Structural bioinformatics of viral replication factory formation and organelle remodeling

1. Introduction

Viral replication factories are specialized intracellular compartments that concentrate viral components and shield replication intermediates from host innate immune detection [1, 2]. These structures arise through extensive remodeling of host organelle membranes and cytoskeletal networks, a process coordinated by virally encoded nonstructural and structural proteins [1, 3]. The study of replication factory architecture has been transformed by structural bioinformatics, which integrates cryo-electron microscopy (cryo-EM), X-ray crystallography, molecular dynamics simulations, and integrative modeling to resolve macromolecular complexes at near-atomic resolution [4, 5, 6]. This review focuses on the structural and computational principles governing double-membrane vesicle (DMV) formation, membrane-anchoring viral proteins, and replication complex assembly in veterinary viral pathogens. The mechanisms described have direct relevance to African swine fever virus (ASFV), bluetongue virus (BTV), rotavirus, orthoreovirus, and piscine orthoreovirus (PRV), among others [7, 8, 9].

2. Double-membrane vesicles and membrane remodeling

Double-membrane vesicles are a hallmark of replication factories for diverse RNA and DNA viruses [1, 10]. These structures arise from the endoplasmic reticulum (ER) or other endomembrane compartments and provide a protected environment for viral RNA synthesis [11, 12]. The ER is the primary source of membrane for DMV biogenesis in many viral systems. For positive-sense RNA viruses, nonstructural proteins insert into the ER membrane, inducing curvature and vesicle budding into the lumen [11, 13]. The resulting DMVs contain viral replication complexes on their interior surfaces, with the double bilayer acting as a physical barrier against cytosolic pattern recognition receptors [10, 2].

For feline calicivirus (FCV), the nonstructural proteins p32, p39, and p30 localize to the ER and function as integral membrane proteins that drive membrane rearrangements [11]. Bioinformatics analysis of these sequences identified transmembrane domains and ER-targeting motifs that direct the formation of vesicular structures resembling replication complexes [11]. Similarly, the ASFV inner envelope protein pE146L induces perinuclear ER aggregation and is essential for viral factory formation [7]. The high-resolution crystal structure of the pE146L soluble region revealed a dimer stabilized by intermolecular disulfide bonds (C103), and disruption of these bonds abrogated ER aggregation and viral replication [7]. These findings illustrate how structural bioinformatics can map functional residues critical for membrane remodeling.

3. Membrane-anchoring viral proteins and replication complex assembly

Membrane anchoring of viral replication proteins is achieved through transmembrane domains, lipid modifications, or monotopic insertion [5, 11]. The alphavirus nsP1 protein, a monotopic membrane-associated capping enzyme, assembles into a dodecameric ring that controls access to the viral replication organelle [5]. Cryo-EM structures revealed that nsP1 oligomerization is coupled to membrane binding and allosteric activation, redefining the replication complex as an RNA synthesis reactor with 12 active sites per complex [5]. This structural arrangement ensures that only properly capped viral RNA exits the membranous niche [5].

In reoviruses, the nonstructural protein μNS (orthoreovirus) or NS80 (aquareovirus) forms the matrix of viral factories [8, 14, 15]. For mammalian orthoreovirus, μNS self-assembles into large cytoplasmic inclusions that recruit core proteins such as μ2, σNS, and λ1 [8, 15]. The NS80 protein of aquareovirus possesses distinct domains for self-aggregation and recruitment of structural proteins VP1, VP2, VP3, VP4, and nonstructural protein NS38 [14]. Bioinformatics dissection of the NS80 sequence identified that residues 506 to 742 are required for cytoplasmic aggregation, while the N-terminal region (aa 1-55) is necessary for NS38 recruitment [14]. These interaction maps are constructed using yeast two-hybrid assays and validated by confocal microscopy, providing a structural interaction network for the factory [14].

For rotavirus, the octameric NSP2 protein serves as an RNA chaperone and ATPase that is essential for viroplasm formation [6, 16, 17]. X-ray crystallography of NSP2 revealed a deep cleft between the C-terminal and N-terminal domains that houses the NTPase active site, with critical histidine residues (His221, His225) analogous to the histidine triad (HIT) hydrolase motif [17]. The K294E mutation in the NSP2 C-terminus stabilizes a rare conformational state and reduces the capacity for robust viroplasm formation, as shown by molecular dynamics simulations [6]. This mutation shifts the conformational ensemble without altering the overall octameric fold, illustrating how single-amino-acid changes impact factory biogenesis [6].

4. Liquid-liquid phase separation and biomolecular condensates

A growing body of evidence indicates that many viral replication factories are biomolecular condensates formed through liquid-liquid phase separation (LLPS) [18, 16, 19]. Rotavirus viroplasms are paradigm examples. The nonstructural proteins NSP2 (an RNA chaperone) and NSP5 (an intrinsically disordered protein) undergo phase separation to form condensates that concentrate viral RNA and other replication factors [16, 20, 19]. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and native mass spectrometry revealed that within these condensates, NSP2 exhibits altered conformational dynamics in its C-terminal region, which regulates its RNA chaperoning activity [16]. The disordered regions of NSP5 also show reduced flexibility within the condensed state, likely facilitating client recruitment and condensate assembly [16].

