Bacteriophage PR772 is a double-stranded DNA (dsDNA) virus with a 70 nm diameter icosahedral protein capsid encapsulating the internal lipid bilayer along with numerous proteins and a 15 kbp long linear genome. It belongs to the Tectiviridae family and infects gram negative hosts like Escherichia coli, Salmonella typhimurium and other bacteria, carrying a R772 plasmid-encoded receptor complex through which DNA can be transported during bacterial conjugation (Coetzee et al., 1979; Coetzee and Bekker, 1979; Mäntynen et al., 2019; Reddy et al., 2017). Much of the functional knowledge of the viral proteins is inferred from previous studies on PRD1, a close relative of PR772 with a genome sequence identity of 97.2% (Lute et al., 2004; Saren et al., 2005). The most striking features of phages from those members of the Tectiviridae family that infect Gram-negative bacteria, is the presence of an inner lipid membrane and lack of a tail in the dormant viral particle. During the process of infection, these viruses produce a membranous tube derived from the inner membrane of the viral particle, lined from the inside by proteins P7, P11 and P32. This tube is used to inject the viral dsDNA into the host (Peralta et al., 2013; Saren et al., 2005).

Genome analysis of PR772 identified 32 open reading frames (ORFs) containing at least 40 codons (Lute et al., 2004). Twenty-eight annotated proteins are known to be expressed from the genome, of which three do not make it into the final assembly (Butcher et al., 2012; Lute et al., 2004). In previous studies on PRD1, it was shown that the capsid is formed by proteins P3 (major capsid protein), P30 (capsid associated protein) and the vertex complex. The vertex complex includes the penton protein (P31), the host-recognition protein (P5) and the receptor-binding protein (P2) along with the infectivity protein (P16) that acts as a cementing protein, holding the vertex complex together and also stabilizing the pseudo-icosahedral capsid of the virus (Saren et al., 2005). Proteins P6, P9, P20 and P22 are involved in DNA packaging and proteins P7, P14, P11, P18, P32 and P34 are responsible for DNA delivery to the host (Grahn et al., 2002; Lute et al., 2004). The Tectiviridae family of viruses are also known to have structural similarities to adenovirus (Saren et al., 2005).

In previous studies on bacteriophage PRD1, many models have been proposed to explain the architecture of the penton base and the vertex complex. Most of these models were speculations based on the observations from gene mutation/knock-out and in-vitro studies of proteins P31, P5 and P2 from PRD1. The in-vitro protein expression studies showed that P31 forms pentamers and P5 form multimers of trimers (i.e, (P5₃)₁, (P5₃)₂, (P5₃)₃) (Sokolova et al., 2001). The gene knock-out studies showed that P31^- mutants produced incomplete PRD1 particles that lacked P31, P5 and P2 functions. They failed to form the vertex complex. A P5^- mutant produced intact viral particle but lacked both P5 and P2 functions (Rydman et al., 1999). These observations led to the hypothesis that the vertex complex appears to be a single spike formed by a pentameric P31 base binds the trimeric P5 spike protein to which P2 is bound (Huiskonen et al., 2007; Caldentey et al., 2000; Rydman et al., 1999; Sokolova et al., 2001). Later, a combination of SAXS modeling of the P5 protein and a low resolution cryoem study of the vertex region, showed that P2 and P5 form two spikes, not one, as previously described. Based on the low-resolution SAXS model of P5, it was speculated that the N-terminal base of P5 could have a size similar to P31 due to sequence similarity (Huiskonen et al., 2007). With the available experimental data, the generally accepted composition of the vertex complex of Tectiviridae is that P31 is a homopentamer and forms the penton base. P5 and P2 are attached to the P31 penton base but their arrangement is not known. (Butcher et al., 2012; Huiskonen et al., 2007). The high resolution x-ray crystallographic structure of the P2 protein is known but the location and orientation with respect to the penton base in its functional form is currently not known (Xu et al., 2003). Putatively, it is suggested that the beta-propeller motif of P2 might be involved in binding to the host receptor.

Here we present the high-resolution structure of bacteriophage PR772 using electron cryo-microscopy (CryoEM). The high-resolution map helped us to resolve subtle variations in the protein conformations and their influence on the formation of a viable viral particle. The N-terminal region of the P3 subunits have three conformations, not two as previously described (Abrescia et al., 2004). The new N-terminal P3 conformation plays an important role in accommodating the P30 protein during particle assembly. The C-terminal region of P3 not only shows the formation of a β-sheet with P30 but also helps in locking the adjacent trisymmetrons through a hinge mechanism, thus facilitating the formation of icosahedral particles and regulating their size. Localized asymmetric reconstruction of the vertex region of PR772 revealed a P5-P31 heteropentameric base and the binding of P2 to P5 in the complex. A combination of high-resolution icosahedral symmetrized single-particle reconstruction, localized asymmetric reconstruction and focused classification has enabled us to answer some of the intriguing questions about the particle architecture, composition of the penton base and arrangement of the vertex complex.

