Multi-homosubunit dsRNA/dsDNA virus genome packaging nanomotors with sequential revolving mechanisms for potent drug development

1 Introduction

The emergence of recent viral pandemics and multidrug resistance presents a critical imperative for developing novel therapeutic strategies (Guo, 2010; Shu et al., 2015). While traditional antiviral approaches have predominantly focused on single drug targets—proteins, RNA, or DNA—leading to rapid resistance development, recent advances in structural biology and biophysics have revealed promising alternatives by targeting multi-subunit molecular machines (Pi et al., 2016a; Hilbert et al., 2015).

A groundbreaking development emerged from discovering that homomeric biological complexes (Figures 1, 2) can be exploited therapeutically via their high stoichiometry and sequential revolving mechanisms (Schwartz et al., 2013a; Pi et al., 2016b). This principle applies to over 30 catalogued ATPase systems across eight functional categories (Table 1), including viral genome packaging motors and ATP-Binding Cassette (ABC) transporters, which share fundamental architectural attributes despite diverse biological functions. Each system employs coordinated ATP hydrolysis by multiple identical subunits, making them ideal candidates for the “Series-Circuit-Christmas-Bulb” therapeutic strategy (Figure 2).

TABLE 1. Structural and functional similarities among various ATPase systems.

Viral packaging motors comprise oligomerized channel complexes and terminase enzymes that translocate viral genomes against enormous pressure gradients through ATP-driven conformational changes (Mancini et al., 2004a; Veesler et al., 2012). These motors, essential for bacteriophages (Zhao et al., 2013), herpesviruses (Yang et al., 2020; Guo, 2020) and other viral families (Mancini et al., 2004b), demonstrate remarkable structural conservation with hexameric ATPase organization. Similarly, ABC transporters contributing to drug resistance operate through comparable sequential mechanisms, with 73% exhibiting hexameric architecture that enables stoichiometric targeting (Gottesman et al., 2002).

Structural studies have revealed striking parallels between these systems’ architectures and operational mechanisms. Both utilize coordinated ATP hydrolysis across multiple subunits to drive conformational changes, creating sequential dependencies exploitable therapeutically—inhibition of one subunit (K = 1) disrupts the entire motor complex, regardless of total subunit number (Z > 1) (Kainov et al., 2004). This vulnerability enables drug potency to increase as a power function of subunit stoichiometry, validated by FDA-approved examples like bedaquiline targeting bacterial F₁F₀ ATPase and experimental RNA nanoparticles achieving 10⁷-10⁸ fold inhibition (Schwartz et al., 2013b).

This review examines how understanding these molecular machines’ structural and functional principles expands drug development possibilities. We explore structure-based targeting, computational approaches, and delivery strategies leveraging sequential motion dependencies to address viral infections and drug resistance simultaneously. The viral genome or motor components both can serve as a therapeutic target or the targeting agent for the treatment of viral diseases or viral drug resistance, following the newly development strategy in the field of RNA therapeutics (Xu et al., 2024; Ho et al., 2024; Beasock et al., 2024; Bolarum and Dobrovolskaia, 2025; Bian et al., 2025).

1.1 Section 1: the structure and mechanism of representative viral genome packaging motors

The conservation of hexameric ATPases among varying biological systems reveals common, fundamental principles underlying molecular motor function (Table 1). The similarities between dsDNA- and dsRNA-packaging motors highlight both the evolutionary convergence of motor architectures and the therapeutic opportunities that arise from targeting conserved structural features. The systematic analysis of different packaging systems—from the well-studied phi29 bacteriophage to complex multi-segmented dsRNA viruses—demonstrates that despite operating in distinct cellular environments, these motors share critical mechanistic principles. This conservation suggests that the Series-Circuit-Christmas-Bulb mechanism accurately models ATP-driven translocation, making it an attractive target for broad-spectrum therapeutic intervention.

The remarkable efficiency of molecular motors fundamentally depends on their ability to convert chemical energy from ATP hydrolysis into mechanical work for substrate translocation. While viral packaging motors share structural similarities that provide the foundation for understanding their operation, the detailed mechanisms of energy coupling and force generation reveal the specific molecular events that enable these powerful biological machines. Understanding these mechanisms is crucial for developing targeted therapeutic strategies, as disruption of energy coupling illustrates a direct approach for halting motor function. The energy transduction mechanisms employed by viral packaging motors necessitate coordination between ATP binding, hydrolysis, and conformational changes that drive nucleic acid translocation. These processes, while sharing fundamental principles across different motor types, exhibit unique adaptations that reflect the specific challenges of packaging different nucleic acid substrates. The comparative analysis of energy coupling mechanisms across dsDNA and dsRNA systems reveals both conserved features, which can be exploited therapeutically, and system-specific vulnerabilities, which enable selective targeting.

1.1.1 dsDNA packaging motors

The energy coupling mechanisms in dsDNA packaging motors represent the most extensively studied examples of ATP-driven nucleic acid translocation, providing the foundational understanding for all viral packaging systems (Guo et al., 1986; Guo PX. et al., 1987; Guo et al., 1987b; Guo et al., 1987c; Jiang et al., 2006; Cerritelli et al., 2003; Maggin et al., 2012). These motors exemplify the fundamental principles of revolving motion demonstrate the fundamental principles of the sequential revolving mechanism while revealing the sophisticated coordination required to achieve efficient DNA translocation against enormous internal pressures.

