Cryo-EM structures of bacteriophage T4 portal-neck assembly intermediates reveal a viral genome retention mechanism

Domain: neuroscience
Published: 2026-02-07
Editorial outcome: minor_revision

Abstract
Bacteriophage T4 has long served as an extraordinary model for tailed phages. During virion assembly, the viral DNA genome is tightly packed into the head, to which tail attaches via a portal-neck connector. Keeping this highly pressurized head leak-proof during these transactions is a challenge, yet the mechanisms remain poorly understood. Here we show that T4 seals its DNA-filled capsid using a double “genome-gate” mechanism. By reconstituting portal (gp20)–neck (gp13/gp14) assembly intermediates in vitro and determining their structures, we find that the gp14 hexamer forms a primary gate that closes the portal-neck opening. This gate is reinforced by a second gate formed by the host protein Hfq, which is hijacked by the virus as an accessory structural component. Hfq also stabilizes neck assembly and prevents its mis-assembly with portal. These studies define a viral genome retention mechanism in which a pre-assembled gp13/gp14/Hfq neck complex seals the pressurized, genome-filled capsid during virion maturation.

Introduction
The bacteriophage (phage) T4 is a contractile tailed phage that infects Escherichia coli bacterium. It is a member of Myoviridae family, which constitutes one the most abundant and widely distributed viruses on Earth1. Historically, phage T4 has served as an important model to elucidate the fundamental principles of molecular biology, and as a prototypical virus to tease out the mechanisms of icosahedral virus assembly and infection2,3. Indeed, many mechanistic parallels were discovered between the prokaryotic T4 phage and the eukaryotic herpes virus assembly and genome packaging4,5. T4 has also emerged as a versatile platform to design next generation vaccines and gene therapies6–9.

T4 first assembles a dodecameric, mushroom-shaped, portal protein (gp20) ring structure that acts as the initiator of head (capsid) assembly through interactions with the major capsid protein gp23 and the major scaffold protein gp22. A prolate icosahedral shell made of gp23 assembles around the scaffolding core made of gp22, with the dodecameric portal forming a distinct vertex. The rest of the eleven vertices are occupied by the pentameric gp24. Later, the portal serves as a docking site for assembling a pentameric DNA packaging motor, which powers translocation of the DNA genome into the capsid through a central channel10–13. After encapsidating ~171-kb (“headful”) genome, packaging is terminated, and the motor is ejected, allowing the portal to interact with the neck proteins gp13 and gp14. Sequential assembly of neck, tail, and tail fibers then generates an infectious virion. During infection, the virion delivers its genome into the host cell through a tunnel formed by portal, neck, and tail13–15.

The structural components of T4 have been extensively investigated. Numerous high-resolution structures have been determined by X-ray crystallography and/or cryo-electron microscopy (EM), including that of the head, the portal, the base plate, and the tail2,15–19. However, the structure of the neck is yet to be resolved. Neck is a critical component because, in addition to connecting the packaged head to the tail, it is thought to act as a plug (seal) to prevent leakage of the packaged DNA20–24. The timing and integrity of this seal are crucial because the highly acidic genome compacted to near crystalline density (~500 mg/ml) creates tremendous pressure, ~25–35 atm25–27 or 5-7 times the pressure in a champagne bottle. Packaging termination, motor ejection, and neck and tail attachment must be seamlessly coordinated while keeping the head leak-proof. Otherwise, the internal pressure could expel the DNA, resulting in abortive assembly. Thus, neck assembly is one of the most vulnerable steps in virion assembly10,28,29. Though several neck structures have been determined30–34, they are all in the mature phage, the finished product. The conformational dynamics of how a plug is created, and then unsealed, and the mechanics of how the genome is fully retained inside the head remain poorly understood in any phage or virus.

In this work, we developed an in vitro assembly system and successfully assembled a variety of neck (gp13/gp14) and portal-neck (gp20/gp13/gp14) complexes that represent the intermediates of virion assembly. We discovered that a Hfq of the E. coli host is associated with the in vitro-assembled neck complexes. Hfq, originally discovered as a host factor involved in phage Qβ replication, is an abundantly expressed hexameric protein that acts as a pleotropic regulator of gene expression in bacteria. Furthermore, we solved a series of high resolution cryo-EM structures, including gp13 (dodecamer)/gp14 (hexamer) complexes with and without Hfq (hexamer), to 2.79 Å and 3.37 Å resolution, respectively, and the gp20 portal dodecamer/gp13 dodecamer/gp14 hexamer/Hfq hexamer complex to 2.91 Å resolution. These structures reveal a phage T4 neck containing two closed genome-gates (“double genome-gate”); a primary gate formed by “stopper loops” of gp14 projected into the lumen of neck channel, which would stop the genome when descended into the neck channel from the pressurized head, and a secondary gate (plug) formed by the binding of an Hfq hexamer to gp14 hexamer at the bottom of the neck, which would reinforce the primary gp14 gate. Besides, Hfq stabilizes the gp13/gp14 complex and prevents its mis-assembly with the portal. Consistent with the structures, our biochemical and genetic studies show that Hfq functions as an accessory protein by transiently associating with the neck to maximize genome retention and then leaving the neck when the tail attaches. These studies lead to a detailed genome retention mechanism that securely contains the packaged genome in the pressurized head and ensures efficient infectious virion assembly. It also highlights how a phage, by co-opting an abundant host protein, evolves a fail-safe mechanism at a critical step in virion assembly, while incurring no genome or metabolic cost to itself.

Results
In vitro assembly of T4 portal-neck connector complexes
To investigate the structure and mechanism of the T4 neck-portal connector complex, we overexpressed gp20, gp13, and gp14 individually in E. coli, purified the proteins by Ni-affinity chromatography and size-exclusion chromatography (SEC) (Fig. 1a, d), and visualized the samples by transmission electron microscopy (TEM). Negative-staining EM showed that while gp20 formed dodecamers as expected (Fig. 1f), gp13 and gp14 existed as monomers (Fig. 1c). In contrast, when we mixed the E. coli lysates to allow the assembly to occur in vitro and then purified the proteins, we observed co-purification of a variety of oligomeric complexes.

Mixing gp13 and gp14 lysates resulted in the assembly of ring structures (Fig. 1a–c), whereas mixing of all three lysates of gp20, gp13, and gp14 resulted in even larger ring structures containing all three proteins (Fig. 1d–f). In contrast, mixing gp13 with gp20 did not produce ring structures but instead resulted in aggregates (Fig. 1f). These results demonstrate in vitro assembly of post-packaging neck connector structures and that the neck protein gp13 preassembles first with gp14 forming stable oligomeric rings before attaching to the dodecameric portal.

Involvement of a host factor, Hfq, in T4 neck assembly
Surprisingly, we observed an additional ~12 kDa protein band in the purified preparations containing gp14 (Fig. 1a, b). LC–MS/MS analysis identified this gp14-associated band as the Hfq protein of E. coli. Hfq is an abundant E. coli protein and originally discovered as a host factor for phage Qβ replication. It was later characterized as a pleiotropic regulator in E. coli and is commonly found in most bacterial genomes35,36. Although Hfq primarily binds to RNA, it can also interact with a wide range of nucleic acids, including DNA, and regulates transcription, genome stability, and mRNA decay37–39.

To assess the physiological importance of Hfq in T4 phage assembly, we first performed in vivo infection assays. The results showed that the phage yield of wild-type (WT) T4 phage on hfq-minus E. coli NM2256540 is ~2-fold lower than that from the isogenic hfq-plus E. coli NM22540 (Fig. 1g).

Second, we tested the ability of the in vitro-assembled gp13/gp14/Hfq complex to attach to the packaged T4 head by developing an in vitro packaging and neck assembly system using purified components. Empty heads were packaged with a ~6 kb linearized plasmid DNA by the packaging motor (gp17) in a defined packaging system and then incubated with the gp13/gp14/Hfq complex. The packaged heads were purified by CsCl density gradient centrifugation and analyzed by cryo-EM (Fig. 1i) and Western blotting (Fig. 1i, j). The images in Fig. 1i show that the heads were packaged with DNA to near headful capacity. Many of the full-heads show protrusions at the portal vertex, likely corresponding to the gp13/gp14/Hfq complex. The Western blotting data show that all the three proteins of the gp13/gp14/Hfq complex are now associated with the packaged heads (Fig. 1j, lanes 5 and 6). Furthermore, most of the packaging motor was dislodged from the head following packaging (compare gp17 band in lane 5 with control lane 2, Fig. 1j), while neck-like protrusions are now seen associated with the head at the portal vertex in the cryo-EM images (blue arrows in Fig. 1i).

Third, we observed that the ratio of Hfq to gp13 to gp14 in the packaged head-neck complexes varied, some experiments showing less than the stoichiometric amount of Hfq (compare lane 5 with control lane 3, Fig. 1j). Furthermore, Hfq is completely absent in the finished virion structure (Fig. 1h).

These results suggest that the Hfq hexamer is transiently incorporated into the neck complex that binds to the packaged head for enhanced genome retention and phage yield and then leaves the neck when tail attaches.