Each trisymmetron of PR772 is composed of 12 trimers of the P3 protein that are in turn bound by P30, an overall arrangement similar to that of the close relative PRD1 (Abrescia et al., 2004). The high-resolution structure of PR772 confirms the previously shown variability of the N and C-terminals of the P3 protein depending on the locations within the trisymmetron. The high-resolution also allows us to extend the analysis of these conformations and show that the N-terminal of P3 can adopt three different conformations, not two as previously described. (Abrescia et al., 2004; Butcher et al., 2012) (Figures 2A and 3B). The newly discovered conformation of the N-terminal region of P3, a long helix with a kink, is structurally critical to accommodate the interlocking region formed by the N-terminal hooks of P30 during the initiation of particle assembly. The N-terminal region of P3 shows plasticity and play an important role in both stabilizing the trimer and anchoring the trimer complex to the membrane. The C-terminal region of P3 also shows differences in its conformation as compared to PRD1. In trimer 1 (Figure 2A,C and H) subunit a and subunit c have elongated C-terminal regions that interact with the adjacent P3 subunits of the neighboring trisymmetrons and in-turn locking on to P30 (Figure 2 right hand side insert; and Figure 6). A similar arrangement can be seen in the C-terminal region from subunit c of trimer two and subunit a of trimer 4 of the neighboring trisymmetrons (Figure 2 left hand side insert). The presence of P16 close to the penton region makes the interaction of all the neighboring trimer one more stable (Figure 6) and also anchors the whole vertex complex to the membrane (Figure 7), emphasizing the role of P30 and P16 in maintaining the size and structure of the icosahedral viral capsid.

Figure 7

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Our studies show that unlike what was predicted for PRD1, the penton base of PR772 is an asymmetric heteropentamer consisting of three copies of P5 and two copies of P31 (Figure 4F). P31 has high sequence similarity with the N-terminal domain of P5. Previously, it was shown that P31 can replace P5 to form the penton and produce an intact viral particle (Bamford and Bamford, 2000). Even though the viral particles were intact, they were non-infectious due to the lack of the viral receptor-binding protein, P2, that is bound to the host recognition protein, P5 (Bamford and Bamford, 2000; Grahn et al., 1999). In another study with the PRD1 sus525 mutant that lacks P31, it was also shown that these particles lacked the vertex complex (Rydman et al., 1999). With the current model, P31 would promote the formation of an intact viral particle by avoiding the steric-hinderances that could occur during the formation of the vertex complex, if the P31 subunits were replaced by P5. A penton with 3 copies of the P5 subunits will support (i) the formation of a stable collagen-like motif (Figure 7) and (ii) the formation of the C-terminal host recognition domain of P5. A heteropentameric base of the vertex complex with three copies of P5 and two copies of P31 address both these issues.

The members of the Tectiviridae family have been shown to have structural similarities with adenoviruses despite infecting different hosts. In adenoviruses, the symmetry mismatch seen in the vertex region is solved by the interaction of the tail region of the trimeric fibre with three of the five grooves formed by the subunits of the penton base (Cao et al., 2012). Unlike in adenoviruses, a similar problem in PR772 is solved by domain swapping. The trimeric P5 protein responsible for the formation of the spike, swaps the N-terminal base domain with three copies of the P31 protein (Figure 4F).

The localized asymmetric reconstruction shows that P2 is bound to P5 (Figure 5). In the current CryoEM data, the orientation of P2 with respect to the penton base is reversed when compared to what was described for PRD1 (Huiskonen et al., 2007; Xu et al., 2003). Here, the beta-propeller motif of P2 with domain I and II, interacts with the N-terminal base of P5 and also with the stalk. This was also speculated on in a previous study, where it was noted that members of the Tectiviridae family with large sequence variation in the beta-propeller motif of P2 also showed variations in the P5 subunit as a compensatory effect (Saren et al., 2005). P2 interacts with both the N-terminal base and the stalk region of P5 but does not interact directly with any other structural protein in PR772. The interaction of P2 with the P5 stalk stabilizes the C-terminal region of P5 (Figure 5). In the case of PRD1, P2 seems to occupy all vertices except for the unique packaging vertex (Butcher et al., 2012; Caldentey et al., 2000; Saren et al., 2005). In this scenario, the special conformation in which the N-terminal residues of P5 are wedged in between the neighboring P3 subunits was found to be prominent (Abrescia et al., 2004). In the current model of PR772, P2 seems to occupy only 2 vertices of the icosahedral shell. The lower quantities of P2 in PR772 were also indicated in a previous study, comparing the abundance of different viral proteins from various members of the Tectiviridae family using western blots (Saren et al., 2005). In PRD1, it was shown that the loss of P2 from a packed viral particle, resulted in spontaneous release of genomic DNA, leading to loss of infectivity and the formation of empty particles (Grahn et al., 1999). In a previous study of PR772, it was shown that after purification, the concentration estimates of the particle by plaque assay and nano-particle tracking analysis were similar (Reddy et al., 2017). PR772 particles remained intact and infectious after purification. The intact PR772 particles can also be observed in the CryoEM micrographs (Supplementary file 3). So, it is unlike that P2 was lost during the purification.

