Structure-Function Analysis of the ϕX174 DNA-Piloting Protein Using Length-Altering Mutations

Abstract

Although the X174 H protein is monomeric during procapsid morphogenesis, 10 proteins oligomerize to form a DNA translocating conduit (H-tube) for penetration. However, the timing and location of H-tube formation are unknown. The H-tube's highly repetitive primary and quaternary structures made it amenable to a genetic analysis using in-frame insertions and deletions. Length-altered proteins were characterized for the ability to perform the protein's three known functions: participation in particle assembly, genome translocation, and stimulation of viral protein synthesis. Insertion mutants were viable. Theoretically, these proteins would produce an assembled tube exceeding the capsid's internal diameter, suggesting that virions do not contain a fully assembled tube. Lengthened proteins were also used to test the biological significance of the crystal structure. Particles containing H proteins of two different lengths were significantly less infectious than both parents, indicating an inability to pilot DNA. Shortened H proteins were not fully functional. Although they could still stimulate viral protein synthesis, they either were not incorporated into virions or, if incorporated, failed to pilot the genome. Mutant proteins that failed to incorporate contained deletions within an 85-amino-acid segment, suggesting the existence of an incorporation domain. The revertants of shortened H protein mutants fell into two classes. The first class duplicated sequences neighboring the deletion, restoring wildtype length but not wild-type sequence. The second class suppressed an incorporation defect, allowing the use of the shortened protein. IMPORTANCEThe H-tube crystal structure represents the first high-resolution structure of a virally encoded DNA-translocating conduit. It has similarities with other viral proteins through which DNA must travel, such as the ␣-helical barrel domains of P22 portal proteins and T7 proteins that form tail tube extensions during infection. Thus, the H protein serves as a paradigm for the assembly and function of long ␣-helical supramolecular structures and nanotubes. Highly repetitive in primary and quaternary structure, they are amenable to structure-function analyses using in-frame insertions and deletions as presented herein. Bacteriophages have evolved several mechanisms to transport hydrophilic genomes through cell walls containing hydrophobic membranes and peptidoglycan. Myoviruses and podoviruses carry tails that, respectively, utilize contractile sheaths (1) and form extensions within the membrane (2). In contrast, siphoviruses, filamentous phage, and other tail-less viruses co-opt host cell membrane channels for penetration (3)(4)(5). However, the tailless microvirus X174 does not require a host-provided conduit for genome transport. Instead, genome translocation is mediated by 10 to 12 copies of the DNA-piloting protein H found inside the virion (6-8).The X-ray structure of H protein's coiled-coil domain (8, 9) shows 10 parallel proteins oligomerized into a tube (Fig. 1) Th...

Finally, a Role Befitting A star : Strongly Conserved, Unessential Microvirus A* Proteins Ensure the Product Fidelity of Packaging Reactions

“…This may explain why the duplications occurred in the noncoding J-F intercistronic region, but it does not address if more DNA, regardless of sequence or location, can assuage XG4J's lethal phenotype. However, there are coding sequences, primarily in gene H, that tolerate in-frame insertions and duplications that do not affect the protein's folding and function (47). Although gene H duplications were not recovered as suppressors, a viable Hϩ42 gene was placed directly in the XGJ background.…”

“…Similar in vivo studies have been conducted to define X174's upper packaging limit. However, in these studies the packaging substrates were phage genomes containing insertions, which ranged in size from a 100.7 to a 103% unit length (47,55,56). The XG4Jϩ210 genome is ϳ104% unit length.…”

“…The XG4Jϩ42H contains a viable insertion in gene H (47). It was constructed as described above using XG4J DNA as a template and abutting primers introducing the gene H insertion as previously described (47). The mutant was recovered in cells expressing the X174 J gene.…”

Mutagenic Analysis of a DNA Translocating Tube’s Interior Surface

“…Gene H mutations can prevent H protein incorporation during morphogenesis or result in H-containing, uninfectious particles [32,44]. To determine whether the H proteins were incorporated at 22 • C, lysis resistant cells expressing the wild-type, T244Q, M251Q, and T244Q/M251Q H genes were infected with am(H)M251.…”

Breaking Symmetry in Viral Icosahedral Capsids as Seen through the Lenses of X-ray Crystallography and Cryo-Electron Microscopy

“…The H “pilot” protein is discreetly packaged inside the capsid until it forms a genome delivery tube at the moment of infection [ 122 , 123 ] ( Figure 2 c). The mechanism by which ФX174 senses cellular contact and builds this apparatus is not yet known, although functionality of the tube is linked to protein length [ 124 ]. Interestingly, the H-protein tube structure is formed by long α-helices and resembles the C-terminal portal protein barrel within Podoviridae ( Figure 1 a and Figure 2 c).…”