Cryo-EM structures of T4 neck complexes
Two types of neck complexes were observed in the cryo-EM images obtained from co-purified gp13 and gp14 fractions; the gp13/gp14 complex comprising ~34% of particles and gp13/gp14/Hfq complex comprising ~66% of particles (Supplementary Fig. 1). Cryo-EM maps were generated for both these complexes without imposing symmetry to 3.70 and 3.10 Å resolution, respectively (Supplementary Fig. 2). In the gp13/gp14 complex, gp13 forms a dodecameric ring and interacts with a hexameric gp14 ring creating a central channel. In the gp13/gp14/Hfq complex, the Hfq hexamer attaches to the gp14 hexamer, essentially covering the lower end of the channel (Supplementary Fig. 2c). Notably, the gp13 ring in the gp13/gp14 complex is incomplete, lacking some of the twelve subunits (Supplementary Fig. 2e). In contrast, the gp13 ring in the gp13/gp14/Hfq complex is complete (Supplementary Fig. 2c), suggesting that Hfq stabilizes the structure of the gp13/gp14 complex.

To further improve the resolution of the cryo-EM structures, C6 symmetry was applied and the structures of gp13/gp14 and gp13/gp14/Hfq complexes were refined to 3.37 Å and 2.79 Å resolution, respectively, allowing the building of atomic models (Fig. 2a, b; Supplementary Fig. 2d and f; Supplementary Table 1). The gp13/gp14/Hfq complex has an inverse bowler hat shape, with gp13 serving as the brim, gp14 as the body, and Hfq as the crown (Fig. 2b). The gp13 dodecamer forms a central tunnel with an inner diameter ranging from 56 Å at the top to 44 Å at the bottom (Fig. 2c). The gp13 subunit can be divided into five parts: domain I, a four-helix bundle (residues 3–51, 239–300); domain II, the “swing” domain (residues 52–192); the fibritin-binding domain III (residues 190–239) which is disordered in the current structure but resolved in the virion41; the gp14-binding adaptor loop (residues 266–279), and the portal-binding C-terminal arm (residues 301-308) (Fig. 2c, d). The helix bundle and the swing domains of gp13 form the core and the peripheral ring, respectively. The helix bundle is made of four helices (α1, α2, α5, α6), with α6 forming the central channel. Three glutamic acids (Glu284, Glu291, and Glu295) on α6 face the channel, creating a negatively charged surface for smooth passage of DNA (Fig. 2e). The swing domain, so named because it is observed to swing upwards in the mature T4 virion (see below), is made of seven β strands and two α helices (β1-7, α3-4) (Fig. 2d). Other than these two main domains, gp13 contain two major loops located before and after α6, named as the adaptor loop and the C-terminal arm, respectively. The 14-aa adaptor loop inserts into gp14, forming extensive interactions (Fig. 3a–c). The C-terminal arms stretch upward, providing a binding site for gp20. The interaction between adjacent gp13 subunits is mainly mediated by hydrogen bonds between the swing and helix bundle domains (Fig. 3e).

Gp14 consists of an N-terminal loop (residues 5-37), a long helix (residues 38-54), a long stopper loop (residues 89-112), and a core β-strands domain (residues 55-88, 113-177) (Fig. 2c, d). The long N-terminal loop and the N-terminal lateral helix (α1) of gp14 wrap around the adaptor loop of gp13, while six core β-strands (β1-6) form an anti-parallel β-barrel. The long loop between β2 and β3 protrudes into the center of the channel. Six such loops, referred to as “stopper loops”, form a gate-like structure that narrows the otherwise open channel (Fig. 3h), likely preventing the release of the tightly packed genome from the pressurized capsid. The C-terminal 80 amino acids are invisible, probably due to their flexibility. The loop between β5 and β6 interact with the adjacent subunits mainly through hydrophobic interactions (I26, N28, Y36 and R54, Q52, Q126). Moreover, residues P157 and F158 form hydrophobic interactions with F140, P141 and M147 of the adjacent subunit (Fig. 3f).

Gp13 and gp14 engage in intensive interactions, with one gp14 interacting with four gp13 subunits (n, n+1, n+2, n+3) (Fig. 3a–c). In addition, the N-terminal loop of gp14 interacts with gp13 (n+3) through both hydrophobic and hydrophilic interactions (Fig. 3c). The N-terminal helix α1 of gp14 spans the adaptor loops of three adjacent gp13 subunits (n, n+1, n+2), forming intensive hydrophobic interactions (M53, L46, V45, L42 from gp14 and I277, L271, I262, I24, I25 from gp13) (Fig. 3b). This explains why neither gp13 nor gp14 could form oligomers when expressed alone, but could do so when mixed.

Neck assembly and energy considerations
The above biochemical and structural data demonstrate that both gp13 and gp14 exist as monomers alone but when mixed together form a stable complex, a phenomenon also observed in the portal-neck assembly of P22 phage42,43. The interface between gp13 dodecamer and gp14 hexamer is extensive with a buried area of 4181 Å, in contrast to the interface area between gp13 subunits (1914 Å) or gp14 subunits (1038 Å). Dissociation free energies (ΔGdiss) calculated by PDBePISA reveal an energetic landscape consistent with cooperative assembly (Supplementary Table 2). Among the gp13 dodecamer, gp14 hexamer, and gp13/gp14 complex, the isolated gp13 dodecamer and gp14 hexamer exhibit moderate thermodynamic stability (ΔGdiss = +46.2 and +45.3 kcal/M, respectively) but are characterized by large entropic penalties for dissociation (TΔSdiss = +158.6 and +66.7 kcal/M, respectively). The gp13/gp14 complex exhibits the lowest entropic penalty (TΔSdiss: +18.1 kcal/M) and highest ΔGdiss (+140.4 kcal/M), suggesting that complex formation is favorable. These are consistent with our experimental observations that gp13 and gp14 monomers do not spontaneously form stable oligomers individually, however mixing gp13 and gp14 monomers facilitates the formation of gp13/gp14 complex.

Thus, the T4 neck complex appears to assemble through a cooperative mechanism in which two metastable components converge to generate a hyper stable structure. This strategy ensures that a strong driving force for assembly is unleashed only upon correct formation of the functional unit, thereby preventing premature or erroneous interactions.

Structure of the neck-Hfq complex
Notably, the E. coli Hfq hexamer fit precisely at the bottom of gp14. The Hfq hexamer engages with the gp14 hexamer primarily through hydrogen bonds, involving residues Q111, D112, E113, E134, K173, I175 of gp14 and K31, T49, Q33, S65, R19 of Hfq, resulting in a substantial buried interface of 3159.2 Å². In addition, the negative charge at the bottom of gp14 and the positive charge at the top of Hfq are likely to facilitate their interaction (Fig. 3d, g). Remarkably, the disordered stopper loop residues (106-113) of gp14 become visible only upon Hfq binding, suggesting that Hfq binding stabilizes the stopper loop (Fig. 2f). Moreover, incorporation of Hfq hexamer constricts the central channel by approximately 2 Å when compared to the gp13/gp14 complex lacking Hfq (Fig. 2f). Thus, Hfq is likely functioning as a secondary genome gate to reinforce the primary gate to prevent DNA leakage (Fig. 2c).

Notably, the gp14 residues that mediate interactions with Hfq and the negatively charged interface are highly-conserved among T4-like phages (Supplementary Fig. 7). In contrast, these residues and the corresponding interface are missing in gp14 orthologs from phages such as SPP1 and lambda (Supplementary Table 4). This suggests that Hfq usage may represent a T4 lineage-specific evolutionary adaptation that co-opts an abundant host protein to install a fail-safe genome retention mechanism without incurring any genome or metabolic cost to phage itself.

Structure of the portal-neck connector complex
Cryo-EM structural analysis of the in vitro assembled gp20/gp13/gp14 complexes revealed that a majority (~90%) of these complexes also contained the Hfq hexamer. Therefore, the structure of this portal-neck connector complex that joins the head and the tail was resolved at 2.91 Å with C6 symmetry (Fig. 4a–c and Supplementary Figs. 3, 4).

The overall structure of the dodecameric gp20 in this complex is similar to that of the recombinant gp20, with a root mean square deviation (r.m.s.d.) of 1.4 Å across 411 (85%) aligned residues (Supplementary Fig. 5a). However, upon gp13 binding, the α6 helix of gp20 clip domain rotates 7° and shifts 4 Å to accommodate the binding of the gp13 (Fig. 4f). The C-terminal arm of gp13 rotates upward by 40° relative to that in gp13-gp14 complex (Fig. 4g), inserting itself between two clip domain α6 helices of adjacent gp20 subunits, and forming extensive hydrophobic interactions with residues on both the helices and with the β-strand of one of the clip domains (Fig. 4d). Additionally, the negatively charged residues at the C-terminus of gp13’s α2 and α4 helices (E47, D51, E295, D301) engage in electrostatic interactions with positively charged residues of gp20’s α6 helix (R295, K296, R339) (Fig. 4d, e). On the other hand, the conformation of the gp14 and Hfq complexes remained unchanged when the neck complex attaches to the portal (Fig. 4h; Supplementary Movie 1).

Hfq ensures correct assembly of portal-neck complex
We identified a class of particles among the portal-neck connector complexes in which gp14, instead of gp13, interacted with gp20 and is positioned between the gp20 portal dodecamer and the gp13 dodecamer (Fig. 5a; Supplementary Figs. 3 and 4). Notably, these gp20/gp14/gp13 complexes are devoid of Hfq and constituted ~14% of the portal-neck complexes (Supplementary Fig. 3). In this structure determined at 3.45 Å resolution, the C-terminal arm of gp14, which normally makes extensive interactions with gp15 subunits, inserts between the clip domains of adjacent gp20 subunits. Similar to the gp20/gp13 complex, gp20 and gp14 mainly interact through hydrophobic interactions and hydrogen bonds (K309, N291, N323, R311 of gp20 and D188, A110, Y187 of gp14) (Fig. 5b, c). Furthermore, residue K309 of gp20 forms a salt-bridge with D188 of gp14. Additionally, electrostatic interactions are also observed between the positively charged clip domain of gp20 (residues of R295, K296, H300 and H303) and the negatively charged C-terminus of gp14 (Fig. 5d).