In the icosahedrally averaged model of PR772, we see that the special conformation of P5 where the N-terminal residues are wedged between the P3 subunits of the adjacent trimer one is not prominent, but these P5 densities are visible at lower RMSD values. The presence of P2 in the vertex complex may coincide with the N-terminal region of P5 adopting the special conformation. It was reported that, compared to the CryoEM map of a wild type PRD1 virion, the CryoEM map of sus690, a mutant PRD1 virion that lacks P2 and P5, showed absence of density in the region where we see the N-terminal of P5 wedged between two neighboring P3 subunits. The lack of this density was attributed to the conformational changes in P31 due to the binding of P5 or the presence of P5 in between P31 and the P3 trimer (Huiskonen et al., 2007). In light of the current findings in PR772, P5 is known to be part of the heteropentameric base forming the penton and the absence of the above-mentioned density confirms that P5 adopts the special N-terminal conformation and not P31.

The above mentioned observations and the quantitative analysis of different proteins from PRD1 and PR772 using western blots (Saren et al., 2005) could explain the discrepancy in P2 packing in PR772. As mentioned earlier, the presence of P2 in the vertex complex may coincide with the N-terminal region of P5 adopting the special conformation. This special conformation of N-terminal region of P5 could be linked to the low copy number of the membrane protein P14 (or higher copy number of P7) observed in PR772 when compared to PRD1 (Saren et al., 2005). In PRD1, gene VII codes for P7 and the 3’ region of the same gene also codes for P14. P14 is smaller and it consists only of the membrane anchor domain and lacks the trans-glycosylase domain that is seen in P7. These proteins, P7 and P14, are proposed to forms a hetero-multimeric complex located under the vertices of the PRD1 particle (Mattila et al., 2015; Rydman and Bamford, 2000). In an intact PRD1 particle, the copy numbers of P7 and P14 are similar but in case of PR772, the copy number of P14 is significantly lower and the copy number of P7 seems higher (Mattila et al., 2015; Saren et al., 2005). In PR772, the copy number of P14, the occurrence of the special conformation of the N-terminal region of P5 protein and the number of P2 proteins packed in an intact viral particle seems to be related. In PR772, the presence of the larger P7 protein in higher numbers at the vertex (along with other proteins in the disordered region) might hinder the formation of the special conformation of the N-terminal region of P5, which could reduce the number of P2 proteins packed in the particle.

The penton has no obvious direct interaction with the neighboring P3 subunits of the capsid, except in the rarely occurring special conformation of P5 where the N-terminus is wedged in between the P3 subunits of trimer one as mentioned above (Figure 4G–H). The difference density map of the vertex region and the modeled penton shows that the disordered region of P16 interacts with the penton (Figure 6—figure supplement 3). These interactions of P16 with P3, P5, P31 along with the membrane seem to play a central role in the formation of the vertex complex and anchoring it to the membrane. In earlier Raman spectroscopy studies on PRD1, it was shown that in the initial stages of viral assembly, P3 and P5 formed a precursor shell assisted by assembly factors and other membrane-associated proteins such as P16 (Bamford et al., 1990; Bamford et al., 1995). Similarly, in PR772 the P5 trimers could be accommodated during the formation of the procapsid and two copies of P31 are later added to the penton region, forming a heteropentameric penton.

From the localized asymmetric reconstruction of the vertex complex, one can notice that there is a variation in the map density below the penton. The map density below the P5 subunits is significantly different when compared to the same region below the P31 subunits. This suggests that, along with the interactions of P16 with P5 and P31, there could also be poorly resolved proteins like P11, P7, P14 or P18, etc; in the region (Luo et al., 1993; Mattila et al., 2015; Rydman and Bamford, 2000). The variation in the map density in this region is also compounded by other interactions, like that of the C-terminal Gly84 of P30 with the Met19 of P5 subunits and lack thereof with respect to the P31 subunits.

