Virus-First Theory Revisited: Bridging RNP-World and Cellular Life
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe current consensus in virology recognizes viruses as obligate intracellular parasites, yet this does not necessarily imply their evolutionary emergence postdated cellular life. RNA, as a central player in the central dogma, uniquely serves dual roles as both genetic material and catalytic machinery—a property that led pioneers like Alexander Rich and Walter Gilbert to propose the RNA World hypothesis as a cornerstone of life's origin (Nature 319, 618; 1986). The formation of ribonucleoprotein (RNP) complexes likely represented a critical evolutionary leap, offering superior protection for unstable RNA genomes. While nucleic acids are constrained by limited base diversity, the side-chain variability of amino acids (e.g., hydrophobic, polar) enabled unprecedented structural and functional complexity in early RNPs. This amino acid-driven diversification facilitated the emergence of progressively sophisticated RNP assemblies, ultimately giving rise to protein-dominated life forms and culminating in cellular organisms. Strikingly, modern ribosomes—archetypal RNP complexes—retain molecular fingerprints of this evolutionary trajectory, while viral RNPs (e.g., influenza polymerase, HIV nucleocapsid) remain prime targets for antiviral therapeutics due to their conserved mechanistic roles in replication.
The manuscript by Prosdocimi et al. presents a novel and thought-provoking theoretical framework that systematically examines the potential roles of ribonucleoprotein (RNP) complexes and viruses in prebiotic evolution. By synthesizing contemporary evidence from virology, structural biology, and evolutionary biology, the authors construct a compelling chronological narrative spanning five key transitions: (1) RNP condensate formation, (2) emergence of a primitive translation system (FUCA), (3) differentiation of progenotes, (4) genesis of virus-like particles, and (5) development of cellular membranes. Particularly noteworthy is their rigorous analysis of simple viral capsid architectures, which offers fresh perspectives on how geometric constraints and symmetry principles could have guided early molecular evolution. While the study remains entirely hypothetical—lacking empirical validations or clearly falsifiable predictions—its conclusions are well-anchored in cited literature, providing valuable insights into critical transitional phases during life's origin. The arguments demonstrate strong scientific coherence and logical consistency throughout. However, as a theoretical contribution, the manuscript would benefit significantly from enhanced visual representations. We recommend: Incorporating schematic diagrams to illustrate proposed RNP-virus evolutionary trajectories Adding comparative models of FUCA systems versus modern translation machinery Including molecular dynamics simulations of prebiotic condensate formation. With these refinements to improve pedagogical accessibility, the work merits publication as a provocative foundation for future experimental testing. The framework's interdisciplinary approach could stimulate new research directions in origins-of-life studies and early virology.
Major comments
- If the virus-first hypothesis holds, the manuscript should address how endogenous viral elements (e.g., Human Endogenous Retroviruses, HERVs) support this model?
- Provided a table or figure comparing key hypotheses (e.g., Patrick Forterre’s virocell concept, RNA world, metabolism-first) would clarify?
- Can the authors elaborate on why certain proteins specifically bind to defined RNA sources? For instance, how viral capsid proteins selectively form RNPs with viral RNA rather than cellular RNA, and whether this specificity relates to RNA structure and chemical modifications.
- Can the authors supplement discussion on experimental validation of key theoretical steps, such as whether the transition from RNP condensates to capsid structures can be observed under simulated early Earth conditions?
- Can the authors discuss how to reconcile the high similarity between viral envelope components and cellular/organelle membranes, given that viruses may have predated or co-emerged with cellular ancestors? Specifically, if viruses borrowed host membranes for envelope formation, how would this mechanism align with a virus-first or co-origin scenario?
- Under the virus-first hypothesis, should non-enveloped viruses theoretically predate enveloped viruses in evolutionary origin? Could the authors provide supporting evidence or theoretical foundations for this claim?
Minor comments
- The capsid self-assembly schematic in Figure 1 could be enhanced with additional molecular-level details.
- Incorporate a time axis and markers for key transitional intermediates in Figure 2.
- Consider revising "In conclusion" since Section 7 already contains a "Conclusion" heading, which may cause confusion for readers
Author Response
> The current consensus in virology recognizes viruses as obligate
> intracellular parasites, yet this does not necessarily imply their
> evolutionary emergence postdated cellular life.
We agree with the reviewer’s observation that the current consensus in virology—recognizing viruses as obligate intracellular parasites—does not inherently imply that viruses originated after cellular life. In fact, our theoretical model was designed precisely to discuss and advance the thoughts on that assumption. We argue that viruses, or virus-like particles, could have originated independently in the prebiotic world as ribonucleoprotein-based assemblies with rudimentary protective and catalytic functions, prior to the emergence of fully cellular organisms.
However, rather than viewing viral parasitism as a defining and ancestral trait, we propose that it is a derived feature resulting from evolutionary specialization. In our framework, early capsid-forming ribonucleoprotein complexes may have played a key role in stabilizing and concentrating early RNA-based genomes in aqueous environments, enhancing replication and molecular cooperation. These structures did not require cellular hosts in the modern sense but could have existed within complex networks of pre-cellular interactions—what we describe as the progenote phase of life’s early evolution.