To further investigate this unexpected phenomenon, and if Hfq played a role in assembling the correct portal-neck complex (gp20/gp13/gp14), we produced recombinant Hfq protein and mixed it in excess first with gp14 extract and then with the gp13 extract, followed by the portal protein extract. SDS–PAGE analysis showed that, under excess Hfq, the portal–neck complex assembled into a homogeneous gp20/gp13/gp14/Hfq complex with an approximate 1:1 molar ratio of gp14 to Hfq, whereas limited Hfq resulted in a heterogeneous mixture containing both gp20/gp13/gp14/Hfq and gp20/gp14/gp13 complexes (~2:1 molar ratio of gp14 to Hfq) (Supplementary Table 3). Cryo-EM analysis confirmed that in the presence of excess Hfq, only correctly assembled gp20/gp13/gp14 complexes were formed and no mismatched gp20/gp14/gp13 complexes detected (Supplementary Fig. 6). Thus, Hfq, by binding to gp14, also inhibits its interaction with gp20, thereby facilitating the correct assembly of the portal-neck complexes.

Conformational changes in portal-neck connector complexes
Comparative structural analysis showed that the structures of gp20, gp13, and gp14 in different in vitro assembled complexes are similar, with r.m.s.d. ranging from 0.68-1.47 (Supplementary Fig. 5). However, comparison of the in vitro assembled gp20/gp13/gp14/Hfq complex with the in situ gp20/gp13/gp14 atomic model of phage virion41 (Fig. 6) shows dramatic conformational changes in each of the neck components, which lead to a dynamic genome retention mechanism (Fig. 6a). Upon completion of headful packaging, a global conformational change occurs in the portal structure and the portal is pushed down by the pressure of the packaged genome exposing the “stem” and “wing” regions of portal for gp13 binding (Fig. 6b). After initial binding with the clip domain, as seen in the current structure, a major conformational transition occurs in the gp13 dodecamer. The swing domain flips upward by approximately 90° and its “anchor” region (aa 90-157) interacts with the now exposed stem and wing domains of the portal and the periphery domains of the major capsid protein gp23 (Fig. 6c). Furthermore, residues 193–238 which are not resolved in the current structure, model into domain III which binds to “fibritin”, a trimer of gpWac (whisker antigen control) (Fig. 6c). Twelve domains III of the gp13 dodecamer interacting with fibritins assemble twelve fibers around the neck, six as “whiskers” and the other six as “collar”. Later in virion assembly, these neck fibers interact with the long tail fibers (LTFs) in ‘up’ conformation in the pre-infection stage (Supplementary Movie 1).

At the other end of the portal-neck connector, gp14 also undergoes a large conformational change. The C-terminal regions of gp14 (aa 178–256), which are disordered in the current structure, form new and extensive interactions with gp15 subunits (residues 203–245), while the β-barrel stopper loops rotate downward by ~90° to dock with the tail sheath terminator protein gp15 located at the tip of the tail (Fig. 6d). These conformational changes open the gp14 genome-gate, while also ejecting the Hfq hexamer.

Quantitative dissociation energy analysis using PDBePISA provides a mechanistic explanation for how the tail protein gp15 triggers Hfq displacement and allow stable docking of the tail. The gp14/Hfq complex exhibits both a low TΔSdiss and low ΔGdiss (+13.6 kcal/M), indicating that it is relatively unstable and can readily dissociate. By contrast, the ΔGdiss of the gp14/gp15 complex (+364.6 kcal/M) is approximately 25 times greater than that of gp14/Hfq, reflecting markedly higher stability. This large difference in stability indicates that, from a thermodynamic perspective, gp15 binding to gp14 is strongly favored.

This is consistent with Hfq’s role, which is restricted to the neck intermediate and dissociates upon tail attachment. Hfq binds gp14, forming a metastable complex. Tail attachment allows gp15 to displace Hfq, forming an ultra-stable gp14/gp15 interface that irreversibly locks the tail onto the neck. Together, these data demonstrate that gp15 provides the energetic driving force to disrupt the gp14/Hfq interaction, ensuring high-fidelity viral assembly (Supplementary Table 2).

These analyses reveal that the current structures represent intermediates trapped by in vitro assembly prior to tail attachment, thus preserving the structure of a transient genome plug formed to leak proof the head and retain the packaged genome while handing it over to the tail.

Methods
Protein expression and purification
The full-length gp13 gene (NC_000866.4), gp14 (NC_000866.4) and Hfq (NC_000913.3) were synthesized (GeneScript) and inserted into the pET-29a, pET28b and pET-28a vectors with N-terminal His tag, N-terminal StrepII tag (Hfq), respectively. Plasmids information and primers Oligonucleotide sequences are provided in Supplementary Data. The protein was expressed in E. coli BL21 (DE3) grown at 37°C, induced by 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at an optical density (OD) of 0.6–0.8 at 16°C. After 4 h, the cells were harvested by centrifugation at 5000 rpm (6240 g) with a JLA-8.1 rotor and resuspended in lysis buffer (20 mM Tris, pH 8.0, 200 mM NaCl, 20 mM imidazole, 1/500 Supernuclease S (Tiandirenhe), 1/10,000 Protease Inhibitor Cocktail (Roche). The mixture was then centrifuged at 40,000 g for 20 min at 4°C and removed fragments by 0.22 µm filter (Millipore USA). The sample was loaded into Ni Smart Beads 6FF (Tiandirenhe) equilibrated in buffer A (20 mM Tris, pH 8.0, 500 mM NaCl), and eluted with buffer A containing 500 mM imidazole. The protein was then purified by Superose 6 Increase 10/300 GL (Cytiva) with buffer B (20 mM Tris, pH 8.0, 500 mM NaCl, and 1 mM TCEP). The elution volume indicated that gp13 and gp14 were monomeric in solution. For further studies, we mixed gp13 and gp14 lysates together from the very beginning in bacterial sonication and centrifugation, and then we used the same purification protocol described above. N-His tagged gp20 (residues 74-524) (NC_000866.4) and untagged gp22 were cloned into the pET-Duel vector for co-expression and purified as previously described1. Finally, we mixed gp13/gp14 neck complex and portal protein gp20 together and incubated for 30 min, and then the mixture was concentrated with 50,000 Da Ultratube (Millipore) into 0.5 ml for size-exclusion chromatography (Superose 6 Increase 10/300 GL, Cytiva).

Identification of proteins in the neck complexes by LC/CL-MS analysis
The gp14 and Hfq bands (the number of samples analyzed is n=1, respectively) were excised from the gel and digested with Trypsin at 37°C for 4 h. The concentrated peptides were analyzed by Orbitrap FusionTM LumosTM TribridTM Mass Spectrometer (Thermo Fisher Scientific). Original data was analyzed by Thermo Proteome Discoverer 2.5.0.400 followed by searching for specific protein sequences in databank.

Analysis of gp14/Hfq ratio based on the SDS-PAGE
The protein bands in the SDS-PAGE were calculated with imageJ software. The SDS-PAGE images were converted to grayscale images. For a more intuitive analysis, we use the inverted grayscale map for numerical calculation. The brightness of the bands indicates the mass of gp14 and Hfq proteins and is finally represented by the Mean value output by the software. The final binding mass and molecular weight were used to calculate the molar ratio of gp14 and Hfq bands on SDS-PAGE.

Phage yield in Hfq-minus E. coli
E. coli strains NM22565 (Hfq-plus) and NM22540 Dhfq::cat-sacB (Hfq-minus) were grown to mid-logarithmic phase (~2 × 10⁸ CFU/mL) in Luria-Bertani (LB) medium at 37°C with shaking at 250 rpm (the strains were provided by Dr. Susan Gottesman from National Institutes of Health). The cells were infected with WT T4 phage at a multiplicity of infection (MOI) of 0.1 and incubated at 37°C for 5 min. Immediately afterward, serial 10²-, 10⁴-, and 10⁶-fold dilutions were prepared in LB medium. The remaining infection mixture (non-diluted) was treated with chloroform to lyse the cells and quantify unbound phage. At 10, 20, 30, 40, and 60 min post-infection, aliquots were taken from the 10⁶-fold dilution and subjected to plaque assays by the soft agar overlay method using E. coli B40. Burst size was calculated as plaque-forming units (PFU) produced per infected cell.

Construction of Hfq⁺ T4 Phage
The Hfq⁺ T4 phage was generated using phage CRISPR-mediated gene insertion, as described previously57. Briefly, E. coli B40 cells were co-transformed with a donor plasmid containing E. coli hfq gene flanked by T4 genomic sequences in the SegF region and a CRISPR-Cas12a-SegF spacer plasmid. The cells were infected with T4-Soc-del phage. Recombinant phages were selected by counter-selection in E. coli B40 expressing the Cas12a-SegF spacer. PCR analysis confirmed successful insertion of the hfq gene under the control of Soc promoter. To verify Hfq expression by the constructed hfq⁺ T4 phage, E. coli NM22565 (hfq⁻) cells were grown to mid-log phase in LB medium at 37°C (~10⁸ CFU/mL) and infected with the Hfq⁺ phage at an MOI of 5. After 5 min of incubation at 37°C, the culture was super-infected with an additional dose of hfq⁺ phage (MOI 5) to prevent lysis. At 30 and 60 min post-infection, ~10⁸ CFU of bacteria were collected for SDS-PAGE and Western blotting using Hfq polyclonal antibodies (see below). The data show that Hfq is well-expressed from hfq+ phage during phage infection.