1.1.1.1 The phi29 dsDNA-packaging motor

The phi29 bacteriophage motor represents the most thoroughly characterized model for understanding dsDNA packaging mechanisms (Guo, 2010) (Figure 1). This nanomachine comprises three essential coaxial components working in precise coordination: a dodecameric connector (portal protein) forming the central channel, a hexameric packaging/prohead RNA (pRNA) ring that serves as a gearing mechanism, and a hexameric ATPase ring that generates the packaging force (Schwartz et al., 2013b; Simpson et al., 2000; Ye et al., 2012; Bourassa and Major, 2002) (Figure 4). The pRNA molecules form a hexameric ring that physically connects the ATPase motor to the connector channel, effectively “gearing” or mechanically coupling the ATPase’s conformational changes to DNA translocation (Bohmer et al., 2025). Although there is controversy on the stoichiometry of the phi29 DNA-packaging motor (five-fold or six-fold), there is biochemical and structural evidence suggesting it is hexameric (for an in-depth review, see Ref. 32) (Bohmer et al., 2025; Bohmer et al., 2023). Each pRNA contains distinct structural domains: one domain binds specifically to the connector channel (Garver and Guo, 1997) while another interacts with the ATPase subunit (Lee et al., 2008; Zhao et al., 2008), creating an essential mechanical linkage that ensures coordinated movement. This gearing function is critical because it translates the ATP-driven conformational changes of the ATPase into the precise revolving motion needed for DNA advancement through the channel.

1.1.1.2 Many revolving DNA translocases share a large, asymmetrical central channel

Motors that exemplify this asymmetry include phi19 gp16 (Zhao et al., 2016), YME1 protease (Puchades et al., 2017), and V1-ATPase (Arai et al., 2013), and many other viruses (Hong et al., 2014; de Haas et al., 1999; Santosh et al., 2020; Lisal and Tuma, 2005) (Figure 3). Revolving dsDNA translocases contain channels that are 1.5- to 1.8-fold larger than the 2-nm-dsDNA, including the phi29 DNA packaging motor (Simpson et al., 2000), T7 DNA-ejection motor (Cuervo et al., 2019; Agirrezabala et al., 2005), TraB plasmid dsDNA-conjugating protein (Amado et al., 2019), Gram-negative bacterial translocase FtsK (Jean et al., 2020), Gram-positive bacterial translocase SpoIIIE (Fiche et al., 2013), herpesvirus DNA-packaging motor (Figures 3, 4) (Yang et al., 2020). These translocases all utilize hexameric motors that revolve the dsDNA, with channel diameters ranging from 2.8 to 3.5 nm (Figure 4) (Guo et al., 2014; De-Donatis et al., 2014). The Acanthamoeba polyphaga mimivirus (APMV) also contains a dsDNA translocation motor that is structurally similar to FtsK (Iyer et al., 2004; Chelikani et al., 2014). Due to this structural similarity, it has been proposed that the APMV motor also uses a revolving mechanism to translocate DNA (De-Donatis et al., 2014).

1.1.1.3 The one-strand contact to the channel wall argues against the rotation model but favors the revolving mechanism

This is because retention would require the contact of both strands (De-Donatis et al., 2014) (Figure 5A). The motor prevents DNA from sliding out by three steps of channel gating (Bohmer et al., 2023; Shu et al., 2025; Wang et al., 2017; Geng et al., 2011) induced by the interaction of the dsDNA with the channel protein internal loops (Figure 5B). Additionally, the sequential transition style supports the revolving motion mechanism and argues against the rotation proposal (Figure 4), with the arginine finger of the motor ATPase regulating the sequential revolving mechanism (Zhao et al., 2016; Liang and Guo, 2021).

1.1.1.4 The 30° left-chirality of the channel wall is commonly found among dsDNA packaging motors

Revolution of one 360° turn of the right-handed dsDNA helix through the 12-subunit channel without rotation requires a 30° left chirality to compensate for the configurational balance and ensure alignment of the electrostatic dsDNA/protein contact. Phi29 (Simpson et al., 2000; Guasch et al., 2002; Bhullar et al., 2022), P22 (Swanson et al., 2022), SPP1 (Lebedev et al., 2007), and Hong Kong 97 phages all exhibit this 30°-tilted left-handed channel wall chirality (De-Donatis et al., 2014) (Figure 6), despite little sequence homology (De-Donatis et al., 2014; Wang et al., 2017), providing compelling structural evidence for the universal adoption of the revolving mechanism across diverse dsDNA packaging systems.

Structural determination of viral DNA-translocation motors has been challenging due to three main factors: limited CryoEM resolution, the difficulty of capturing rapidly moving objects with varying asymmetrical phases, and the complexity of reconstructing asymmetric structures from collective data. The phi29 dsDNA packaging motor exemplifies these challenges. When reconstructed using CryoEM technology, it displayed an asymmetrical structure under one-fold symmetry enforcement, appeared as a five-fold pentamer when five-fold symmetry was enforced, and showed no distinct subunits under six-fold enforcement due to its inherently asymmetrical hexameric nature (Morais et al., 2001).