For norovirus, the RNA-dependent RNA polymerase (RdRP) forms amyloid-like fibrils via an LLPS-to-solid transition, as shown by Thioflavin-T fluorescence, Congo red binding, and transmission electron microscopy [21]. Bioinformatics analysis of the RdRP sequence using multiple web-based servers identified aggregation-prone hot spots that promote amyloid formation [21]. These findings suggest that the RdRP may provide a platform for sequestering nonstructural proteins and viral RNA to form replication factories during infection [21].

5. Cytoskeletal reorganization and organelle subversion

Viral replication factories are often enmeshed in a cage of cytoskeletal elements, including actin, microtubules, and intermediate filaments [1, 22]. For mammalian reovirus, the μ2 protein functions as a microtubule-associated protein (MAP) that stabilizes microtubules and determines whether inclusion bodies assume filamentous or globular morphology [23]. A single amino acid difference at position 208 of μ2 (Pro208 versus Ser208) dictates MT association and inclusion morphology, as demonstrated by reassortant analysis and site-directed mutagenesis [23].

In ASFV infection, vimentin intermediate filaments undergo rearrangement through retrograde transport along microtubules, a process dependent on phosphorylation by calcium calmodulin kinase II [22]. The resulting cage surrounds viral factories and is thought to provide structural support [1, 22]. Super-resolution STED microscopy combined with Click IT chemistry revealed that the ASFV factory contains a reticular network of newly synthesized structural proteins (p54, p34) embedded in a complex of membrane assembly intermediates [3]. Electron tomography showed that these intermediates progress from small membrane fragments to linked structures and finally to icosahedral virions [3].

Gamma-carrageenan, a sulfated polysaccharide, inhibits ASFV infection through multiple mechanisms, including suppression of viral factory formation [24]. Structural analysis using NMR and electrospray mass spectrometry identified that the trisulfated disaccharide units of lambda-carrageenan are required for binding to the outer envelope protein CD2v, thereby disrupting factory formation [24].

6. Three-dimensional visualization of replication complex architecture

Integrative structural bioinformatics combines cryo-EM, X-ray crystallography, and molecular modeling to visualize the macromolecular organization of replication factories [4, 5, 25]. For the piscine orthoreovirus μNS protein, expression in fish cells results in dense globular cytoplasmic inclusions that recruit σNS, μ2, and λ1 proteins, as shown by confocal microscopy [8]. The mammalian orthoreovirus σNS structure, solved by X-ray crystallography and cryo-EM, forms helical filaments stabilized by N-terminal arm domain swapping [4]. These filaments bind RNA within an interior channel and possess RNA chaperone activity that is essential for genome replication [4]. Bile acids disrupt the helical assembly by binding at the N-terminal arm interface, providing a potential mechanism for factory disassembly [4].

For bluetongue virus, the nonstructural protein NS2 forms helical oligomers in the presence of calcium ions [25]. Cryo-EM structures revealed that the N-terminal domain wraps around the C-terminal domain during oligomerization, and calcium binding triggers a helix-to-coil transition that enhances NS2 phosphorylation and VIB assembly [25]. Mutations in the EF-hand-like calcium-binding motif prevent recovery of viable virus by reverse genetics [25].

A decision tree for the structural bioinformatics workflow used to study viral factory formation is presented below.

flowchart TD
    A[Viral protein sequence], > B[Homology modeling / AlphaFold2]
    B, > C{Structural prediction}
    C, >|Membrane protein| D[TM domain prediction / lipid interaction]
    C, >|Soluble protein| E[Crystallography / cryo-EM]
    D, > F[Molecular dynamics simulation]
    E, > F
    F, > G[Conformational ensemble analysis]
    G, > H[LLPS propensity / aggregation prediction]
    H, > I[Validation in cell culture]
    I, > J[Factory morphology assessment]
    J, > K[Structure-based antiviral targeting]

7. Implications for antiviral targeting

Understanding the structural basis of replication factory formation opens avenues for antiviral intervention [7, 24, 26]. The host cyclophilin A (CypA) stabilizes the ASFV capsid protein p72 by reducing its ubiquitination and proteasomal degradation [26]. Cyclic CypA inhibitors destabilize p72, disrupt viral factory formation, and impair virion assembly [26]. The ASFV thioredoxin A151R is expressed early during infection and participates in factory formation, and monoclonal antibodies targeting the epitope 84KIWSGEPR91 provide tools for diagnostic detection [27].

For BTV, the calcium-sensing function of NS2 represents a druggable vulnerability, as mutations in the EF-hand motif abrogate viral viability [25]. Similarly, targeting the clathrin-binding motif of reovirus μNS (711-LIDFS-715) could restore host membrane trafficking functions that are disrupted during infection [28].

8. Conclusion

Structural bioinformatics has provided unprecedented detail into the molecular architecture of viral replication factories. From the dodecameric nsP1 gate of alphaviruses to the phase-separated rotavirus viroplasm, unifying principles of membrane remodeling, protein oligomerization, and cytoskeletal coupling are emerging [1, 5, 19]. The application of integrative structural methods to veterinary viruses such as ASFV, BTV, and PRV promises to accelerate the development of vaccines and antivirals for animal health [7, 3, 8].

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