We constructed tetracycline-inducible cell lines expressing SNAP-tagged glycosylphosphatidylinositol (GPI)-anchored proteins (SNAP-GPI) and β2 adrenergic receptors (SNAP-β2AR), labeled these proteins with donor (Tb2+) and acceptor fluorophores, and measured FRET while varying diffusion. The FRET signal (sensitized emission) between SNAP-GPI proteins decreased by 62 ± 3% (P<0.001; n=4; mean ± S.D.) when these proteins were immobilized with aldehyde fixation. Similarly, immobilization decreased FRET between SNAP-β2AR by 76 ± 2% (P<0.001; n=4). Fixation did not change acceptor emission after direct excitation, nor did it change donor emission in the absence of an acceptor. Similar FRET decreases were observed when mobility was decreased by crosslinking with biotin and avidin, or by decreasing temperature. The effect of decreased mobility on FRET was evident for both proteins across a wide range of expression levels. These results suggest that lateral diffusion significantly enhances FRET between membrane proteins. Because diffusion can only enhance FRET between donors and acceptors that diffuse independently (i.e. are not part of the same complex), these results further suggest that a substantial fraction of GPI-anchored proteins and β2 adrenergic receptors exist on the cell surface as rapidly-diffusing monomers.Supported by NIH grants GM078319 and GM096762.

Cleft Closure Mechanism in Gating of Acid Sensing Ion Channel 1A

Acid sensing ion channels (ASICs) are trimeric proton activated channels which share sequence similarities with Degenerin/Epithelial sodium channels. They are responsible for proton evoked currents in central nervous system of most vertebrates. Their physiological functions include synaptic transmission, sensory perception, learning, and pathophysiology of stroke. Crystal structure of the low pH state of chicken ASIC shows thumb and palm like domains in the extracellular part of each subunit connected through hydrogen bonds between carboxylate residue pairs, thought to be part of proton sensors in ASIC. Based on the low pH structure it's been hypothesized that these domains would be further apart at high pH due to negative charges and protonation would allow the two domains to move closer, which in turn could trigger changes in the transmembrane segments, causing channel opening. We have used Fluorescence Resonance Energy Transfer(FRET) to study the high pH resting structure of ASIC and determined conformational changes between the thumb and palm domains. Cysteine residues were introduced on these domains and tagged with thiol reactive donor and acceptor fluorophores. The recognition site for protease Xa was introduced on either side of the cysteine on the palm domain. This allowed us to perform the FRET measurements in oocytes and HEK-293 cells without purification, as addition of the protease allowed us to quantitively characterize the background FRET. The FRET constructs were characterized using electrophysiology to establish that they were functional. The FRET based distances showed 3A decrease in distance between thumb domain residue 340 and palm domain residues 130 and 139 upon changing the pH from 8 to 6. This decrease in distance provides evidence for the hypothesized movement in the extracellular domain of ASIC and also supports the protonation of the carboxylate pairs across these domains.

Mechanisms in Co-Translational Protein Folding Elucidated using Single Molecule FRET

In nature, protein folding mechanisms are determined by both amino acid sequence AND physiological context. Inside living cells the transient and vectorial nature of protein synthesis, the presence of molecular chaperones, and macromolecular crowding can each have a profound effect on the paths nascent chains take en route to their native states. This adds a daunting level of complexity to the study of in-vivo protein folding mechanisms. To begin to address these issues we have developed methods which enable the detection of ribosome-bound nascent chain (RNC) conformational distributions and dynamics using single molecule fluorescence resonance energy transfer. Our approach employs a purified and reconstituted E. coliin-vitro translation system to generate stalled RNCs, the site-specific incorporation of chemically-reactive alkyne-bearing unnatural amino acid tags into nascent chains, and post-translational bioconjugation of pairs of single-molecule donor and acceptor fluorophores to the RNCs using a highly-efficient ligand accelerated copper-click chemistry reaction. We apply our novel RNC labeling approach to explore the role of helix stabilization by and propagation from the exit tunnel of the ribosome as a general means by which the ribosome may direct the co-translational folding of nascent polypeptides.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Biophotonic logic devices based on quantum dots and temporally-staggered Förster energy transfer relays