Thus, we emphasize that the intracellular parasitism seen in modern viruses should not be projected backward as a requirement for viral origin. Even if this is not the current consensus in virology, it is indeed somehow defended by many researchers. Instead, our virus-first proposal considers the possibility that viral-like compartments evolved prior to cells and contributed structurally and functionally to the formation of cellular lineages, particularly through their influence on the evolution of proteolipidic encapsulation.
> RNA, as a central player in the central dogma, uniquely serves dual roles as
> both genetic material and catalytic machinery—a property that led pioneers
> like Alexander Rich and Walter Gilbert to propose the RNA World hypothesis
> as a cornerstone of life's origin (Nature 319, 618; 1986).
> The formation of ribonucleoprotein (RNP) complexes likely represented a
> critical evolutionary leap, offering superior protection for unstable RNA
> genomes. While nucleic acids are constrained by limited base diversity, the
> side-chain variability of amino acids (e.g., hydrophobic, polar)
> enabled unprecedented structural and functional complexity in early RNPs.
> This amino acid-driven diversification facilitated the emergence of
> progressively sophisticated RNP assemblies, ultimately giving rise to
> protein-dominated life forms and culminating in cellular organisms.
> Strikingly, modern ribosomes—archetypal RNP complexes—retain
> molecular fingerprints of this evolutionary trajectory, while viral RNPs
> (e.g., influenza polymerase, HIV nucleocapsid) remain prime targets
> for antiviral therapeutics due to their conserved mechanistic roles in replication.
We thank the reviewer for this insightful synthesis, which captures the central logic and motivation behind our theoretical framework. We are fully in agreement with the idea that RNA’s dual role as both genetic material and catalytic molecule was a critical feature of the earliest stages of life, as highlighted in the RNA World hypothesis. Our model directly builds upon this premise by proposing that ribonucleoprotein (RNP) complexes constituted a key transitional stage in prebiotic evolution.
As the reviewer correctly points out, the combinatorial richness of amino acid side chains allowed for the structural and functional diversification of RNP assemblies, ultimately leading to increased molecular complexity and the emergence of systems on which the nucleic acids and peptides perform a molecular symbiosis. In our view, this structural innovation was not only essential for the stabilization of fragile RNA genomes but also laid the foundation for the evolution of self-organizing compartments—including capsids and later proteolipidic membranes.
We particularly appreciate the reviewer’s reference to modern ribosomes and viral RNPs, which indeed serve as molecular “fossils” of this evolutionary trajectory. These examples reinforce the notion that RNP systems remain central to the continuity of biological processes, from the earliest stages of life to current antiviral strategies. This perspective aligns deeply with the arguments we present throughout the manuscript.
> Major comments
>
> If the virus-first hypothesis holds, the manuscript should address
> how endogenous viral elements (e.g., Human Endogenous Retroviruses, HERVs)
> support this model?
We thank the reviewer for raising this thoughtful point. While Human Endogenous Retroviruses (HERVs) offer fascinating insights into virus-host co-evolution, we believe they are not directly relevant to the context of life’s origins or the virus-first hypothesis as we propose it.
HERVs are found in humans—a highly derived and complex eukaryotic lineage that is temporally and biologically distant from any pre-cellular or prebiotic stage of evolution. As such, they reflect evolutionary processes that occurred long after the establishment of cellular life and do not bear directly on the fundamental question of how viruses may have originated prior to or alongside cells.
Therefore, we do not consider the relationship between HERVs and the virus-first hypothesis to be conceptually appropriate. Nonetheless, in order to acknowledge the reviewer’s comment and to offer context for interested readers, we have added the following clarifying sentence to the end of Section 6:
“An important consequence of the long-standing relationship between viral and cellular lineages is the existence of endogenous viral elements (EVEs), such as Human Endogenous Retroviruses (HERVs), which now constitute over 8% of the human genome. These elements are molecular fossils of ancient viral infections and provide compelling evidence for the deep integration of viral functions within cellular genomes. Their persistence and co-evolution with host genomes reinforce the plausibility that viruses were present from the earliest stages of cellular evolution, consistent with the virus-first model. However, while endogenous retroviruses are often cited as evidence of virus-host genetic integration, most of them probably represent relatively recent events in genomic evolution and are not directly informative about pre-cellular stages or the origin of viruses themselves.”
This distinction helps avoid conflating late-stage genomic fossilization events with early evolutionary innovation.
> Provided a table or figure comparing key hypotheses (e.g., Patrick Forterre’s
> virocell concept, RNA world, metabolism-first) would clarify?
Thank you for this valuable suggestion. In response, we have added a concise overview of the major alternative hypotheses at the end of the Introduction section, highlighting their main propositions and limitations. To complement this discussion, we also included a new comparative table (Table 1) summarizing the core features, strengths, and weaknesses of the Virus-First, RNA World, Metabolism-First, and Virocell hypotheses. We hope this addition enhances the clarity and contextual grounding of our work.
> Can the authors elaborate on why certain proteins specifically bind to defined
> RNA sources? For instance, how viral capsid proteins selectively form RNPs with
> viral RNA rather than cellular RNA, and whether this specificity relates to
> RNA structure and chemical modifications.