1.1.1.5 Mechanism of phi29 dsDNA packaging

The hexameric ATPase ring operates through a coordinated mechanism where adjacent subunits undergo sequential conformational changes in a “hand-off” pattern (Chistol et al., 2012). This process creates the power that drives DNA translocation with forces exceeding 57 pN, making it one of the strongest known biological motors (Smith et al., 2001). Single-molecule studies have revealed that each ATPase subunit undergoes distinct conformational states during the ATP hydrolysis cycle (Chistol et al., 2012). The transition between these states drives DNA translocation through a complex network of protein-DNA interactions (Zhao et al., 2016). Motor activity is initiated when two gp16 subunits are bridged by binding of an arginine finger to the Walker A motif of an adjacent subunit, forming an ATP-binding pocket (Liang and Guo, 2021). When bound to ATP, gp16 exhibits high affinity for dsDNA. Upon ATP hydrolysis by the Walker B domain, the gp16 subunit adopts a conformation with lower affinity for dsDNA, which transfers the dsDNA strand to the adjacent ATP-bound subunit (Bohmer et al., 2023; Schwartz et al., 2012) The motor’s remarkable efficiency stems from the tight coupling between ATP hydrolysis and mechanical motion, with one ATP molecule consumed per 1.7 base pairs of DNA packaged (Chistol et al., 2012).

1.1.1.6 Herpesvirus dsDNA-packaging motor

Similar to phi29, possibly due to convergent evolution, the eukaryotic herpesvirus also uses a hexameric ATPase biomotor to package dsDNA through a dodecameric connector into a pre-formed capsid (Yang et al., 2020). The motor experiences substantial conformational change upon ATP binding, which is driven by a trans-arginine finger that bridges two subunits (Zhao et al., 2016; Liang and Guo, 2021). ATP hydrolysis in one subunit is triggered when the arginine finger of an adjacent subunit is inserted into its ATPase active site (Yang et al., 2020). These conformational changes of the motor drive dsDNA translocation in a revolving motion (Guo et al., 2014). Since this system relies on the cooperation of multiple subunits, inactivation of any one subunit would render the whole system inoperative, thus making it a potential target for antiviral therapies (Shu et al., 2015; Pi et al., 2016a).

The comprehensive mechanistic evidence from dsDNA packaging motors establishes the fundamental principles of the sequential revolving mechanism that underlies the therapeutic vulnerability of these systems. The conservation of these features across diverse DNA packaging systems, combined with the similar principles observed in dsRNA motors (Mancini and Tuma, 2012) (see below), provides strong support for the universal applicability of the Series-Circuit-Christmas-Bulb mechanism targeting strategy. This mechanistic foundation sets the stage for understanding how these same principles extend to other ATP-driven systems, particularly the ABC transporters that contribute to drug resistance.

1.1.2 Packaging motors of dsRNA viruses

Multi-segmented dsRNA viruses, including cystoviruses, employ specific packaging mechanisms to accommodate multiple genomic segments during viral assembly (Borodavka et al., 2018). The P4 packaging system of dsRNA cystoviruses, such as phi12, demonstrates the mechanistic principles of hexameric molecular motors (Juuti et al., 1998; Johnson et al., 2017). In these viruses, the motors package three ssRNA segments and an RNA polymerase replicates them to produce dsRNA (Mancini et al., 2004a). These ATPases operate through mechanisms that couple energy from ATP hydrolysis to drive RNA translocation (Kainov et al., 2006; Travis et al., 2025) and exhibit structural similarities to RecA-type helicases, reflecting evolutionary relationships among different packaging systems (El Omari et al., 2013).

The hexameric P4 system of cystovirus phi12 is believed to bind ATP in a pocket between two adjacent monomers, and this process appears to be regulated by two arginine fingers of each P4 subunit (Mancini et al., 2004a). Six ATP molecules are used to package eleven ssRNA bases (Mancini et al., 2004a), which is similar to the numbers reported for analogous motors such as those of phi29 (Guo et al., 1987c), T3 (Morita et al., 1993), and T7 (Kim et al., 2002). The hexameric P4 undergoes iterative conformational changes around the ring in response to ATP binding, hydrolysis, and unbinding in an anticlockwise fashion due to the right-handed RNA (Figure 1D) (Mancini et al., 2004a). These coordinated actions explain how the P4 motor packages ssRNA without rotation. Additionally, similar to the lysine rings of phi29 (Fang et al., 2012), the phi12 P4 also contains a conserved lysine that interacts with the RNA and is essential for packaging activity (Mancini et al., 2004a).

Similarly, the bacteriophage phi6 genome is also packaged into the procapsid via segmented strands (Mindich, 2004; Poranen and Bamford, 1999). The segmented strands include large (L), middle (M), and small (S) ssRNAs. Upon packaging, the other strand is synthesized by P2 RNA polymerase within the procapsid, resulting in dsRNA. To initiate RNA packaging, the L, M, and S RNAs contain an 18-nt sequence at the 5′ end that binds to the procapsid (Qiao et al., 1997). Like phi12 and other Cystoviruses, it contains the hexameric P4 ATPase that sits at the pentameric procapsid portal of the procapsid. While the sequences of the P4 ATPase domains are dissimilar among Cystovirus species, they share common secondary structural features, specifically a Rossmann-type nucleotide-binding domain (El Omari et al., 2013). Despite little sequence homology, the similar structural features indicate a common mechanism (El Omari et al., 2013; Mantynen et al., 2018). However, some differences remain; phi6 requires ATP/ADP binding to stabilize the P4 hexamer, while the phi12 P4 hexamer is instead stabilized by an α-helix (Juuti et al., 1998; Gottlieb and Alimova, 2022). Overall, the similar structural features of the Cystovirus P4 ATPase hexamer illuminate a conserved mechanism of RNA packaging.