Hfq western blotting
E. coli strains [B40, NM22565 (Hfq-plus) and NM22540 Dhfq::cat-sacB (Hfq-minus)] were grown to mid-logarithmic phase in LB medium at 37°C with shaking at 250 rpm. Bacterial cells (approximately 10⁸ CFU) were harvested by centrifugation at 5000 xg for 5 min, resuspended in 200 µL 1X SDS-PAGE loading buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.02% bromophenol blue), and denatured by boiling for 10 min. The samples were then electrophoresed on a 4-20% gradient Mini-PROTEAN TGX precast gel (Bio-Rad) and transferred to nitrocellulose membranes using a Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with 5% BSA in PBS-T (PBS containing 0.05% Tween-20, pH 7.4) for 1 h at room temperature with gentle shaking, then incubated overnight at 4°C with anti-Hfq rabbit polyclonal antibodies (provided by Dr. Susan Gottesman from National Institutes of Health) at 1:1000 dilution in 5% BSA/PBS-T. After five 5-minute washes with PBS-T, membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:10,000, Abcam) for 1 h at room temperature. Following five additional PBS-T washes, stained protein bands were visualized using an enhanced chemiluminescence substrate (BioRad) and imaged using a Bio-Rad Gel Doc XR+ imaging system with exposure times optimized for each blot.

Binding of gp13/gp14/Hfq complex to in vitro packaged heads
The empty heads were purified from 10am13am-9DE mutant phage infected cells as described previously9. The heads were packaged with the linearized ~6kb AAV-GFP plasmid DNA in a defined packaging system containing the purified packaging motor protein (gp17) and ATP according to the protocol described earlier58. After packaging for 5 min, the gp13/gp14/Hfq complex purified as above was added to the packaging reaction mixtures at a molar 3:1 ratio of neck complexes to packaging motors. After 40 min incubation, the packaged heads were separated from empty heads and unbound complexes by CsCl density gradient centrifugation. The DNA-full heads that migrated to near the bottom of the tube were extracted by puncturing the side of the tube and dialyzed first against 50 mM Tris-HCl pH 7.4 buffer containing 5 mM MgCl2 and 200 mM NaCl for 1 h and then for another 1 h against the same buffer containing 100 mM NaCl. The packaged head-neck complexes were centrifuged at 36,000 xg for 1 h and resuspended in 50 mM Tris-HCl pH 7.4 buffer containing 5 mM MgCl2 and 50 mM NaCl. The samples were then analyzed by cryo-EM and Western blotting using anti-His tag monoclonal antibody (Invitrogen). The motor protein gp17 and each of the three proteins of the gp13/gp14/Hfq complex contain His tag fused to either N- or C-terminus of the recombinant protein.

Cryo-EM sample preparation and data collection
All samples were evaluated by negative stain before preparing cryo-grids. 5 µL purified samples were applied to the glow-discharged copper grids (Electron Microscopy China) for 1 min then blotted with filter paper. The grids were then negatively stained with 2% (w/v) uranyl acetate, blotted and air-dried. Images were recorded with Talos L120C TEM (Thermo Fisher Scientific) equipped with a CCD camera at a nominal magnification of 73,000× or 92,000×, defocus between −0.5 µm and −3.5 µm.

All cryo-grids were prepared with the same condition. Here, we took gp20/gp13/gp14 as an example and more details are shown in the Supplemental Information. Quantifoil holey carbon grids (R20/20 Ni-Ti-Au 300) were glow-discharged under the atmosphere of argon and oxygen mixture. 3 µL sample containing 0.05% β-OG was loaded on the grids, and then blotted and vitrified using a Vitrobot Mark IV (Thermo Fisher Scientific). All cryo-EM data were collected at the Center for Cryo-Electron Microscopy (Fudan University) with Titan Krios TEM (Thermo Fisher Scientific) operated at 300 kV, equipped with Bio-Quantum energy filter (Gatan).

Cryo-EM images processing
For gp13/gp14/Hfq complex, a total data set of 12,169 images were collected with the defocus range of −1.2 μm to −2.2 μm, in super-resolution mode (magnification 81,000×) using K3 Summit direct electron detection device with a physical pixel size of 1.064 Å/pixel. The total exposure time was 3 s with 40 frames giving an accumulated dose of 58.3 e-/Å². Automated data acquisition was performed with SerialEM software through the beam-image-shift method59. More details are listed in Supplementary Table 1. All super resolution images were binned 2, dose weighted, and motion corrected using MotionCor260 and subsequent CTF (contrast transfer function) of micrographs was estimated using Gctf61. Bad images were excluded upon ice condition, defocus range and estimated resolution. Remaining 11,557 good images were imported into cryoSPARC62 for further patched CTF-estimating, blob-picking and 2D classification. Good 2D classes were selected as the template for template picking. From the 2D classes, good particles from blob-picking and template-picking were merged and deduplicated. The whole particle stacks were separated into subsets to accelerate processing. 3D classification in Relion of the first subset of 1,873,444 particles shows two major conformations: gp13/gp14/Hfq and gp13/gp14, these two conformations were selected and merged and after another round of 3D classification, followed by 3D auto-refinement with C6 symmetry, CTF Refinement and Bayesian Polish, a map of 2.79 Å gp13/gp14/Hfq was obtained from 381,536 particles. The whole dataset was used for iterative 3D classification to yield a 3.37 Å map of gp13/gp14 complex.

The gp20/gp13/gp14/Hfq dataset was collected at magnification 130,000x using K2 Summit direct electron detection device with a physical pixel size of 1.046 Å/pixel, total exposure time 8 s, 36 frames, at an accumulated dose of 53 e−/Å. A total of 4252 images were collected and 4136 good ones were selected. Particles were Laplacian and templated autopicked in Relion, and 297,605 particles were used for iterative 3D classification. The best resolved classes were selected and yielded a 2.91 Å map of gp20/gp13/gp14/Hfq complex.

The reported resolutions are all based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion. All the visualization and evaluation of 3D density maps were performed with UCSF Chimera63. The above procedures of data processing are summarized in (Supplementary Figs. 1, 3 and 6). These sharpened maps were generated by DeepEMhancer64.

Model building and refinement
Model building of gp13/14 was performed de novo in COOT65, while Hfq was fitted and refined using previous crystal model (PDB: 1HK9). After structure refinement in PHENIX66, the model was fit into the gp20/gp13/gp14/Hfq map. Gp20 was built from the crystal model (PDB: 3JA7) and manually adjusted in COOT. Statistics associated with data collection, 3D reconstruction and model refinement can be found in Supplementary Table 1.