Thank you for this thoughtful and insightful question. Viral capsid proteins often display high specificity for viral RNA due to the presence of conserved packaging signals—such as stem-loop motifs, unique sequence elements, or chemically modified bases—that are recognized by the protein’s RNA-binding domains (Aksyuk & Rossmann, 2011). These interactions are typically mediated through electrostatic complementarity, hydrogen bonding, and spatial conformations that have co-evolved with the viral genome. This co-evolution ensures that capsid proteins preferentially encapsulate self-RNA over host RNA, contributing to efficient and selective viral assembly (Twarock et al., 2018).
That said, we believe that such specificity is likely a later evolutionary refinement. During the earliest stages of molecular evolution—especially in a pre-cellular or RNP-dominated context—RNA-binding proteins likely exhibited broader, less discriminating affinities. The high specificity observed in modern viruses probably emerged alongside increasingly complex interactions with host organisms, where selective packaging became advantageous for survival and replication.
We chose not to include a detailed discussion of this topic in the manuscript itself, as we feel it may introduce unnecessary complexity and potentially confuse readers regarding the scope and aims of our theoretical proposal. Nevertheless, we are grateful for the suggestion, which highlights an important evolutionary consideration in the virus-cell transition.
Aksyuk, A. A., & Rossmann, M. G. (2011). Bacteriophage assembly. Viruses, 3(3), 172–203. https://doi.org/10.3390/v3030172
Twarock, R., Bingham, R. J., Dykeman, E. C., & Stockley, P. G. (2018). A modelling paradigm for RNA virus assembly. Current Opinion in Virology, 31, 74–81. https://doi.org/10.1016/j.coviro.2018.07.003
> Can the authors supplement discussion on experimental validation of key
> theoretical steps, such as whether the transition from RNP condensates
> to capsid structures can be observed under simulated early Earth conditions?
Thank you for raising this important point. As this manuscript presents a theoretical framework, we hope it will inspire future experimental efforts to test some of the proposed transitions, particularly the emergence of capsid-like structures from ribonucleoprotein (RNP) condensates under prebiotic conditions.
Recent progress in in vitro systems and computational modeling has provided encouraging results that support the plausibility of such transitions. For example, self-assembling peptides and RNA have been shown to form phase-separated condensates through liquid-liquid phase separation (LLPS), which can serve as functional, membrane-less compartments (Franzmann et al., 2018; Dignon et al., 2020). Additionally, simulations and experimental models demonstrate that these condensates can adopt organized, shell-like architectures, suggesting a potential pathway for the emergence of capsid-like enclosures (Perlmutter & Hagan, 2015).
While these systems do not yet replicate early Earth conditions in their entirety, they offer promising avenues for experimental validation of the transitions we propose—from RNP condensates to primitive capsids.
We have added a brief mention of this potential in Section 4 to acknowledge the feasibility of such investigations. We hope that our model encourages researchers to pursue experimental approaches that could test these ideas under prebiotically relevant settings.
Dignon, G. L., Best, R. B., & Mittal, J. (2020). Biomolecular Phase Separation: From Molecular Driving Forces to Macroscopic Properties. Annual review of physical chemistry, 71, 53–75. https://doi.org/10.1146/annurev-physchem-071819-113553
Franzmann, T. M., Jahnel, M., Pozniakovsky, A., Mahamid, J., Holehouse, A. S., Nüske, E., Richter, D., Baumeister, W., Grill, S. W., Pappu, R. V., Hyman, A. A., & Alberti, S. (2018). Phase separation of a yeast prion protein promotes cellular fitness. Science (New York, N.Y.), 359(6371), eaao5654. https://doi.org/10.1126/science.aao5654
Perlmutter, J. D., & Hagan, M. F. (2015). Mechanisms of virus assembly. Annual review of physical chemistry, 66, 217–239. https://doi.org/10.1146/annurev-physchem-040214-121637
> Can the authors discuss how to reconcile the high similarity between viral
> envelope components and cellular/organelle membranes, given that viruses
> may have predated or co-emerged with cellular ancestors?
> Specifically, if viruses borrowed host membranes for envelope formation,
> how would this mechanism align with a virus-first or co-origin scenario?
Thank you for this thoughtful and challenging question. We believe that the reviewer’s formulation reflects the traditional paradigm in which viruses are thought to have originated from cells, hence assuming that viral envelopes must have been derived by borrowing host membranes. However, from a virus-first or co-origin perspective, the direction of inheritance is reversed: it is the cells that may have inherited membrane features from ancient virus-like entities.
In our view, viral capsids may have initially evolved to bind and stabilize RNA through ribonucleoprotein interactions, and later, certain capsid proteins may have developed affinity for hydrophobic molecules such as lipids. This capacity to recruit lipids could have conferred selective advantages—e.g., in forming more stable compartments or facilitating interactions with prebiotic environments—thereby leading to the evolution of proteolipidic structures before fully developed cellular membranes.
We have revised Section 6 to clarify this perspective with the following paragraph:
“While many modern viruses acquire their lipid envelopes by budding through host membranes, it is plausible that ancestral capsid-like structures evolved the ability to bind lipid components independently. Such proteolipidic intermediates may have predated fully cellular membranes and represent a transitional form.”