High-resolution crystallographic structures of phi12 P4 in apo and nucleotide-bound conformations (El Omari et al., 2013) define it as a RecA/AAA-like hexameric ATPase, with conserved motifs suggesting possible arginine-finger-like inter-subunit interactions. This structural homology supports a plausible parallel with well-characterized dsDNA motors. X-ray crystallography uncovered two arginine fingers per P4 monomer that are positioned to interact with the adjacent monomer and the γ-phosphate of an ATP molecule (or AMPcPP, the non-hydrolyzable ATP analog used in the study) (Mancini et al., 2004a). This γ-phosphate is bound by an asparagine residue, which is conserved among cystoviruses.

While dynamic rearrangements in RNA-binding loops (such as L2) are supported by structural data (Mancini et al., 2004a), their direct role in translocation remains unclear. Notably, no single-molecule packaging kinetics studies have been reported for P4 motors specifically. However, based on their structural data, Mancini et al. proposed a coordination mechanism, in which at any given moment, five sites contain ATP and one site contains ADP. The ADP-bound state is stabilized by RNA binding the L2 loop. As a result, the arginine finger inserts into the catalytic site of the adjacent subunit, promoting ATP hydrolysis at the adjacent site. Upon ATP hydrolysis at the adjacent site, the RNA strand is transferred to the L2 of the adjacent subunit, which, at the original subunit, causes the release of ADP and binding of ATP. This process is repeated between adjacent subunits until the RNA is packaged (Mancini et al., 2004a).

dsRNA motors can exhibit nonspecific nucleoside triphosphatase activity that allows utilization of various NTPs for RNA packaging (Kainov et al., 2004; Pirttimaa et al., 2002). P4 motors demonstrate this nucleotide-substrate non-specificity while maintaining precise control over translocation through their hexameric structure. The P4 motor’s central channel is optimized for single-stranded RNA translocation during ssRNA packaging, with the motor subsequently serving as a passive channel during transcription when dsRNA synthesis occurs within the viral core (Kainov et al., 2004). This dual functionality, active packaging followed by passive export, distinguishes dsRNA virus packaging systems from their dsDNA counterparts.

The Reoviridae family, including rotaviruses with eleven-segment dsRNA genomes, demonstrates complex genome packaging mechanisms (McDonald and Patton, 2011). Rotavirus packaging entails the formation of RNA complexes through specific inter-molecular interactions between different genomic segments during viral assembly (McDonald and Patton, 2011). Cryoelectron tomography has revealed the structural organization of these complex viral particles (Shah et al., 2023). Rotavirus particles contain three concentric protein layers: the inner VP2 core, the intermediate VP6 layer, and the outer VP7 layer, which together form the complete viral particle (Shah et al., 2023). The coordination required to package eleven distinct genomic segments demonstrates the complexity of segmented virus assembly processes (McDonald and Patton, 2011).

The packaging mechanisms in dsRNA viruses exemplify how different viral families approach the challenge of genome organization and assembly. Understanding these packaging processes provides insights into the strategies used by segmented RNA viruses to achieve successful genome packaging (Borodavka et al., 2018).

1.1.3 Assembly motors and translation ATPase of ssRNA viruses and related ATPases

ssRNA viruses use different genome packaging strategies compared to dsDNA and dsRNA viruses, with many achieving genome encapsidation through simultaneous capsid formation and RNA packaging (Butler, 1999; Klug, 1999). The energy requirements for ssRNA virus assembly and replication differ from those involved in packaging genomes against the high internal pressure observed in dsDNA viruses (Perlmutter et al., 2015), making viral self-assembly a process that coordinates capsid and genome organization (Bruinsma et al., 2003). Furthermore, ssRNA viruses can appropriate host machinery for viral genome translocation and replication. For example, the Ded1p DEAD-box RNA helicase of yeast, a translation factor, has been shown to enhance positive strand synthesis in Tomato bushy stunt virus (TBSV) (Kovalev et al., 2012).

Single-stranded RNA viruses have evolved ATP-coupling mechanisms that differ from the packaging motors of dsDNA and dsRNA viruses. ssRNA virus ATPases couple ATP hydrolysis to mechanical processes such as RNA remodeling and conformational regulation (Fairman-Williams et al., 2010; Singleton et al., 2007). Superfamily 2 helicases demonstrate this diversity of ATP-dependent mechanisms used by different viral and cellular systems (Byrd and Raney, 2012). In the DECH-Box Cysteine helicase of the hepatitis C virus, ATP hydrolysis is coupled with helicase conformational change and RNA unwinding. Kinetic studies suggested that ATP binding to the helicase reduced its affinity to the RNA substrate. Upon ATP hydrolysis, helicase affinity to the RNA strand is restored (Levin et al., 2003; Yerukhimovich et al., 2018). This mechanism of coupling NTP hydrolysis to oligo substrate procession has been documented in other helicases such as vaccinia virus NPH-II (Jankowsky et al., 2000) and eukaryotic eIF4A (Lorsch and Herschlag, 1998). The underlying ATP coupling mechanisms in helicases share coordinated conformational changes and allosteric regulation, illustrating conserved principles across different helicase superfamilies (Byrd and Raney, 2012).