Interface and dissociation energy analysis using PDBePISA
To analyze the protein–protein interfaces and assess the thermodynamic stability of the complexes, we utilized the PDBePISA (Protein Interfaces, Surfaces and Assemblies) web server (http://www.ebi.ac.uk/pdbe/pisa/). PISA was employed to calculate the buried surface area, dissociation free energy (ΔGdiss), and entropic contribution (TΔSdiss) for each complex. These parameters provide insights into the stability and assembly energetics of the gp13 adjacent subunits, gp14 adjacent subunits, gp13/gp14, gp14/Hfq, and gp14/gp15 complexes. Hydrogen bonds and salt bridges were identified based on geometric criteria (distance ≤ 4.0 Å), and only interactions meeting these thresholds were included in the final analysis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In order to elucidate how the T4 genome is kept inside the capsid during the later stages of assembly despite the strong internal pressure, Han et al. have studied the in vitro assembly of the gp13-gp14-gp20 portal neck complexes. They observe the unexpected presence of an additional protein that they could identify as the host protien Hfq. They determine reconstructions of the entire and partially assembled complex and show that this Hfq protein stabilizes the neck and facilitates its correct docking to the phage head. This work provides the first detailed mechanism of viral genome retention in tailed bacteriophages.\n\nMajor point : The authors have shown here that Hfq can bind to gp14, which leads to a closure of the channel. However the experiments on hfq-minus E.coli strain only show a 50% reduction in burst size even if it is significant. A larger effect could have been expected if Hfq was essential for DNA retention. Moreover, given that Hfq is involved in several major bacterial processes (transcription regulation, genome stability, mRNA decay), it is possible that impairing these functions would have an effect on the phage biology and thus on the burst size. The authors should thus discuss this apparent contradiction between the structural data that suggest a major role of Hfq in genome retention and the in vivo data that suggest a not that important role. In particular, is there a significant conservation of the gp14 aminoacids that are binding to Hfq ?\n\nMinor points\n1- Line 73 : \"To our surprise, we discovered 73 that a previously unknown protein, identified as the E. coli Hfq\". \"Unknown\" is incorrect, the protein itself was known but its interaction with the T4 neck was unknown\n2- line 93-94 : \"purified the proteins (Fig. 1A and 1D)\" the test should refer to figure 1B as well (for gp13 and for gp14). An SDS-PAGE analysis of gp20 alone would be necessary to confirm that this sample has also been correctly purified.\n3- Line 149-150 \"Three glutamic acids (Glu284, Glu291, and Glu295) on α6 face the channel, creating a negatively charged surface for smooth passage of DNA\" An image of the electrostatic potential would be useful to confirm this point\n4- Fig 3B : Several labels are hardly readable. This includes R265 in blue ans I262 in green\n5- Line 175-179, about the relative stability of the interfaces between gp13 and gp14 : the text is ambiguous. 10675 Å² seems to correspond to the entire surface of interaction between the gp13 12-mer and the gp14 6-mer, while 1725.6 Å² seems to correspond to the interface between two gp13 protomers. If it is correct, the latter value is not that small for a ring complex. As a comparison, the adaptor protein in HK97 assembles as stable rings despite a significantly smaller surface of interaction (1250 Å² per interface as calculated from the PDB entry 3JVO) than T4 gp13 (Cardarelli et al., JMB 2010). PISA not only calculates the surface of interaction but also calculates a dissociation energy of the complex, which is at least as important at the surface of interaction itself. Providing these values would be useful here.\n6- Figure 3C,3H and 4D : several hydrogen bonds or salt bridges seem to have unusual geometries (K171/N143, K13/E13, N169/T142, R295/D51). Especially, the distance between R295/D51 is larger than 4Å which is very large for a salt bridge. The Material&Methods section indicate that Pisa has been used for calculating the interface areas, has it also been used for having a non-biased identification of polar contacts between chains ?\n7- Supplementary Movie 2 : the movie suggests that the gp13 conformational change to stabilize its interaction with gp20 occurs after the tail has attached to gp14, which is extremely unlikely.\n8- The stability of the gp14-Hfq complex is not discussed. A surface of interaction is provided, but there is no corresponding dissociation energy. In the SDS-page analysis on figure 1B, the amount of Hfq seems to be extremely small in comparison with gp14. This suggests that Hfq slowly detaches from gp14 and that it could have been lost during the size exclusion chromatography. A weak interaction is not surprising, given that Hfq has to be expelled in the latter stages of assembly. It would thus be interesting to discuss this point and its possible implication for the assembly mechanism.\n9- Line 353 : \"the cells were harvested by centrifugation at 5,000 rpm\". The author should either provide the centrifugation speed and the name of the rotor, or provide the number of g.\n10- Line 642, legend of figure 2F : the author should precise which proteins/domains have been used for the structural alignment", "reviewer_2": "This is a well-written paper describing the assembly of the T4 portal protein complex and the function of an endogenous factor, identified as a plug, which prevents genome leakage at the end of packaging before tail assembly. The paper is clear and easy to understand. Although not groundbreaking, the authors present an interesting discovery that could have implications for other T4-like phages. Also, is a ~2-fold decrease in phage yield in hfq-plus E. coli sufficient to conclude that Hfq is physiologically important? What would the phage yield be if a common chaperone were deleted in E. coli? Overall, I like the paper but find its scope and significance somewhat limited to stand alone as a full article in Nature Communications."}, "author_response": "We thank the reviewers for their insightful comments and valuable suggestions,\nwhich prompted additional analyses and experiments that improved the manuscript\nsignificantly and also resulted in greater illumination of the viral genome retention\nmechanism.\nWe acknowledged this point in the Acknowledgment section of the revised\nmanuscript.\nReviewer #1\nIn order to elucidate how the T4 genome is kept inside the capsid during the later stages\nof assembly despite the strong internal pressure, Han et al. have studied the in vitro\nassembly of the gp13-gp14-gp20 portal neck complexes. They observe the unexpected\npresence of an additional protein that they could identify as the host protein Hfq. They\ndetermine reconstructions of the entire and partially assembled complex and show that\nthis Hfq protein stabilizes the neck and facilitates its correct docking to the phage head.\nThis work provides the first detailed mechanism of viral genome retention in tailed\nbacteriophages.\nWe thank the reviewer for a positive and highly enthusiastic assessment of our\nwork.\nWe greatly appreciate the reviewer’s recognition that our work provides “the first\ndetailed mechanism of viral genome retention in tailed bacteriophages”.\nThe thoughtful and detailed comments below by the reviewer have been invaluable\nin refining the manuscript. We have addressed each point carefully and the\ncorresponding revisions are implemented in the revised manuscript, which we believe\nhave significantly improved the clarity, accuracy, and quality of the manuscript.\nMajor point : The authors have shown here that Hfq can bind to gp14, which leads to a\n1\nclosure of the channel. However the experiments on hfq-minus E.coli strain only show\na 50% reduction in burst size even if it is significant. A larger effect could have been\nexpected if Hfq was essential for DNA retention. Moreover, given that Hfq is involved\nin several major bacterial processes (transcription regulation, genome stability, mRNA\ndecay), it is possible that impairing these functions would have an effect on the phage\nbiology and thus on the burst size. The authors should thus discuss this apparent\ncontradiction between the structural data that suggest a major role of Hfq in genome\nretention and the in vivo data that suggest a not that important role.\nWe thank the reviewer for making these insightful points. We agree, as the reviewer\nnoted, that this is an “apparent contradiction” regarding the role of Hfq, and we\nappreciate the opportunity to clarify this, as follows:\nI. A 50% reduction in phage burst size when grown on E. coli Hfq mutant appears\nmodest if Hfq were an “essential” function. However, Hfq is not an essential function.\nRather, it is an accessory function.\nMultiple structures of the neck assemblies described in our manuscript demonstrate\nthat Hfq is not the primary genome gate. It is a secondary gate that reinforces the\nprimary genome gate formed by the phage-encoded gp14 protein. We should have made\nthis distinction more explicit in the original manuscript.\nThe gp14 gate is the one to contact the genome and stop it, while the secondary\nHfq gate merely supports it. In principle, the gp14 genome gate might be sufficient\n(mutants of gp14 are lethal) but having a second gate would make it fail-safe. Hence,\nHfq is an accessory to the phage neck.\nConsistent with its accessory function, Hfq’s role is transient as it leaves the neck\ncomplex once the tail approaches for docking onto the neck through interaction with\ngp15 situated at the top of the tail structure. Clearly, Hfq is not found in the virion as\ndemonstrated by Western blotting and structural analyses. Our energy calculations\ngiven below in response to another comment (another insightful comment) show that\nthe gp14-Hfq interaction is weak (ΔGdiss =13.6 kcal/M) and driven by modest enthalpic\n2\nand entropic changes, indicative of a transient or regulatory association rather than a\ncore structural contact. It dissociates easily when tail attaches.\nIn response to this comment (and a comment by Reviewer #2 below), we have\ndeveloped a new assay to test the ability of our in vitro-assembled gp13/gp14/Hfq\ncomplex to bind to in vitro-packaged heads. Data from these experiments show,\nconsistent with the energy calculations, that the gp13/gp14/Hfq complex binds to\npackaged heads demonstrating its functional significance. Additionally, the data show\nthat Hfq stoichiometry varied and lower than expected 1:1 ratio indicating that Hfq\ninteraction might weaken when attached to pressurized head and some of it dissociates\neven when the tail is not attached.