This explanation supports the idea that the resemblance between viral envelopes and cellular membranes is not necessarily due to borrowing, but rather due to shared ancestry or evolutionary convergence in encapsulation strategies.
> Under the virus-first hypothesis, should non-enveloped viruses theoretically
> predate enveloped viruses in evolutionary origin? Could the authors provide
> supporting evidence or theoretical foundations for this claim?
Thank you for raising this important point. Under the virus-first hypothesis, we indeed consider that non-enveloped viruses likely predate enveloped ones. Non-enveloped viruses are structurally simpler and energetically more feasible in early prebiotic contexts.
In contrast, enveloped viruses depend on lipid bilayers, which either derive from host membranes or require lipid-binding protein domains and biosynthetic pathways. Such dependencies suggest a more advanced molecular context—likely reliant on pre-existing host-like systems or proteolipidic intermediates. Therefore, non-enveloped viruses are more consistent with a primordial stage in the emergence of biological compartmentalization.
That said, we acknowledge that viral evolution is dynamic and ongoing. New enveloped and non-enveloped viral forms continue to arise through mutation, recombination, and horizontal gene transfer. Structural simplicity or complexity alone does not determine evolutionary timing in current biosystems. Hence, while our model proposes that non-enveloped architectures are ancestral, modern non-enveloped viruses are not necessarily ancient lineages, but may include relatively recent innovations (Koonin et al., 2020).
A paragraph with this discussion was added to Discussion.
Reviewer 1 – Minor Comments:
> The capsid self-assembly schematic in Figure 1 could be enhanced with
> additional molecular-level details.
Thank you for this comment. We agree that such details would improve the figure; however, at this stage we are not equipped to produce a more detailed molecular-level illustration. We hope the current scheme conveys the conceptual progression of the text effectively.
> Incorporate a time axis and markers for key transitional intermediates in Figure 2.
We considered this thoughtful suggestion, but ultimately decided not to include a time axis, as any attempt to assign temporal estimates to these transitions would be highly speculative and may not strengthen the theoretical framework we propose.
> Consider revising "In conclusion" since Section 7 already contains a "Conclusion"
> heading, which may cause confusion for readers
Thank you for pointing this out. We have removed the phrase “In conclusion” and incorporated the excerpt into the final sentence of the manuscript to improve clarity and avoid redundancy.
We sincerely thank Reviewer 1 for the exceptional depth, clarity, and scholarly insight offered in their comments. Their suggestions greatly enriched our manuscript—from conceptual refinements in our virus-first framework to the addition of comparative models, evolutionary references, and experimental perspectives. In particular, we appreciated the reviewer’s emphasis on the RNA–peptide world, ribonucleoprotein evolution, and the integration of viral endogenous elements, all of which helped us refine and expand key sections of the text. Their encouragement to clarify mechanistic details and contextualize our proposal alongside current virology paradigms significantly strengthened both the structure and scientific value of our work. We are grateful for this thoughtful and constructive engagement.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis essay addresses the origin of modern cells as one of the key challenges in abiogenesis. Unfortunately, much of the work consists of self-citations. Nearly every claim in the article is followed by references to the same authors' previous publications, while the number of novel propositions not discussed previously remains limited and no new data is presented. While a few arguments are presented in support of the proposed hypothesis, there is no comparison with competing theories or similiar but different concepts, no discussion of their contradictions, and no exploration of potential methods to test the hypothesis.
The paper hypothesizes a role for T=3 symmetry capsids, yet it fails to examine or dismiss alternative possibilities. For instance, why not consider T=1 capsids? Some of discussed folds (SJR) are hypothetized previously to originate from cellular ancestors, do authors disagree with these hypothesis or argue that the ancestral capsids are compltely extinct? Were these primordial capsids stable or metastable, how did they help RNA to replicate? Are the intermediates between protein capsids given as examples and lipid bilayers possible despite the different monomer binding principles?
Author Response
> This essay addresses the origin of modern cells as one of the key challenges
> in abiogenesis. Unfortunately, much of the work consists of self-citations. Nearly
> every claim in the article is followed by references to the same authors'
> previous publications, while the number of novel propositions not discussed
> previously remains limited and no new data is presented.
The reviewer is partially right. We acknowledge that several references in the manuscript are indeed to our own previous work. This is primarily because our theoretical framework on pre-cellular evolution has been developed progressively over a series of studies, building a coherent model over time.
However, we would like to emphasize that the manuscript contains 99 references, with 15 being ours, meaning that approximately 85% of all citations refer to works by other authors. In this new version, we have included additional references, incorporating relevant contributions from many other renown authors in the field.
> While a few arguments are presented in support of the proposed hypothesis, there
> is no comparison with competing theories or similiar but different concepts,
> no discussion of their contradictions, and no exploration of potential methods
> to test the hypothesis.
To cope with this critique, we have now added a new comparative section in Introduction as well as a table (Table 1) summarizing the main features, strengths, and limitations of competing theories, including the RNA World, Metabolism-First, Virocell, and Virus-First models. This addition provides a more balanced perspective and helps contextualize our approach within the broader landscape of origin-of-life research.