Picornavirus 2C proteins represent another class of ATP-dependent viral proteins, with poliovirus protein 2C demonstrating both ATPase and GTPase activities (Rodriguez and Carrasco, 1993). A ssRNA picornavirus, foot-and-mouth disease virus (FMDV), AAA + ATPase 2C forms hexameric complexes when in the presence of ATP and ssRNA (Sweeney et al., 2010). Without ATP and RNA, FMDV 2C multimerizes in a concentration-dependent manner, forming a maximum of tetramers. Furthermore, FMDV 2C uses the Series-Circuit-Christmas-Bulb mechanism of coordinated ATP hydrolysis. When wild-type and mutant 2C subunits were mixed at different ratios, ATPase activity decreased non-linearly, which indicates coordinated hydrolysis (Sweeney et al., 2010). These proteins are examples of RNA helicases with ATP-dependent activity (Xia et al., 2015).

Coronavirus NSP13 helicase exemplifies ATP-dependent RNA processing in ssRNA virus systems, as its structure and mechanism have been characterized (Newman et al., 2021). NSP13 has been identified as a potential therapeutic target for COVID-19 treatment due to its considerable conservation in the SARS-CoV-2 genome (White et al., 2020; Romeo et al., 2022).

The ESCRT (endosomal sorting complex required for transport)-VPS4 system is another host machinery component utilized by viruses for membrane scission during viral release. VPS4 is an AAA + ATPase involved in membrane budding and scission processes that are appropriated during viral replication (Hur et al., 2010). The asymmetric ring structure of VPS4 is required for ESCRT-III disassembly (Caillat et al., 2015).

These diverse viral systems demonstrate the importance of ATP-dependent processes in viral assembly and replication across different virus families, representing potential targets for therapeutic intervention.

1.2 Section 2: structure and function of ATP-binding cassette, the ABC drug transporters

The transition from viral packaging motors to ABC transporters represents a critical shift in our understanding of how the Series-Circuit-Christmas-Bulb mechanism extends beyond viral systems to cellular machinery involved in drug resistance. While viral packaging motors translocate nucleic acids for viral assembly, ABC transporters utilize remarkably similar ATP-driven mechanisms to export drugs and other substrates from cells, creating therapeutic challenges that can be addressed using the same stoichiometric targeting principles (Table 1). Genomic RNA, pRNA, or other RNA can serve as either the therapeutic target or the targeting agent for therapeutic purposes of viral disease or anti-drug resistance, following the current strategy in the booming RNA therapeutic field (Zhou and Chen, 2024; Binzel et al., 2025; Rychahou et al., 2024; Gupta et al., 2024; Pranger et al., 2024).

1.2.1 Background on ABCs

ATP-binding cassettes (ABCs) are a family of transporters that play a critical role in drug resistance. They are ATP-dependent drug efflux pumps or, simply, ATPase biomotors. For example, the P-glycoprotein (P-gp) is an ABC transporter in the hepatocytes of the liver normally involved in the excretion of toxins from cells. The failure of many therapeutic interventions is often attributed to the multi-faceted nature of drug resistance, which includes drug efflux by ABC transporters. Strategies to counter these mechanisms, such as ABC transporter inhibition and targeted drug delivery, are continuously investigated (Choi and Yu, 2014).

1.2.2 Structural conservation and functional adaptation

Several ABC transporters demonstrate critical hexameric arrangements that are essential for their function. The DevBCA system shows that efficient substrate binding and export activity depends on stable DevB hexamers (Staron et al., 2014). The MlaD system revealed a unique “asymmetric protomer movement” (APM) mechanism, where half of the EcMlaD hexamer rises on the periplasmic side while its pore loops move outward, significantly altering the central channel geometry (Dutta and Kanaujia, 2024) (Figure 7C).

1.2.3 Complex assembly and substrate transport

The bacterial MacAB-TolC multidrug efflux complex exhibits a sophisticated tripartite multidrug efflux pump architecture, with MacA forming distinctive interactions with TolC through its α-hairpin tip region. The conserved residues at the tip of the α-hairpin are important for establishing proper interaction with TolC in this ABC-type system. Based on structural data, it has been proposed that this system couples ATP hydrolysis to conformational change to translocate its substrates (Fitzpatrick et al., 2017). The molybdate/tungstate binding protein Mop from Sporomusa ovata shows structural features that enable binding of multiple oxyanions, demonstrating how protein organization can enhance substrate binding capacity (Wagner et al., 2000).

The bacterial EcMlaD system’s phospholipid transport mechanism illuminates the structural features that facilitate phospholipid transportation. This system exemplifies the APM mechanism via coordinated conformational changes during the transport cycle. The structural organization facilitates substrate transportation through systematic domain movements, with the cycle consisting of symmetric resting states, asymmetric protomer movement, ligand capture, and reset phases (Dutta and Kanaujia, 2024).

1.3 Section 3: comparison of structure and mechanism of viral DNA packaging motors with ABCs: Universal principles and their applications

Viral dsDNA translocation motors translocate double-stranded DNA genomes via revolving hexameric ATPases that use asymmetric motor mechanisms (Bohmer et al., 2023). These systems utilize ATP hydrolysis to power DNA translocation during viral packaging, and the hexameric organization allows for coordinated subunit action during translocation.