\nFinally, it is also worth noting that a 50% reduction in phage yield is significant,\ngiven that the yield for the wild-type (WT) phage is only ~100 phages/infected cell. A\n2-fold enhancement by Hfq therefore does provide a significant survival advantage in\nthe highly competitive phage universe.\nIn fact, curiously, phage T4 evolved by incorporating many such accessory\nfunctions that provide modest ~50% enhancement of phage yield. Examples include\nMotB, gp5.4, gp45.2, and so on (Mattenberger et al., 2025; Patterson-West et al., 2018);\n(Fokine et al., in preparation). Referred to as “nonessential” genes, such accessory\nfunctions constitute a significant portion of the T4 genome. What is novel and\ninteresting here is co-opting a host protein for enhancing genome retention and virion\nassembly with no genome or metabolic cost to phage itself. This to our knowledge has\nnot been observed in any other phage system so far.\nTherefore, our structural, genetic, biochemical, and binding/dissociation energy\nanalyses are consistent with Hfq serving in an accessory role through transient\nassociation with the neck complex for maximizing the efficiency of neck assembly and\ngenome retention.\nII. With regard to Hfq-minus E. coli metabolism, it should be noted that E. coli has\nredundant regulatory systems that compensate for Hfq. The most impact Hfq has is\n3\nwhen E. coli is grown in nutrient-deficient and other stress conditions. Under nutrient-\nrich conditions, the main effect observed is that the E. coli rods become longer because\nHfq does inhibit cell division. If anything, this phenotype might actually skew our\nresults towards higher phage yield because equal number of cells were used for\ninfection in our experiments to determine the phage yield. Bulkier cells may provide\nmore nutritional resources to support phage infection.\nFurthermore, phage T4, unlike most other phages, completely shuts down host\nmachineries immediately after infection and is not much dependent on E. coli\nmetabolism. T4 then repurposes the existing intracellular resources to maximize its\nreplicative capacity and phage production. Therefore, there is no evidence that Hfq-\nminus E. coli metabolic state as such will have an impact on phage T4 yield.\nAs suggested by the reviewer, we have incorporated the above points in various\nsections of the revised manuscript, particularly Discussion (Pages 17-21).\nAdditionally, in view of the comments by the reviewers regarding Hfq’s role, we\nhave modified the title by removing the “host component” and centering the focus on\nthe genome retention mechanism.\nIn particular, is there a significant conservation of the gp14 amino acids that are binding\nto Hfq?\nWe thank the reviewer for this insightful comment. To address this, we performed\nsequence alignments of gp14 homologs from T4-like phages.\nSequence alignments show that the residues involved in Hfq binding are indeed\nwell-conserved among the T4-like phages with various degrees of genome divergence\n(Supplementary Fig. 7). In contrast, these residues and the corresponding interface are\nmissing in gp14 orthologs from non-T4-like phages such as SPP1 and lambda\n(Supplementary Table. 4, see below).\ngp15. (A) Sequence alignment of gp14 proteins from T4 and T4-like phages. Residues 90–189 of T4 gp14\nare shown, corresponding to the region primarily involved in Hfq binding. Conserved positions are\nhighlighted in red, with darker shades indicating complete identity. Residues participating in Hfq and gp15\ninteractions are indicated by magenta and green arrows, respectively, while residues contributing to both\ninteractions are marked by red arrows. PID: percentage of identity (compared to T4). (B) Structural views of\ngp14 in complex with Hfq or gp15. Left: gp14/Hfq complex. Right: gp14/gp15 complex. gp14 is shown as a\nyellow cartoon; Hfq and gp15 are displayed as surface representations (Hfq in purple; the two gp15 subunits\nin cyan and blue). (C) Two orientations of an individual gp14 subunit from the gp14/gp15 complex. The C-\nterminal domain, observed only in the gp14/gp15 structure, is shown in grey. gp14 residues involved in Hfq\nand gp15 interactions are colored magenta and green, respectively, and those participating in both interactions\nare highlighted in red.\nSupplementary Table 4: Surface charge of T4 gp14 bottom and its orthologs from other phages\nshowing surface charges.\nPhage T4 SPP1 Lamda\nProtein gp14 gp16 gpFII\nPDB This study 7z4w 8XOW\nSurface\ncharge\ninnovation unique to T4-like phages, providing a novel feature to enhance (maximize)\ngenome retention and phage yield by coopting an abundantly present host protein\nhexamer without incurring any genome or metabolic cost to phage itself.\nWe have incorporated the above points into the main text (Page 12) and added a\nnew Figure (Supplementary Fig 7) and a new table (Supplementary Table 4) in the\nrevised manuscript.\nMinor points\n1-Line 73: \"To our surprise, we discovered that a previously unknown protein,\nidentified as the E. coli Hfq\". \"Unknown\" is incorrect, the protein itself was known but\nits interaction with the T4 neck was unknown.\nWe have revised this sentence to: “To our surprise, we discovered that a 12 kDa\nprotein that was not previously identified as part of the T4 neck complex, determined to\nbe the Hfq of E. coli, is associated with the in vitro-assembled T4 neck\ncomplexes.”(Page 4)\n2-line 93-94 : \"purified the proteins (Fig. 1A and 1D)\" the test should refer to figure 1B\nas well (for gp13 and for gp14). An SDS-PAGE analysis of gp20 alone would be\nnecessary to confirm that this sample has also been correctly purified.\nAs the reviewer suggested, we have also referred to Figure 1B in the revised\nManuscript. Additionally, an SDS gel image of gp20 alone has also been added as\nFigure 1E for better clarity (Page 35).\nFig 1 (E) SDS-PAGE analysis of the SEC peak fraction of gp20 and gp20/13/14 complex.\n3-Line 149-150 \"Three glutamic acids (Glu284, Glu291, and Glu295) on α6 face the\nchannel, creating a negatively charged surface for smooth passage of DNA\" An image\nof the electrostatic potential would be useful to confirm this point.\nThank you for this suggestion. We have added a new panel (Fig. 2E) in the revised\nmanuscript showing the electrostatic potential of the channel interior, specifically\nhighlighting the region formed by α-helices containing Glu284, Glu291, and Glu295\n(Page 37). This clearly shows the strong negative charges lining the channel, which is\nconducive to repulsion and smooth passage of the negatively charged DNA.\nFig 2 (E) Three glutamic acids (Glu284, 291, and 295) lining on α6 of gp13, forming a negatively\ncharged channel surface for DNA passage.\n4-Fig 3B : Several labels are hardly readable. This includes R265 in blue and I262 in\ngreen\n5.\nFig 3 (B) Close-up view of gp13 and gp14 interfaces. Residues involved in hydrophobic and\nelectrostatic interactions are shown as sticks.\n5- Line 175-179, about the relative stability of the interfaces between gp13 and gp14 :\nthe text is ambiguous. 10675 Å² seems to correspond to the entire surface of interaction\nbetween the gp13 12-mer and the gp14 6-mer, while 1725.6 Å² seems to correspond to\nthe interface between two gp13 protomers. If it is correct, the latter value is not that\nsmall for a ring complex. As a comparison, the adaptor protein in HK97 assembles as\nstable rings despite a significantly smaller surface of interaction (1250 Å² per interface\nas calculated from the PDB entry 3JVO) than T4 gp13 (Cardarelli et al., JMB 2010). PISA\nnot only calculates the surface of interaction but also calculates a dissociation\nenergy of the complex, which is at least as important at the surface of interaction itself.\nProviding these values would be useful here.\nWe thank the reviewer for this insightful suggestion. The dissociation energy\n(ΔGdiss) is indeed an important parameter for analyzing complexes. We have now\nperformed a comprehensive dissociation energy analysis for all relevant complexes (see\nbelow) using PDBePISA, which provides energetic insights.\nSupplementary Table 2. Dissociation energy analysis of gp13, gp14, and their complexes.\n8\n△Gdiss (kcal/M) T△Sdiss(kcal/M)\ngp13 dodecamer 46.2 158\ngp14 hexamer 45.3 66.7\ngp13/gp14 140.4 18.1\ngp14/Hfq 13.6 14.5\ngp14/gp15 364.6 84.9\nΔGdiss: free energy of assembly dissociation (kcal/M), the free energy of dissociation corresponds to the\nfree energy difference between dissociated and associated states; TΔSdiss: rigid-body entropy change at\ndissociation (kcal/M). Positive values of ΔGdiss indicate that an external driving force should be applied\nin order to dissociate the assembly, therefore assemblies with high ΔGdiss are thermodynamically stable.\nHigh TΔSdiss indicates the binding is entropically costly and dissociation favorable. The Dissociation\nenergy was analyzed by PDBePISA.\nAmong gp13 dodecamer, gp14 hexamer, and gp13/gp14 complex, the isolated\ngp13 dodecamer and gp14 hexamer exhibit moderate thermodynamic stability (ΔGdiss\n= +46.2 and +45.3 kcal/M, respectively) but are characterized by large entropic\npenalties for dissociation (TΔSdiss = +158.6 and +66.7 kcal/M, respectively). The\ngp13/gp14 complex exhibits the lowest entropic penalty (TΔSdiss: +18.1 kcal/M) and\nhighest ΔGdiss (140.4 kcal/M), suggesting that complex formation is favorable. It is\nconsistent with our observation that gp13 and gp14 monomers do not spontaneously\nform stable oligomers individually, however mixing gp13 and gp14 monomers\nfacilitates the formation of gp13/gp14 complex.\nThus, the T4 neck complex appears to assemble through a cooperative mechanism\nin which two metastable components converge to generate a hyperstable structure. This\nstrategy ensures that a strong driving force for assembly is unleashed only upon correct\nformation of the functional unit, thereby preventing premature or erroneous interactions.\nIn this context, gp13 and gp14 homomeric assemblies are individually unstable, but\nwhen associated, form a robust complex. Notably, the cooperative mode of assembly,\nrather than the intrinsic stability of the individual components, underlies the fidelity and\nstability of the final structure.\nmodel into Results and Discussion sections of the revised manuscript (Pages 11 and 18\nin Main Text). The above table showing the thermodynamic parameters of various\ncomplexes is added to the Supplementary section (Supplementary Table 2).\n6-Figure 3C,3E and 4D : several hydrogen bonds or salt bridges seem to have unusual\ngeometries (K171/N143, K13/E13, N169/T142, R295/D51). Especially, the distance\nbetween R295/D51 is larger than 4Å which is very large for a salt bridge. The\nMaterial&Methods section indicate that Pisa has been used for calculating the interface\nareas, has it also been used for having a non-biased identification of polar contacts\nbetween chains?