We also added in Chapter 4 some ideas about how a specific hypothesis, i. e., the transition between LLPS-based systems to capsids could be better tested and explored.
“Recent progress in in vitro systems and computational modeling has provided encouraging results that support the plausibility of the transition from LLPS-like compartments to capsid structures. For example, self-assembling peptides and RNA have been shown to form phase-separated condensates through liquid-liquid phase separation (LLPS), which can serve as functional, membrane-less compartments (Franzmann et al., 2018; Dignon et al., 2020). Additionally, simulations and experimental models demonstrate that these condensates can adopt organized, shell-like architectures, suggesting a potential pathway for the emergence of capsid-like enclosures (Perlmutter & Hagan, 2015). While these systems do not yet replicate early Earth conditions, they offer promising avenues for experimental validation of the transitions from RNP condensates to primitive capsids.”
> The paper hypothesizes a role for T=3 symmetry capsids, yet it fails to
> examine or dismiss alternative possibilities. For instance, why not
> consider T=1 capsids?
We sincerely thank the reviewer for this insightful observation. Until now, we had not considered the relevance of T=1 icosahedral capsids, and we greatly appreciate the opportunity to expand our discussion. Based on this suggestion, we revised Section 4.1 to include a discussion of T=1 capsids, such as those found in satellite viruses and Circoviridae. These represent some of the simplest and most genetically economical viral architectures known. We now acknowledge their evolutionary plausibility as early encapsulation systems and contrast them with T=3 capsids, which—despite being structurally more complex—offer increased internal volume and architectural stability, potentially making them more suitable for the emergence of larger and functionally diverse prebiotic compartments. This addition strengthens our theoretical model and highlights the potential continuum between T=1 and T=3 capsid-based encapsulation strategies during the early stages of biological evolution.
> Some of discussed folds (SJR) are hypothetized previously to originate from
> cellular ancestors, do authors disagree with these hypothesis or argue that the
> ancestral capsids are compltely extinct?
We thank the reviewer for raising this important and thought-provoking question. We fully agree that many protein folds, including SJR (Single Jelly-Roll), likely emerged before the origin of cells. Regarding the possibility that ancestral capsids are now completely extinct, we believe this cannot be definitively answered. However, the persistence of extant viral families that utilize simple capsid architectures built from a single gene product—particularly those with T=1 and T=3 icosahedral symmetries—strongly suggests that some of these ancient structural strategies have survived and remain in use across diverse viral lineages.
As for the question of which specific proteins may have constituted the earliest proteolipidic membranes, we recognize this as a fascinating and challenging area of research. Identifying such ancestral membrane proteins would require extensive comparative genomics and bioinformatic analyses, and even then, may not yield conclusive results due to the vast evolutionary distances and potential for convergent evolution. Nonetheless, we agree that it would be a valuable and worthwhile line of investigation.
With respect to the SJR fold specifically, we have clarified in Section 4.1 that we do not assume these folds are extinct. Rather, we suggest the possibility of multiple independent origins of simple capsid-like proteins, possibly including SJR folds.
> Were these primordial capsids stable
> or metastable, how did they help RNA to replicate?
We thank the reviewer for raising this important question, which touches on the central functional role of early capsid-like structures in prebiotic systems. In response, we have added a clarifying sentence to the revised manuscript, noting that primordial capsids were most likely metastable rather than rigidly stable structures. Their primary evolutionary advantage may not have been long-term structural integrity, but rather the transient protection and compartmentalization of RNA genomes in fluctuating environments.
These metastable protein shells could have shielded RNA from degradation and environmental fluctuations during transit between microenvironments. Upon arrival at locations containing FUCA-like progenotes with rudimentary translational machinery, the metastable nature of the capsid could have facilitated spontaneous disassembly, thereby releasing the encapsulated RNA at the appropriate site for replication and translation. In this view, early capsids would have functioned not only as protective vessels but also as delivery systems—contributing to a primitive spatial regulation of molecular processes.
Additionally, by increasing the local concentration of genetic material and associated factors, these primitive capsids could have enhanced the kinetics of template replication or interactions with ribonucleoprotein partners. Although these early structures likely lacked sophisticated control over genome release, their selective advantage would have emerged from improving the chances of successful replication cycles in the highly dynamic and heterogeneous prebiotic environment.
We have created Section 4.3 (Metastable Capsids, RNA delivery and the Spatial Regulation of Prebiotic Replication) to include this explanation.
> Are the intermediates
> between protein capsids given as examples and lipid bilayers possible
> despite the different monomer binding principles?
We thank the reviewer for this question. We understand the concern refers to the apparent disparity between the assembly principles of protein-based capsids—typically relying on specific protein-protein interactions and symmetrical geometry—and lipid bilayers, which are based on amphipathic self-assembly driven by hydrophobic interactions. At first glance, the biophysical mechanisms behind these two systems seem incompatible.
However, we propose that plausible intermediates can exist between these two types of compartments, particularly in the form of capsid-like structures that gradually incorporated additional proteins with new functional domains. This process would have involved: (i) the co-option of accessory proteins acting as channels, effectors, or receptors (as already seen in modern viruses); and (ii) the evolution of lipid-binding proteins, which enabled the interaction between the proteinaceous shell and amphipathic molecules available in the environment. This progressive acquisition of lipid-binding capability could have gradually transformed fully proteinaceous shells into proteolipidic enclosures, leading toward membrane-bound systems.