ATP-binding cassette (ABC) transporters represent another class of ATP-dependent transport systems, and are commonly involved in drug efflux and resistance. These transporters utilize ATP binding and hydrolysis to drive substrate transport across cellular membranes. Moreover, ABC transporters contribute to multidrug resistance through their ability to export various pharmaceutical compounds from cells (Choi and Yu, 2014). Based on the global structure of ABCs, most are composed of two protein subunits. However, by looking carefully at the detailed structure, ABCs contain a channel with six or twelve sub-domains that form a channel embedded in the membrane as a membrane channel (Jones and George, 2000).

Both viral packaging motors and ABC transporters are examples of ATP-dependent biological systems that translocate substrates, though they operate in different cellular contexts and transport distinct substrates. Viral motors are involved in genome packaging during viral assembly, while ABC transporters are involved in cellular drug efflux and resistance processes. Understanding the mechanisms of these different ATP-dependent systems provides insights into diverse biological transport processes (Figure 7).

1.3.1 Mechanism for highly potent inhibition with therapeutic targeting and resistance prevention

Researchers have investigated the development of drugs that target multi-subunit biological complexes through stoichiometric approaches. This new approach has been developed for creating ultra-high inhibitory drugs using the power function of the stoichiometry of targeted nanomachines or biocomplexes (Shu et al., 2015). This approach demonstrates how the number of subunits in a complex can affect drug potency through mathematical relationships.

The mathematical framework for this approach involves the relationship between stoichiometry and inhibition efficiency. When targeting multi-subunit complexes, the inhibition of individual subunits can lead to disruption of the entire complex’s function. The power function relationship shows that higher numbers of subunits can enhance drug effectiveness through coordinated targeting strategies (Shu et al., 2015). Binomial distribution principles have been applied to quantify protein subunits in biological nanoassemblies and functional nanomachines (Figures 8A,B) (Fang et al., 2014). This mathematical approach provides a framework for understanding how subunit organization affects complex function and can inform strategies for targeting multi-subunit systems.

In the binomial mathematical expression (p + q)^Z, p is the fraction of inactive (drugged) subunits, q is the fraction of active (undrugged) subunits, and Z is the stoichiometry (Pi et al., 2016a). Assume a population contains 60% drugged subunits. If Z = 1 (monomer), then p is 0.4, meaning that the number of active motors is equal to the fraction of undrugged subunits 40%). If Z = 6 (hexamer), then (p + q)⁶ equals p6 + 6p⁵q + 15p⁴q² + 20p³q³ + 15p²q⁴ + 6pq⁵ + q⁶. For the hexameric motor, the probability of a motor containing 6 copies of undrugged subunit is q⁶ = 0.4 = 0.004 or 0.4% of all motors in the population remaining active (Figure 8). This is because if one or more subunits are inactive, the whole motor is nonfunctional. Therefore, it is more efficient to target high-stoichiometry machines.

The development of nanoparticles as delivery vehicles for antiviral therapeutic drugs represents another approach to enhancing drug efficacy (Maus et al., 2021). These delivery strategies can improve drug targeting and reduce side effects through controlled delivery mechanisms. As researchers continue to investigate how understanding the stoichiometric properties of biological complexes can inform drug development strategies, the mathematical principles underlying multi-subunit targeting provide insights into potential approaches for developing more effective therapeutic interventions.

1.3.2 A novel method for potent drug development targeting multi-homosubunit biomachines

Recent advances in structural biology, computational methods, and delivery systems have revolutionized our approach to targeting multi-homo subunit molecular machines. These developments enable precise exploitation of the series-circuit vulnerability inherent in both viral packaging motors and ABC transporters, leading to novel therapeutic strategies with enhanced potency and reduced resistance potential.

1.3.3 Structure-based targeting of sequential motor mechanisms

Advanced structural biology techniques have provided detailed insights into the organization and function of various molecular machines. Early sequence analysis identified potential ATPases involved in viral DNA packaging through comparative studies of viral proteins (Koonin et al., 1993). These foundational studies helped establish the presence of conserved ATPase domains across different viral packaging systems.

Multiple molecular machines demonstrate conserved architectural principles across biological systems. Early sequence analysis identified potential ATPases involved in viral DNA packaging through comparative studies of viral proteins (Koonin et al., 1993). The hexameric DnaB helicase, which is essential for bacterial DNA replication, utilizes coordinated mechanisms for DNA processing (Fernandez and Berger, 2021). RecA recombinase plays crucial roles in DNA repair processes in bacteria. It forms a continuous filament around the DNA with six monomers per turn (Cox, 1999). Viral packaging motors, such as the phi29 phage, employ hexameric pRNA rings in their assembly and function (Bohmer et al., 2025; Prokhorov et al., 2024; Zhang et al., 1998). The FtsK DNA translocase performs essential functions in bacterial cell division through its translocation mechanisms (Graham et al., 2010; Lowe et al., 2008).