Integrating photonic inputs/outputs into unimolecular logic devices can provide significantly increased functional complexity and the ability to expand the repertoire of available operations. Here, we build upon a system previously utilized for biosensing to assemble and prototype several increasingly sophisticated biophotonic logic devices that function based upon multistep Förster resonance energy transfer (FRET) relays. The core system combines a central semiconductor quantum dot (QD) nanoplatform with a long-lifetime Tb complex FRET donor and a near-IR organic fluorophore acceptor; the latter acts as two unique inputs for the QD-based device. The Tb complex allows for a form of temporal memory by providing unique access to a time-delayed modality as an alternate output which significantly increases the inherent computing options. Altering the device by controlling the configuration parameters with biologically based self-assembly provides input control while monitoring changes in emission output of all participants, in both a spectral and temporal-dependent manner, gives rise to two input, single output Boolean Logic operations including OR, AND, INHIBIT, XOR, NOR, NAND, along with the possibility of gate transitions. Incorporation of an enzymatic cleavage step provides for a set-reset function that can be implemented repeatedly with the same building blocks and is demonstrated with single input, single output YES and NOT gates. Potential applications for these devices are discussed in the context of their constituent parts and the richness of available signal.

Simple Estimation of Förster Resonance Energy Transfer (FRET) Orientation Factor Distribution in Membranes

Because of its acute sensitivity to distance in the nanometer scale, Förster resonance energy transfer (FRET) has found a large variety of applications in many fields of chemistry, physics, and biology. One important issue regarding the correct usage of FRET is its dependence on the donor-acceptor relative orientation, expressed as the orientation factor κ2. Different donor/acceptor conformations can lead to κ2 values in the 0 ≤ κ2 ≤ 4 range. Because the characteristic distance for FRET, R0, is proportional to (κ2)1/6, uncertainties in the orientation factor are reflected in the quality of information that can be retrieved from a FRET experiment. In most cases, the average value of κ2 corresponding to the dynamic isotropic limit (<κ2> = 2/3) is used for computation of R0 and hence donor-acceptor distances and acceptor concentrations. However, this can lead to significant error in unfavorable cases. This issue is more critical in membrane systems, because of their intrinsically anisotropic nature and their reduced fluidity in comparison to most common solvents. Here, a simple numerical simulation method for estimation of the probability density function of κ2 for membrane-embedded donor and acceptor fluorophores in the dynamic regime is presented. In the simplest form, the proposed procedure uses as input the most probable orientations of the donor and acceptor transition dipoles, obtained by experimental (including linear dichroism) or theoretical (such as molecular dynamics simulation) techniques. Optionally, information about the widths of the donor and/or acceptor angular distributions may be incorporated. The methodology is illustrated for special limiting cases and common membrane FRET pairs.

Conformational Dynamics of Prothrombin Dictate Its Ordered Cleavage by Prothrombinase

Abstract 3359The prothrombinase complex (membranes/Xa/Va) binds prothrombin through exosite interactions which orient and present the substrate to the protease active site for cleavage. Although both cleavage sites within the substrate are accessible, prothrombin is preferentially activated by prothrombinase through sequential cleavage at R320 followed by cleavage at R271. We investigated the exosite-dependent interaction between prothrombin and prothrombinase using fluorescence resonance energy transfer (FRET). An acceptor fluorophore was placed onto the prothrombin cleavage site by introducing the mutation R271C and fluorescently modifying the introduced thiol (IIC271*). The active site of Xa was covalently modified with a chloromethyl ketone conjugated to a donor fluorescent compound. Titrations of the fluorescently labeled prothrombinase complex with IIC271* revealed a maximum energy transfer efficiency of 70–80 % observed with two different donor probes. Resonance energy transfer arising from the substrate docking interaction was confirmed by equivalent changes in the excited state lifetime of the donors. The calculated distance between the probe at C271 and the probe tethered at the active site of Xa ranged between 34 and 40 Å. Variation in the measured distance reflects the different lengths of the tethers used to connect the donor probes. The FRET measurements, validated with two donor-acceptor pairs, indicate that the 271 site within prothrombin docked to prothrombinase is positioned distant from the active site of Xa within the enzyme complex. This distant constraint prevents R271 from effectively engaging the active site of Xa within prothrombinase, thereby providing a physical explanation for the ordered action of prothrombinase on prothrombin. It follows that initial cleavage at R320 must reorient the substrate to allow R271 access to the Xa active site. We pursued an intramolecular FRET approach in which both donor and acceptor probes were placed on the prothrombin molecule to investigate the putative conformational change of prothrombin as a result of initial cleavage. Orthogonal labeling of prothrombin was accomplished using a prothrombin variant containing C271 and a LPETG extension at its C terminus. The optimized C271 label was used for the first modification. For the second, the sequence-specific transpeptidation activity of bacterial Sortase A was used to incorporate a fluorophore at the C-terminus (IICterm*). To assess the new fluorescent modification, FRET studies of prothrombin binding to prothrombinase were repeated with the new IICterm* construct. Titrations revealed a maximal energy transfer of 16 %, confirmed by corresponding changes in excited state lifetime and yielded a Kd = 39 nM indistinguishable from the Kd= 41 nM seen for IIC271* binding. Thus, two different approaches illustrate that the affinity of prothrombin for prothrombinase is independent of active site docking by the substrate. To examine the intramolecular dynamics of prothrombin during activation, the C271 site was labeled with BADAN (donor) and the Sortase labeling method placed the Alexa532 (acceptor) at the C terminus. The orthogonal placement of BADAN and Alexa532 on the prothrombin molecule resulted in extensive energy transfer between the two probes. The cleavage of doubly-labeled prothrombin to produce meizothrombin was associated with a change in intramolecular energy transfer. The variation of the energy transfers observed between the zymogen and protease forms can be interpreted as global rearrangement of the prothrombin molecule renders the second cleavage site accessible of active site docking. The FRET studies provide a comprehensive physical explanation for the ordered cleavage of prothrombin by prothrombinase. Initial cleavage occurs at the spatially accessible cleavage site R320. The prothrombin molecule then significantly re-orients itself making the second R271 cleavage site accessible to prothrombinase in order for activation to proceed and produce thrombin. Disclosures:No relevant conflicts of interest to declare.