Indeed, many lipid-binding protein families exist today and play key roles in modern biological membranes. These include annexins, BAR domain proteins, PH domain proteins, START domain proteins, fatty acid-binding proteins (FABPs), and caveolins, among others (Sezgin et al., 2017). Such proteins illustrate the evolutionary feasibility of bridging protein-only compartments and lipid-based systems.
Additionally, we now mention in two paragraphs added to Section 5 that modern enveloped viruses (e.g., poxviruses and certain archaeal viruses) use both capsid proteins and lipid envelopes, which may reflect evolutionary intermediate states. These viruses demonstrate that protein-lipid hybrids are not only structurally possible, but biologically functional.
Thus, while the principles behind monomeric assembly in proteins and lipids differ, they are not mutually exclusive. We propose that the emergence of proteolipidic compartments was a gradual and modular process, facilitated by the evolution of proteins capable of interfacing with lipids. This reconciles the mechanistic gap between rigid capsids and fluid membranes, supporting the plausibility of intermediates between the two systems.
We would like to sincerely thank Reviewer 2 for their critical and detailed assessment of our manuscript. Their comments helped us clarify the scope of our theoretical contribution, improve the scholarly balance of our citations, and strengthen the manuscript’s connections with broader origin-of-life research. In particular, we appreciated the reviewer’s insistence on incorporating comparative perspectives, alternative hypotheses, and more diverse references—points that prompted substantial improvements, including the addition of a new comparative section and table in the Introduction.
The reviewer’s suggestions regarding capsid symmetry (T=1 vs. T=3), the plausibility of intermediate encapsulation systems, and the biochemical logic behind membrane evolution all contributed significantly to refining our central arguments. Their questions also pushed us to better articulate the functional role of metastable capsids and the transitions from RNP condensates to proteolipidic compartments. We are grateful for the opportunity to expand on these key topics and for the insights that helped enhance both the theoretical depth and clarity of the work.
Overall, we found Reviewer 2’s thoughtful feedback to be both challenging and constructive, and we thank them sincerely for their contribution to improving the manuscript.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis is an interesting paper that indicates that the first viruses, translation systems, coding, metabolism and coacervates (membraneless organelles) coevolved. The suggestion is made that cell envelopment in membranes may have occurred as a late stage in forming the first cells. The evidence for membranes late is that membrane systems are very different for Archaea and Bacteria.
I have a few suggestions for the authors consideration.
In the discussion of pseudoicosahedral virus capsid evolution, the authors should take an example from Archaea early in the discussion. Late in the discussion, a reference is given indicating that Archaea utilize such viruses. Archaea appear to be closest organisms to LUCA, so an example from Archaea would be useful to a reader to indicate that spherical virus coats could be as old as LUCA.
This reviewer thinks a reference to the Muller paper (reference 4 below) would be appropriate in discussing the RNA-peptide world.
The authors might consider other polymers (i.e., peptidoglycan) to encapsulate the first cells.
There are models for coevolution of coding and coacervates (references are below).
The paper does not appear to stress the relationship of coacervates to concentration of metabolism and coding factors.
How do the authors account for evolution of protein synthesis: tRNA, AARS enzymes, protoribosomes, first proteins, ribosomes?
Coacervates can concentrate metabolism and coding components, partially delaying the need for membrane (or virus capsid) encapsulation.
Are the authors suggesting that a small spherical viral capsid could enclose a functional protocell unit or a complex autocatalytic cycle?
Minor points:
Abstract: should be understood as a distinct class of ribonucleoprotein (RNP) systems, some of them
Viruses appear to be as old as LUCA (Moody paper referenced in paper draft)
Line 162: together with other ions ans inorganic molecules necessary for primitive metabolism.
Since an icosahedron has 219 12 vertices and 20 faces, the proteins distribute as follows: Because would be better English style
Author Response
> This is an interesting paper that indicates that the first viruses, translation systems,
> coding, metabolism and coacervates (membraneless organelles) coevolved. The
> suggestion is made that cell envelopment in membranes may have occurred as a late
> stage in forming the first cells. The evidence for membranes late is that membrane
> systems are very different for Archaea and Bacteria.
We are grateful for this summary and fully agree with the reviewer’s interpretation. Our intention has been to provide a cohesive scenario in which the stepwise evolution of compartmentalization—from coacervates to protein-based capsids to proteolipid membranes—is integrated with the coevolution of metabolism, genetic coding, and translation. The late emergence of membranes is supported by phylogenomic evidence showing independent biosynthetic pathways and structural features in archaeal and bacterial membrane systems.
> I have a few suggestions for the authors consideration.
> In the discussion of pseudoicosahedral virus capsid evolution, the authors
> should take an example from Archaea early in the discussion. Late in the discussion
> , a reference is given indicating that Archaea utilize such viruses. Archaea appear
> to be closest organisms to LUCA, so an example from Archaea would be useful
> to a reader to indicate that spherical virus coats could be as old as LUCA.