Preliminary results for the whole particle asymmetric reconstruction using symmetry relaxation in EMAN2 of the wild type PR772 do not show the presence of a unique packaging portal in the dormant particle in contrast to PRD1. All the vertices of PR772 show the heteropentameric arrangement of the penton with three subunits showing the stalk and the other two without the stalk (Figure 4C–D, Supplementary file 2).

Figure 7 shows our model of the vertex complex in PR772. Using this model, we propose a mechanism for the initiation of infection. In analogy to studies on PRD1, it is known that P5 is needed for host recognition, but the binding of P5 to the host is transient (Grahn et al., 1999). The host binding is stabilized by the high-affinity interaction of P2 and its receptor, locking the viral particle to the host (Grahn et al., 1999). The relative changes between P5 and P2 upon binding to the receptor on the host could trigger the disruption of the vertex complex by pulling the N-terminal region of the P5 trimers that are wedged between the P3 subunits. This will in turn disrupt the interaction between the P5 proteins and P30 as seen in Figure 6. This disruption cascades further and disturbs the interaction between P30 and P16, rendering the whole vertex complex unstable. P30 dimers could also transduce these effects to the neighboring vertices. The disruption of the vertex complex exposes the viral membrane and membranous proteins like P18, P11/P7, P32 etc; to the host surface to facilitate the formation of the membranous tube for DNA transport. P16, that holds the vertex complex, along with other membranous proteins, could act as a protein tether that would help in moving the lipid membrane closer to the host (Figure 6—figure supplement 4).

CryoEM Density maps and atomic models that support the findings of this study have been deposited in the Electron Microscopy Database and the Protein Databank with the accession codes EMD-4461 (Whole particle reconstruction), EMD-4462 (Vertex Complex), EMD-10237 (Localized reconstruction of the penton region), EMD-10238 (Focused Classification of the penton region) and PDB ID 6Q5U (Atomic model of the asymmetric unit).

1. 1. Hemanth KN Reddy
   2. Marta Carroni
   3. Janos Hajdu
   4. Martin Svenda

   (2019) Electron Microscopy Data Bank

   ID EMD-4462. Vertex Complex of Bacteriophage PR772.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4462
2. 1. Hemanth KN Reddy
   2. Marta Carroni
   3. Janos Hajdu
   4. Martin Svenda

   (2019) Protein Data Bank

   ID 6Q5U. High resolution electron cryo-microscopy structure of the bacteriophage PR772.

   https://www.rcsb.org/structure/6Q5U
3. 1. Hemanth KN Reddy
   2. Marta Carroni
   3. Janos Hajdu
   4. Martin Svenda

   (2019) Electron Microscopy Data Bank

   ID EMD-4461. High resolution electron cryo-microscopy structure of the bacteriophage PR772.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4461
4. 1. Hemanth KN Reddy
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   (2019) Electron Microscopy Data Bank

   ID EMD-10238. Focused Classification of the vertex region of Bacteriophage PR772 showing the heteropentameric penton.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-10238
5. 1. Hemanth KN Reddy
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   3. Janos Hajdu
   4. Martin Svenda

   (2019) Electron Microscopy Data Bank

   ID EMD-10237. Localized Reconstruction of the vertex region of Bacteriophage PR772 showing the heteropentameric penton.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-10237

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Author details

1. Hemanth KN Reddy

   Department of Cell and Molecular Biology, Laboratory of Molecular Biophysics, Uppsala University, Uppsala, Sweden

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   hemanth.kumar@icm.uu.se

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4698-8005

2. Marta Carroni

   Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

   Contribution

   Resources, Data curation, Validation, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7697-6427

3. Janos Hajdu

   1. Department of Cell and Molecular Biology, Laboratory of Molecular Biophysics, Uppsala University, Uppsala, Sweden
   2. Institute of Physics, ELI Beamlines, Academy of Sciences of the Czech Republic, Prague, Czech Republic

   Contribution

   Resources, Supervision, Funding acquisition, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3747-2760

4. Martin Svenda

   1. Department of Cell and Molecular Biology, Laboratory of Molecular Biophysics, Uppsala University, Uppsala, Sweden
   2. Department of Applied Physics, Biomedical and X-Ray Physics, KTH Royal Institute of Technology, Stockholm, Sweden

   Contribution

   Conceptualization, Resources, Supervision, Validation, Investigation, Project administration, Writing—review and editing

   For correspondence

   Martin.Svenda@icm.uu.se

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1162-8285

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