\nOur analysis was indeed guided by the PDBePISA software, which provides an\nunbiased identification of potential polar and ionic interactions based on geometry and\nenergy criteria. PISA can suggest potential stabilizing interactions even when distances\nare slightly beyond the strictest cutoffs, as the local chemical environment and\ndynamics can influence bonding. However, in view of the reviewer’s comment, we have\nre-evaluated the hydrogen bonds and salt bridges and performed further refinement of\nthe corresponding residues, including adjustment and optimization of side-chain angles.\nIn the revised figures, only hydrogen bonds and salt bridges within 4 Å are shown.\nBelow are the updated Figures 3C, 3D, 3E, and 4D:\nelectrostatic interactions are shown as sticks. (D) Close-up view of the gp14-Hfq interface. Residues\ninvolved in salt-bridge and hydrogen bonding interactions are shown as sticks. (E) Top view of the gp13\noligomeric ring, with each subunit in a different color. The boxed regions highlight the specific\ninteraction interfaces between monomers.\nFig 4 (D) Interface between the C-terminal arm of gp13 and two neighboring subunits of gp20.\nHighlighted residues are involved in hydrophobic and electrostatic interactions.\n11\n7-Supplementary Movie 2 : the movie suggests that the gp13 conformational change to\nstabilize its interaction with gp20 occurs after the tail has attached to gp14, which is\nextremely unlikely.\nWe apologize for this error. In the revised manuscript, we have updated the movie\nto show that the gp13 conformational change occurs upon its docking to the portal\nprotein (gp20), accurately reflecting the events during head-neck assembly prior to tail\nattachment. Additionally, we have added a note in the movie legend to clarify that the\nanimation is for illustrative purpose only.\n8- The stability of the gp14-Hfq complex is not discussed. A surface of interaction is\nprovided, but there is no corresponding dissociation energy. In the SDS-page analysis\non figure 1B, the amount of Hfq seems to be extremely small in comparison with gp14.\nThis suggests that Hfq slowly detaches from gp14 and that it could have been lost\nduring the size exclusion chromatography. A weak interaction is not surprising, given\nthat Hfq has to be expelled in the latter stages of assembly. It would thus be interesting\nto discuss this point and its possible implication for the assembly mechanism.\nWe thank the reviewer for these insightful comments. We conducted several additional\nanalyses to address this issue, providing deeper insights into the Hfq assembly/dis-\nassembly mechanism.\nI. About the dissociation energy and the stability of the gp14-Hfq complex:\nQuantitative analysis using PDBePISA provides a clear mechanistic explanation\nfor how the tail protein gp15 triggers Hfq displacement and the stable docking of the\ntail. The gp14–Hfq complex exhibits both a low TΔSdiss and low ΔGdiss, indicating that\nthe complex is not stable and it can dissociate easily. By contrast, the ΔGdiss of the\ngp14/gp15 complex is approximately 25 times greater than that of gp14/Hfq, reflecting\nmarkedly higher stability. This large difference in stability indicates that, from a\nthermodynamic perspective, gp15 binding to gp14 is strongly favored.\nThis is consistent with Hfq’s role, which is restricted to the neck intermediate and\ndissociates upon tail attachment. Hfq binds gp14 (ΔG_diss: +13.6 kcal/M), forming a\n12\nmetastable complex. Tail attachment allows gp15 to displace Hfq, forming an ultra-\nstable gp14/gp15 interface (△Gint of +364.6 kcal/M) that irreversibly locks the tail onto\nthe neck. Together, these data demonstrate that gp15 provides the energetic driving\nforce to disrupt the gp14/Hfq interaction, ensuring high-fidelity viral assembly.\nThis energy analysis table has been added to the revised manuscript to clarify this\nkey mechanistic step (Supplementary Table 2).\nSupplementary Table 2. Dissociation energy analysis of gp13, gp14, and their complexes.\n△Gdiss (kcal/M) T△Sdiss (kcal/M)\ngp13 dodecamer 46.2 158\ngp14 hexamer 45.3 66.7\ngp13/gp14 140.4 18.1\ngp14/Hfq 13.6 14.5\ngp14/gp15 364.6 84.9\nΔGdiss: free energy of assembly dissociation (kcal/M), the free energy of dissociation corresponds to the\nfree energy difference between dissociated and associated states; TΔSdiss: rigid-body entropy change at\ndissociation (kcal/M). Positive values of ΔGdiss indicate that an external driving force should be applied\nin order to dissociate the assembly, therefore assemblies with high ΔGdiss are thermodynamically stable.\nHigh TΔSdiss indicates the binding is entropically costly and dissociation favorable. The Dissociation\nenergy was analyzed by PDBePISA.\nII. Analysis of gp14/Hfq ratio based on the SDS-PAGE in Figure 1B:\nThe band intensity in SDS-PAGE primarily reflects the mass of the target proteins.\nThe molecular weight of gp14 (~34 kDa) is approximately three times of the Hfq (~11\nkDa), and larger proteins bind proportionally more Coomassie dye, resulting in a darker\nband for gp14 even in a 1:1 molar complex.\nThe sample shown in SDS-PAGE in Figure 1B was purified from overexpressed\ngp20, gp13, and gp14 proteins, with Hfq pulled down from E. coli, resulting in limited\nHfq availability. Cryo-EM analysis indicated that this sample contained\ngp20/gp13/gp14/Hfq and gp20/gp14/gp13 complexes, and also possibly\n13\ngp20/gp13/gp14. Quantification of gp14 and Hfq bands using ImageJ revealed a mass\nratio of gp14:Hfq approximately 7:1, corresponding to a molar ratio of ~2:1 (see below).\nTo supply sufficient Hfq, we overexpressed Hfq and co-purified it with gp20, gp13,\nand gp14. In this case, the assembled portal–neck complex contained only the\ngp20/gp13/gp14/Hfq complex, essentially complete occupancy of gp14. SDS-PAGE\nshowed that the Hfq band in this complex was darker than in the sample with limited\nHfq. Quantification using ImageJ revealed a mass ratio of gp14:Hfq of approximately\n3:1, corresponding to a molar ratio of ~1:1.\nSupplementary Table 3: Quantification of portal-neck complex by SDS-PAGE and\nImageJ.\nSDS PAGE Mean from imageJ Molar ratio\ngp14 Hfq gp14:Hfq gp14:Hfq\n36 12 3:1 1:1\nSufficient Hfq\n49 7 7:1 ~2:1\nLimited Hfq\nThe purification of portal-neck complex with excess Hfq yielded a homogeneous gp20/gp13/gp14/Hfq\ncomplex with an approximate 1:1 molar ratio of gp14 to Hfq, in contrast to the heterogeneous assembly\nobserved with limited Hfq supply.\nWe have added the following statement in the revised manuscrip t Page 14)：“SDS–\na homogeneous gp20/gp13/gp14/Hfq complex with an approximate 1:1 molar ratio of\ngp14 to Hfq, whereas limited Hfq resulted in a heterogeneous mixture containing both\ngp20/gp13/gp14/Hfq and gp20/gp14/gp13 complexes (~2:1 molar ratio of gp14 to\nHfq).”\nIII. The gp13/gp14/Hfq complex binds to packaged heads\nWe have conducted a new series of experiments to determine the functional\nimportance of the newly discovered gp13/gp14/Hfq complex by testing its ability to\nbind to in vitro packaged heads. Empty (emptied) heads were purified from\n10am13am23-DE mutant infections as described earlier (Zhang et al., 2011). The heads\nwere then filled in vitro with a ~6 kb linearized plasmid DNA using a defined packaging\nsystem (Zhu et al., 2023), and then incubated with purified gp13/gp14/Hfq complex\npurified by mixing overexpressed gp13, gp14, and Hfq extracts. The packaged heads\nwere then separated from unpackaged empty heads and unbound gp13/gp14/Hfq\ncomplex by CsCl density gradient centrifugation. The full-heads band at the bottom of\nthe gradient was extracted and analyzed by cryo-EM and Western blotting (see Figure\n1I & J below).\nCryo-EM images (panel I) showed that most of the heads appear full or nearly full,\nand many of the full-heads showed neck-like protrusions at the portal vertex likely\ncorresponding to the gp13/gp14/Hfq complex. Often, the heads were clustered through\ninteraction of these exposed necks in the absence of tail attachment.\nWestern blotting analysis showed that the gp13/gp14/Hfq complex is associated\nwith the packaged heads (panel J), demonstrating the functional importance of the\ngp13/gp14/Hfq complex. Closer examination of the intensity of the bands indicates that\nthe ratio of Hfq to gp13 and gp14 varied in different experiments and is lower than the\nexpected stoichiometry. This means that some of the Hfq might be falling off the neck\ncomplex because of its relatively weaker interaction. These results are consistent with\nthe energy calculations described above, and with its functional role, transiently\ninteracting with gp14 to reinforce the primary gp14 genome gate but leaving the neck\n15\nreadily when tail (gp15) attaches.\nThe Results (Page 7) and Discussion (Page 20) sections are expanded in the revised\nmanuscript by incorporating the above points and data.\n9- Line 353: \"the cells were harvested by centrifugation at 5,000 rpm\". The author\nshould either provide the centrifugation speed and the name of the rotor, or provide the\nnumber of g.\nWe have provided the centrifugal force (6240g）and the name of the rotor JLA-\n8.1 in the Methods section (Page 22).\n10- Line 642, legend of figure 2F: the author should precise which proteins/domains\nhave been used for the structural alignment.\nThe α6 helix of gp13 C-terminal has been used for the structural alignment. This\nstatement has been included in the revised manuscript (Page 38).\nThis is a well-written paper describing the assembly of the T4 portal protein complex\nand the function of an endogenous factor, identified as a plug, which prevents genome\nleakage at the end of packaging before tail assembly. The paper is clear and easy to\nunderstand. Although not groundbreaking, the authors present an interesting discovery\nthat could have implications for other T4-like phages.\nWe thank the reviewer for stating that our paper reports an “interesting discovery“,\nand for an overall positive assessment of our work and its presentation.\nWe should note that this paper reports the first atomic structures of portal-neck\nassembly intermediates, and the first complete viral genome retention mechanism of\nany tailed phage. This is a major advancement because the post-genome packaging\nmechanisms, particularly the highly vulnerable genome retention in a pressurized head,\nremained elusive despite decades of intensive research. Additionally, the paper reports\nthe discovery that a host component, Hfq, is coopted by T4 phage to enhance genome\nretention and phage production. \nAlso, is a ~2-fold decrease in phage yield in hfq-plus E. coli sufficient to conclude that\nHfq is physiologically important? What would the phage yield be if a common\nchaperone were deleted in E. coli? Overall, I like the paper but find its scope and\nsignificance somewhat limited to stand alone as a full article in Nature Communications.