1.4 Section 4: practical examples of multi- target inhibitors with potent effect

1.4.1 Complete inhibition of bacterial virus phi29 assembly by targeting the hexameric motor pRNA

The phi29 motor operates through a sequential inchworm-like mechanism where each gp16 ATPase subunit must anchor its C-terminal domain to the pRNA’s CCA bulge before its N-terminal domain can engage with DNA (Guo et al., 1986; Prokhorov et al., 2024). As demonstrated in Figures 2D,E, this creates a coordinated hand-over-hand sequential action where DNA must be transferred from one subunit to the next in an obligatory cascade (see Section 1A for mechanism) (Zhao et al., 2016; Liu et al., 2015; Trottier et al., 1996; Chen and Guo, 1997). Therefore, when one subunit is unable to properly bind to pRNA, the DNA cannot be transferred to the next subunit in the sequential chain (Bohmer et al., 2023; Trottier et al., 1996). This dependency is structurally enforced by the asymmetric ring configuration where each subunit must be in the correct conformational state to receive DNA from its neighbor (Guo et al., 1986). Unlike parallel processing systems, this sequential mechanism creates an obligatory dependency between subunits, explaining why inhibition at any point in the cycle results in complete motor stalling rather than just reduced efficiency.

Studies have revealed that while certain pRNA mutants retain their ability to bind to procapsids, they can completely disrupt the packaging mechanism by preventing proper subunit coordination, resulting in complete inhibition of phage infection with 0 plaque-forming units (PFU)/mL compared to wild-type controls showing substantial viral output (up to 2.5 × 10⁴ PFU/mL) (Figure 8F) (Schwartz et al., 2013a; Pi et al., 2016b; Zhou et al., 2006). PFU is the number of infectious phages that form visible plaques in bacterial culture. The potency of this inhibition correlates directly with the stoichiometry of the homo-subunit machine (Figure 8B) (Pi et al., 2016a), with hexamer targeting proving more effective than monomer targeting, and ATP-based inhibition showing exceptionally high potency due to its thousand-fold stoichiometry.

1.4.2 Therapeutic implications of dsRNA packaging motors

The unique features of dsRNA packaging motors display distinct advantages for development of antiviral drugs. The requirement for precise sequential packaging of multi-segmented viral genomes creates numerous potential points of intervention (Borodavka et al., 2018; Gottlieb and Alimova, 2022), while the coupling of packaging and replication processes presents the possibility of simultaneously disrupting multiple aspects of viral assembly (Gottlieb and Alimova, 2022).

The hexameric organization of dsRNA motors, specifically the P4 proteins that form hexameric rings at procapsid vertices (Mancini et al., 2004a; Kainov et al., 2006), makes them potential targets for drug development. Targeting multisubunit complexes with sequential actions has been proposed as an approach for developing potent antiviral drugs (Pi et al., 2016a).

Moreover, the substrate specificity mechanisms of dsRNA motors—such as the purine nucleotide preference observed in P4 proteins (Pirttimaa et al., 2002; Makeyev and Grimes, 2004)—provide opportunities for developing virus-specific inhibitors that would have minimal impact on host cellular processes. The structural differences between viral RNA packaging motors and host RNA-processing machinery offer a therapeutic window for selective targeting.

1.4.3 Targeting ssRNA virus ATPases

AAA + ATPase protein 2C presents an attractive antiviral target that has been successful through the discovery of several drugs that target it. A small molecule screen against enterovirus 2C identified fluoxetine, a selective serotonin reuptake inhibitor, as an effective inhibitor (Zuo et al., 2012). Cryo-EM studies found that fluoxetine works by locking the 2C subunits in a single conformation, disallowing the sequential action required for motor function (Hurdiss et al., 2022). A later study designed fluoxetine derivatives with higher selectivity against CV-B3 and EV-D68 using X-ray crystallographic structures and computational docking (Khemiri et al., 2026). 2C is highly conserved among enteroviruses, plays many essential roles throughout the viral life cycle, and follows the Series-Circuit-Christmas-Bulb mechanism, making it an ideal target for potent antiviral therapy. This is currently an unmet need because the only FDA-approved enterovirus vaccine is for poliovirus, and there are no antiviral drugs to treat enterovirus infections (Hurdiss et al., 2022; Khemiri et al., 2026).

1.4.4 A therapeutic example is bedaquiline to target the F₁F₀ ATPase homo-subunit hexamer for tuberculosis treatment

The therapeutic example of an FDA-approved drug for targeting multiple-homosubunit complexes is the treatment of multidrug-resistant tuberculosis, which targets bacterial F₁F₀ ATPase (Figures 3F, 7D) (Vestergaard et al., 2022; Noji et al., 2017). Mycobacterium tuberculosis is responsible for tuberculosis, which is the global leading cause of mortality from an infectious agent. This bacteria has a unique cell wall that consists of a thick waxy coat with tightly packed layers of complexed sugars, lipids, and peptides, making chemical drug penetration challenging. It requires a very potent drug to penetrate and kill the bacteria. The success of bedaquiline in inhibiting this pathogen demonstrates the power of exploiting high-stoichiometry targets.