We appreciate this excellent suggestion. We have now revised the section discussing the origin of viral capsids to include early examples of archaeal viruses, such as those from the families Globuloviridae and Sphaerolipoviridae. These archaeal viruses, which infect thermophilic and acidophilic hosts, often exhibit pseudoicosahedral morphology and demonstrate that structurally conserved capsids are not restricted to bacterial systems.
The following paragraph has been added to end of the section 4.1:
Additionally, we must acknowledge that viruses infecting Archaea also exhibit pseudoicosahedral capsids and might provide examples of simple encapsulation systems. Members of the Sulfolobus spindle-shaped virus family, while non-icosahedral in morphology, encode capsid proteins homologous to the single jelly-roll (SJR) fold found in many icosahedral viruses (Krupovic & Bamford, 2008). Furthermore, archaeal viruses such as Sulfolobus turreted icosahedral virus (STIV) adopt T=3 capsids built from a double β-barrel fold, considered homologous to the SJR fold found in bacterial and eukaryotic viruses (Khayat et al., 2005). These examples underscore that pseudoicosahedral or icosahedral architectures were also present in viruses infecting the archaeal domain, reinforcing the hypothesis that such structural strategies may have evolved prior to the divergence of the primary domains of life
> This reviewer thinks a reference to the Muller paper (reference 4 below) would
> be appropriate in discussing the RNA-peptide world.
Thank you for highlighting this. We have added a reference to H.J. Muller (1922), who first proposed the concept of primitive “genetic enzymes” and emphasized the role of nucleic acids in the origin of life. We acknowledge his contribution as an early proponent of a gene-centered model that anticipated the RNA-peptide coevolution framework later elaborated by others. This historical context enriches the discussion of the RNP world and supports our integration of capsid evolution into this perspective.
> The authors might consider other polymers (i.e., peptidoglycan) to
> encapsulate the first cells.
We thank the reviewer for this valuable point. While our model focuses on capsid-based and proteolipidic encapsulation, we now briefly mention peptidoglycan-like polymers as alternative or supplementary structural components that could have contributed to early cell wall formation in specific lineages.
First peptidoglycan biosynthesis is not conserved across Archaea and Bacteria, it likely represents a later adaptation postdating the initial emergence of protocells. Furthermore, in the early stages of prebiotic evolution, the molecular toolkit was limited primarily to RNA and peptides (RNPs) — products of relatively straightforward abiotic synthesis or simple biopolymerization. The emergence of more complex molecules like peptidoglycan would have required the prior evolution of an enzymatic network capable of generating sugars, activating them, and linking them to amino acids in specific arrangements. Thus, while cell walls based on such polymers may have arisen later to provide structural support and environmental resistance, their complexity makes them less plausible candidates for initial encapsulation compared to protein-based coats.
> There are models for coevolution of coding and coacervates (references are below).
We agree entirely and thank the reviewer for emphasizing this. We did not see your references, but we have added references to recent models that explore the coevolution of coding systems and coacervate compartments. These studies reinforce the plausibility that phase-separated membraneless condensates offered a selective environment for the emergence of coding molecules, such as tRNAs and ribozymes, prior to the advent of lipid-bound cells.
A new paragraph was added to Section 3:
“Beyond their structural roles, coacervates may have played a functional role in the origin of coding mechanisms. As proposed in recent studies (Prosdocimi & Farias, 2025a), the dynamic nature of liquid-liquid phase-separated compartments—formed from intrinsically disordered peptides and RNAs—could have promoted the co-localization of essential prebiotic components such as ribozymes, proto-tRNAs, and aminoacylating peptides. This spatial proximity would have enhanced the emergence of early coding rules, favoring interactions between specific RNA sequences and amino acid residues. In such environments, peptide-RNA symbioses likely advanced through cycles of selective stabilization and feedback, reinforcing functional assemblies. Recent theoretical and experimental models also suggest that coacervates can increase the local concentration of molecules involved in translation-like processes, including tRNA mimics and ribozymes with peptide bond-forming capacity (Franzmann & Alberti, 2019; Bose et al., 2022). Consequently, the coevolution of compartmentalization and coding might not have been coincidental but interdependent. This view is consistent with the chemical symbiosis model, where RNA and peptides reciprocally enhanced each other’s evolutionary potential, driving the system toward higher levels of molecular organization and ultimately paving the way for translation-capable progenotes.”
> The paper does not appear to stress the relationship of coacervates to concentration
> of metabolism and coding factors.
We appreciate this observation and have revised Section 2 to emphasize that LLPS-based coacervates served as dynamic microenvironments capable of concentrating nucleotides, amino acids, catalytic RNAs, and primitive enzymes. These compartments could enhance reaction kinetics and promote the emergence of autocatalytic cycles and primitive translation, consistent with their modern analogs in stress granules and nucleoli. Actually this topic has been extensively worked elsewhere (Prosdocimi and Farias, 2025a)
Prosdocimi, F., & Farias, S. T. (2025a). Coacervates meet the RNP-world: liquid-liquid phase separation and the emergence of biological compartmentalization. Bio Systems, 105480. https://doi.org/10.1016/j.biosystems.2025.105480
> How do the authors account for evolution of protein synthesis: tRNA, AARS
> enzymes, protoribosomes, first proteins, ribosomes?