\nWe appreciate the reviewer’s concern. We apologize for not clearly explaining the\nrole of Hfq in the original manuscript. This was largely because we focused on the\nmechanism, not enough on dissecting the functional role of Hfq. We therefore thank the\nreviewer for the above comments which allowed us to address this concern in detail.\nThese comments also motivated us to perform additional experiments to demonstrate\nthe functional significance of the gp13/gp14/Hfq neck complex, as follows:\nI. We understand the reviewer’s point that a 50% reduction in phage burst size when\ngrown on E. coli Hfq mutant appears modest, if Hfq were an “essential” function.\nMultiple structures of the neck assemblies described in our manuscript demonstrate\nthat Hfq is not the primary genome gate. It is a secondary gate that reinforces the\nprimary genome gate formed by the phage-encoded gp14 protein. We should have made\nthis distinction more explicit in the original manuscript.\nThe gp14 gate is the one to contact the genome and stop it, while the secondary\nHfq gate merely supports it. In principle, the gp14 genome gate might be sufficient\n(mutants of gp14 are lethal) but having a second gate would make it fail-safe. Hence,\nHfq is an accessory to the phage neck.\nConsistent with its accessory function, Hfq’s role is transient as it leaves the neck\ncomplex once the tail approaches for docking onto the neck through interactions with\ngp15 situated at the top of the tail structure. Clearly, Hfq is not found in the virion as\ndemonstrated by Western blotting and structural analyses.\nOur energy calculations given above (Supplementary Table 2) in response to\nReviewer #1’s comment show that the gp14-Hfq interaction is weak (ΔGdiss =13.6\nkcal/M) and driven by modest enthalpic and entropic changes, indicative of a transient\nor regulatory association rather than a core structural component. It also dissociates\neasily when the tail attaches.\nFurthermore, as describe below, we have developed a new assay to test the ability\nof our in vitro-assembled gp13/gp14/Hfq complex to bind in vitro-packaged heads.\nData from these experiments show, consistent with the energy calculations, that the\ngp13/gp14/Hfq complex binds to packaged heads demonstrating its functional\nsignificance. Additionally, the data show that Hfq stoichiometry is variable and lower\nthan expected indicating that Hfq interaction might weaken when attached to the\npressurized head and some of it dissociates even when the tail is not attached.\nFinally, it is also worth noting that a 50% reduction in phage yield is significant,\ngiven that the yield for the wild-type (WT) phage is only ~100 phages/infected cell. A\n2-fold enhancement by Hfq therefore provides a significant survival advantage in the\nhighly competitive phage universe.\nIn fact, curiously, phage T4 evolved by incorporating many such accessory\n18\nfunctions that provide modest ~50% enhancement of phage yield. Examples include\nMotB, gp5.4, gp45.2, and so on (Patterson-West et al., 2018, doi: 10.3390/v10070343;\nMattenberger et al., 2025, doi: 10.1101/2025.08.28.672839; Fokine et al., in\npreparation). Referred to as “nonessential” genes, such accessory functions constitute\na significant portion of the T4 genome. What is novel and interesting here is co-opting\na host protein for enhancing genome retention and virion assembly with no genome or\nmetabolic cost to phage itself. This to our knowledge has not been observed in any other\nphage system so far.\nTherefore, our structural, genetic, biochemical, and binding/dissociation energy\nanalyses are consistent with Hfq serving in an accessory role through transient\nassociation with the neck complex for maximizing neck assembly and genome retention.\nII. With regard to Hfq-minus E. coli metabolism, it should be noted that E. coli has\nredundant regulatory systems that compensate for Hfq. The most impact Hfq has is\nwhen E. coli is grown in nutrient-deficient and other stress conditions. Under nutrient-\nrich conditions, the main effect observed is that the E. coli rods become longer because\nHfq does inhibit cell division. If anything, this phenotype might actually skew our\nresults towards higher phage yield because equal number of cells were used for\ninfection in our experiments to determine the phage yield. Bulkier cells may provide\nmore nutritional resources to support phage infection.\nFurthermore, phage T4, unlike most other phages, completely shuts down host\nmachineries immediately after infection and is not much dependent on E. coli\nmetabolism. T4 then repurposes the existing intracellular resources to maximize its\nreplicative capacity and phage production. Therefore, there is no evidence that Hfq-\nminus E. coli metabolic state as such will have an impact on phage T4 yield.\nIII. To address the question of physiological relevance, we have conducted a new series\nof experiments to determine if the newly discovered gp13/gp14/Hfq complex exhibit\nthe predicted function, assembly on packaged heads. Empty (emptied) heads were\npurified from 10am13am23-DE mutant infections as described earlier (Zhang et al.,\n19\n2011). The heads were then filled in vitro with a ~6 kb linearized plasmid DNA using\na defined packaging system (Zhu et al., 2023), and then incubated with gp13/gp14/Hfq\ncomplex purified by mixing over-expressed gp13, gp14, and Hfq extracts. The\npackaged heads were then separated from unpackaged empty heads and unbound\ngp13/gp14/Hfq\ncomplex by CsCl density gradient centrifugation. The full-heads band at the bottom of\nthe gradient was extracted and analyzed by cryo-EM and Western blotting (Figure\nbelow).\nCryo-EM images (panel I) showed that most of the heads are filled to near headful\ncapacity and many of the full-heads show neck-like protrusions at the portal vertex\nlikely corresponding to the gp13/gp14/Hfq complex. Often, the heads are clustered\nthrough interaction of these exposed necks in the absence of tail attachment.\nWestern blotting analysis showed that the gp13/gp14/Hfq complex is associated\nwith the packaged heads (panel J), demonstrating the functional importance of the\ngp13/gp14/Hfq complex. Closer examination of the intensity of the bands indicates that\nthe amount of Hfq associated varied in different experiments and is lower than the\nexpected stoichiometry. This means that some of the Hfq might be falling off the neck\ncomplex because of its relatively weaker interaction. These results are consistent with\nthe energy calculations described above, and with its functional role, transiently\ninteracting with gp14 to reinforce the primary gp14 genome gate but leaving the neck\nreadily when tail (gp15) attaches.\nFig 1: (I, J) Binding of gp13/gp14/Hfq neck complex to packaged heads. Binding of in vitro-\n20\nassembled and purified gp13/gp14/Hfq complex to in vitro-packaged heads was tested as described in\nMaterials and Methods. (I) Cryo-EM of the DNA-full heads purified by CsCl density gradient\ncentrifugation. Blue arrows point to neck-like protrusions at the portal vertex likely corresponding to the\nbound gp13/gp14/Hfq complex. (J) SDS-gel electrophoresis and Western blotting of the samples using\nHistag-specific antibodies. Gp17, gp13, gp14, and Hfq contain a Histag either at N- or C-terminus of the\nrecombinant protein. In lanes 2 and 3, gp17 and gp13-gp14-Hfq complex corresponding to 1:1 ratio of\npackaging motor (gp17 pentamer) or gp13/gp14/Hfq complex to head particles, respectively, were loaded\nfor comparison of the same in lane 5. Lanes 2-5 correspond to samples taken from the same experiment\nand lane 6 corresponds to the packaged head-gp13/gp14/Hfq complex isolated from an independent\nexperiment. Lanes 5 and 6 correspond to CsCl-purified packaged head-gp13/gp14/Hfq complexes from\ntwo independent experiments.\nWe believe that the above data and analyses thoroughly address the reviewer’s\nconcern.\nWe have incorporated the above points and the new data in various sections of the\nrevised manuscript, particularly Results and Discussion.\nAdditionally, in view of the comments by the reviewers regarding Hfq’s role, we\nhave modified the title by removing the “host component” and centering the focus on\nthe genome retention mechanism.\nOur sincere thanks to both the reviewers for the comments and questions, which\nresulted in greater illumination of the viral genome retention mechanism in\nbacteriophage T4.\nReferences:\nMattenberger, Y., Knyazhanskaya, E.S., Shneider, M.M., Buth, S.A., Nazarov, S., Robins, W.P.,\nLeiman, P.G., and Belin, D. (2025). The spike tip protein of bacteriophage T4. bioRxiv,\n2025.2008.2028.672839.\nPatterson-West, J., Arroyo-Mendoza, M., Hsieh, M.L., Harrison, D., Walker, M.M., Knipling, L.,\nand Hinton, D.M. (2018). The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves\nPhage Fitness. Viruses 10.\nZhang, Z., Kottadiel, V.I., Vafabakhsh, R., Dai, L., Chemla, Y.R., Ha, T., and Rao, V.B. (2011). A\npromiscuous DNA packaging machine from bacteriophage T4. PLoS Biol 9, e1000592.\nZhu, J., Batra, H., Ananthaswamy, N., Mahalingam, M., Tao, P., Wu, X., Guo, W., Fokine, A., and\nRao, V.B. (2023). Design of bacteriophage T4-based artificial viral vectors for human genome\nremodeling. Nat Commun 14, 2928.\n21"}, {"round": 2, "reviewer_comments": {"reviewer_1": "This article by Han et al. has been significantly improved after this revision. The main issue in the first submission was about the importance of the host protein Hfq in the phage channel closure, which was only weakly supported by the data. The new data are useful and they clarify the role of Hfq as an accessory rather than essential protein for this process. The corresponding parts in the Results and Discussion section have been rewritten and now the conclusions are well supported by the data.\nThe different minor points highlighted after the previous prevision have also been taken into account, which improves the quality of the article\nAfter this revision, The Han et al. provide robust, interesting and unexpected data about the genome retention mechanism by bacteriophage T4 during particle assembly.", "reviewer_2": "The authors have responded to the reviewers' comments and made significant revisions to the manuscript. They present evidence that Hfg is hijacked from the host and pre-assembled to gp14, the primary genome-gating factor in T4.\nThe paper is certainly improved, especially in terms of clarity and readability. However, I still find the scope to be quite limited. The discovery of a non-essential host factor (Hfg) that aids in retaining DNA within the phage with gp14 is relevant but largely specific to phage T4. While this is interesting, I do not see the conceptual significance or broad advancement I would expect for a Nature Communications paper. In this regard, the paper may be more suitable for a specialized journal focused solely on microbiology or structural biology."}, "author_response": ""}]

Decision letter