1.4.5 Targeting the ABC efflux pump P-gp to inhibit liver cancer

Hepatocellular carcinoma (HCC) is a liver cancer that is the third leading cause of cancer-related death worldwide. The liver’s unique role as a biological filter, combined with cancer cells’ overexpression of drug exporters, has historically complicated HCC treatment. Analogous to a border custom, the liver functions as a filter to detoxify chemicals and metabolites administered orally or intravenously by removing them from the liver cells via efflux pumps. Some liver cancer cells highly overexpress ABC drug exporters that efficiently expel therapeutic drugs from liver cancer cells, leading to chemoresistance and a diminished chemotherapeutic effect. RNA nanotechnology has been used to target the ABC drug efflux pump P-gp. Multivalent RNA nanoparticles, a 6-way junction (6WJ) scaffold (Figure 9), were used to deliver miRNA-122 to treat liver cancer by targeting the P-gp ABC (Wang H. et al., 2021). These nanoparticles can carry multiple therapeutic cargoes while maintaining structural stability, making them especially valuable for treating challenging conditions like HCC by inhibiting P-gp. The authors incorporated three hepatocyte-targeting ligands, one copy of miR122 and 24 copies of paclitaxel, creating a synergistic approach to overcome drug resistance.

The therapeutic impact of this system is particularly evident in the role of miR-122, which is significantly downregulated in liver cancers with intrahepatic metastasis. Restoration of miR-122 substantially reduces tumor progression through multiple mechanisms, including the regulation of ADAM10 (A Disintegrin And Metalloprotease 10) (Wang H. et al., 2021). In vivo studies have demonstrated that RNA nanoparticles effectively accumulate in HCC tumor sites, with an IC50 of 460 nM, while significantly inhibiting tumor growth after multiple IV injections (Wang H. et al., 2021).

Figure 9C demonstrates the efficacy of a novel targeted delivery system combining RNA nanotechnology with engineered exosomes (Ellipilli et al., 2023). The system uses a 4-way junction (4WJ) RNA nanoparticle designed to carry 24 copies of paclitaxel (PTX) and one copy of miR122, which is then loaded into exosomes decorated with GalNAc ligands (Figure 9C). This design enables specific targeting of liver cancer cells through GalNAc binding to overexpressed asialoglycoprotein receptors (ASGP-R). The stepwise assembly process, shown through gel electrophoresis, confirms the successful construction of both the 4WJ/PTX/miR122 complex and the Gal/3WJ surface modifications. This multi-specific approach demonstrated enhanced therapeutic efficacy in both cell culture and mouse xenograft studies, with reduced effective drug doses indicating improved efficiency and lower toxicity, particularly for treating challenging cancers like HCC.

2 Concluding remarks and perspectives

This comprehensive analysis of over 30 ATPase systems across eight categories reveals noteworthy parallels between dsDNA/dsRNA viral genome packaging motors and ABC transporters in both structure and function (Table 1). Targeting homosubunits of these motors achieves potent inhibition through the Series-Circuit-Christmas-Bulb mechanism (Z > 1, K = 1), where disrupting one subunit terminates the entire functional chain. Clinical validation of this approach includes FDA-approved bedaquiline, which targets bacterial F₁F₀ ATPase to treat tuberculosis (Vestergaard et al., 2022; Courbon et al., 2023). The mathematical framework of X = (p + q)^z demonstrates how stoichiometric targeting, compared to traditional single-target approaches, dramatically enhances the potency of drugs while reducing the potential for resistance.

Advances in structural biology and computational methods have revolutionized selective targeting potential, with studies unveiling conserved features persisting despite sequence mutations. RNA nanotechnology, particularly the stable three-way junction (3WJ) motif from bacteriophage phi29 ¹, imparts versatile delivery platforms that can be engineered into various geometries for multifunctional therapeutic delivery. Machine learning algorithms trained on structural and functional data have identified common sequential activation patterns, facilitating the design of sophisticated inhibitors targeting multiple circuit aspects simultaneously (Piazza et al., 2020; Haas et al., 2023).

Critical challenges include understanding conformational changes during motor activity and predicting resistance pathways. Dual-targeting strategies could generate synergistic effects by simultaneously addressing viral packaging and drug efflux systems, particularly with RNA nanotechnology-based delivery, distinguishing host from pathogen machinery. Modern antiviral development increasingly exploits sequential activation mechanisms, exemplified by Allosteric HIV-1 Integrase Inhibitors (ALLINIs) disrupting normal processes through induced aggregation (Kessl et al., 2012).

Notably, ABC transporter homosubunit organization may differ from other classes of ATPase motors. While ABC channels contain multiple similar domains, most consist of two subunits with each contributing multiple targetable domains. However, some, such as MacAB–TolC and EcMlaD, contain hexameric rings as part of their assemblies (Dutta and Kanaujia, 2024; Fitzpatrick et al., 2017). Motors with this arrangement have the potential to become high-efficiency drug targets utilizing the series-circuit mechanism.

Correction note

A correction has been made to this article. Details can be found at: 10.3389/frnar.2026.2009050.

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Footnotes

Edited by: Feng Jiang, Stanford University, United States

Reviewed by: Ismael Bustos-Jaimes, National Autonomous University of Mexico, Mexico

Kasturika Shankar, University at Buffalo, United States

Footnotes

ABC, ATP-binding cassette; pRNA, packaging/prohead RNA; PFU, plaque-forming unit; APM, asymmetric promoter movement; 3WJ, 3-way junction; 4WJ, 4-way junction; 6WJ, 6-way junction; PTX, paclitaxel; ASGP-R, asialoglycoprotein receptor; ALLINI, allosteric HIV-1 integrase inhibitor; EcMlaD, Escherichia coli MlaD; FtsK, filament temperature sensitive mutant K; APMV, Acanthamoeba polyphaga mimivirus.