Surface‐Immobilized Monolayers of Conjugated Oligomers as a Platform for Fluorescent Sensors Design: The Effect of Exciton Delocalization on Chemosensing Performance

Surface-immobilized monolayers of fluorescent molecular sensors consisting of a short conjugated oligo(p-phenylene ethynylene) core end-capped with an acceptor fluorophore (analyte receptor) display significant signal amplification due to enhanced intermolecular energy transfer within the monolayer. This general phenomenon offers a superior platform for designing ratiometric fluorescent sensors. An example of how this can be used to convert a narrow-range threshold fluorescent pH indicator (fluorescein) to a broad-range ratiometric fluorescent chemosensor is described.

Overview of Live‐Cell Imaging: Requirements and Methods Used

Overview of Live‐Cell Imaging: Requirements and Methods Used

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium, cAMP, inositol phosphates and cGMP. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker.

Frequency of close positioning of chromosomal loci detected by FRET correlates with their participation in carcinogenic rearrangements in human cells

It has been well established that genes participating in oncogenic rearrangements are non-randomly positioned and frequently close to each other in human cell nuclei. However, the actual distance between these fusion partners has never been determined. The phenomenon of fluorescence resonance energy transfer (FRET) is observed when a donor fluorophore is close (<10 nm) to transfer some of it energy to an acceptor fluorophore. The aim of this study was to validate the use of FRET on directly labeled DNA molecules to assess the frequency of positioning at <10 nm distances between genes known to be involved in rearrangement and to correlate it with their probability to undergo rearrangement. In the validation experiments, the frequency of FRET-sensitized emission (SE) was found to be 93-96% between probes for the immediately adjacent chromosomal regions as compared to 0.1-0.2% between probes for the random loci located on large linear separation. Further, we found that the frequency of FRET-SE between four pairs of genes that form rearrangements in thyroid cancer was 5% for RET and CCDC6, 4% for RET and NCOA4, 2% for BRAF and AKAP9, and 2% for NTRK1 and TPR. Moreover, the frequency with which FRET was observed showed strong correlation (r = 0.9871) with the prevalence of respective rearrangements in thyroid cancer. Our findings demonstrate that FRET can be used as a technique to analyze proximity between specific DNA regions and that the frequency of gene positioning at distances allowing FRET correlates with their probability to undergo chromosomal rearrangements.

Fluorescence Resonance Energy Transfer-Based Intracellular Assay for the Conformation of Hepatitis C Virus Drug Target NS5A

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and assembly and is a critical drug target. Biochemical data suggest large parts of NS5A are unfolded as an isolated protein, but little is known about its folded state in the cell. We used fluorescence resonance energy transfer (FRET) to probe whether or not different regions of NS5A are in close proximity within the cell. Twenty-three separate reporter constructs were created by inserting one or more fluorophores into different positions throughout the three domains of NS5A. FRET efficiency was maximal when donor and acceptor fluorophores were positioned next to each other but also could be observed when the two fluorophores flanked NS5A domain 1 or domain 3. Informatic and biochemical analysis suggests that large portions of the carboxy terminus of NS5A are in an unfolded and disordered state. Quercetin, a natural product known to disrupt NS5A function in cells, specifically disrupted a conformationally specific domain 3 FRET signal. Intermolecular FRET indicated that the NS5A amino termini, but not other regions, are in close proximity in multimeric complexes. Overall, this assay provides a new window on the intracellular conformation(s) of NS5A and how the conformation changes in response to cellular and viral components of the replication and assembly complex as well as antiviral drugs.