We thank the reviewer for raising this fundamental point. The origin and evolution of the genetic code and the translation machinery—including tRNAs, aminoacyl-tRNA synthetases (AARS), protoribosomes, and ribosomes—constitute a vast and ongoing field of investigation that lies beyond the immediate scope of this manuscript. However, we fully recognize that the emergence of translation was a critical step in the transition from prebiotic chemistry to biological systems.
In previous publications, we and others have discussed the evolution of translation within the framework of an RNP-world, where peptides and RNAs coevolved through mutual reinforcement (Di Giulio, 1997; Farias & Prosdocimi, 2022). In this view, early coacervates could have concentrated RNA fragments and peptide-like molecules, favoring the development of specific RNA–peptide interactions and eventually leading to rudimentary coding systems. Protoribosomes likely emerged as dynamic RNP condensates capable of catalyzing primitive peptide bond formation (Root-Bernstein & Root-Bernstein, 2015), and were later stabilized by the addition of structured proteins, evolving into modern ribosomes.
While our current work focuses primarily on encapsulation strategies, the framework we present is compatible with these broader models of translation evolution, particularly by situating the emergence of capsids and membranes within a biochemical context shaped by RNP–peptide coevolution. We hope future research will continue to bridge these complementary dimensions.
> Coacervates can concentrate metabolism and coding components, partially
> delaying the need for membrane (or virus capsid) encapsulation.
We thank the reviewer for highlighting this important point. Indeed, the capacity of coacervates to concentrate nucleic acids, peptides, and small metabolites likely played a crucial role in the early stages of molecular evolution. This property may have delayed the evolutionary pressure for the emergence of membrane-based or capsid-based compartmentalization by offering a primitive yet effective means of spatial organization and proto-compartmentalization.
We now briefly elaborate on this point in Sections 3 and 4.3 of the revised manuscript. Additionally, we have addressed this topic in greater detail in that recent dedicated publication (Prosdocimi & Farias, 2025a), where we explore the interplay between LLPS-based coacervates and the RNP-world as a foundational step toward biological compartmentalization.
Prosdocimi, F., & Farias, S. T. (2025a). Coacervates meet the RNP-world: Liquid-liquid phase separation and the emergence of biological compartmentalization. BioSystems, 105480.
> Are the authors suggesting that a small spherical viral capsid could enclose a
> functional protocell unit or a complex autocatalytic cycle?
Our hypothesis is that early icosahedrical capsids likely enclosed simplified, but functionally relevant, RNP assemblies—possibly including short replicative RNAs, ribozymes, and cofactors—but not full-fledged metabolic networks. These entities would represent a metastable capsids as transitional form between progenotes and cells, allowing for RNA delivery, enhancing genetic stability and mobility while relying on interaction with FUCA-like translational systems. Thus, we do not suggest that those capsids could enclose functional protocells. The metabolic complexity necessary for a true protocell required subsequent steps, including ribosome integration and membrane evolution.
> Minor points:
> Abstract: should be understood as a distinct class of ribonucleoprotein (RNP)
> systems, some of them
Done, thank you.
> Viruses appear to be as old as LUCA (Moody paper referenced in paper draft)
We clarified in the last paragraph of Section 5 that “Here we propose that viral strategy probably emerged before the prokaryotic ancestor (LUCA) and therefore before cells.”
> Line 162: together with other ions ans inorganic molecules necessary
> for primitive metabolism.
Ok, done.
> Since an icosahedron has 219 12 vertices and 20 faces, the proteins distribute
> as follows: Because would be better English style
Great! Done. Thank you.
We would like to sincerely thank Reviewer 3 for their thoughtful, constructive, and well-informed feedback. We greatly appreciated the clarity with which they summarized the core contributions of our manuscript, as well as their insightful suggestions that helped refine and expand our theoretical framework.
In particular, we are grateful for the recommendation to include examples of pseudoicosahedral viruses from Archaea early in the discussion. This suggestion allowed us to better connect our model to archaeal systems, strengthening the plausibility that such viral architectures predate LUCA. The reviewer also encouraged us to acknowledge the historical contributions of early theorists such as H.J. Muller, which we have now done to enrich the conceptual lineage behind the RNA–peptide world.
Moreover, the suggestion to consider alternative polymers such as peptidoglycan, and to better explore the role of coacervates in concentrating metabolic and coding components, prompted important clarifications and additions throughout the manuscript.
Each of these contributions has helped us build a more nuanced and coherent narrative, grounded in evolutionary plausibility. We thank Reviewer 3 for engaging so deeply with our work and for helping us improve its scientific clarity and relevance.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI am impressed by how much more comprehensive, detailed, and well-balanced this manuscript has become after peer review. I now consider this work worthy of publication.
Lines 294, 303, 311 - missing italics.
Author Response
> I am impressed by how much more comprehensive, detailed, and well-balanced this manuscript has
> become after peer review. I now consider this work worthy of publication.
We thank very much the editor and reviewers for their encouraging feedback.
We truly appreciate the reviewing process, which helped us to improve the manuscript significantly.
> Lines 294, 303, 311 - missing italics.
The missing italics on lines 294, 303, and 311 have now been added as suggested.
