Efficient DNA Packaging of Bacteriophage PRD1 Requires the Unique Vertex Protein P6

Abstract

The assembly of bacteriophage PRD1 proceeds via formation of empty procapsids containing an internal lipid membrane, into which the linear double-stranded DNA genome is subsequently packaged. The packaging ATPase P9 and other putative packaging proteins have been shown to be located at a unique vertex of the PRD1 capsid. Here, we describe the isolation and characterization of a suppressor-sensitive PRD1 mutant deficient in the unique vertex protein P6. Protein P6 was found to be an essential part of the PRD1 packaging machinery; its absence leads to greatly reduced packaging efficiency. Lack of P6 was not found to affect particle assembly, because in the P6-deficient mutant infection, wild-type (wt) amounts of particles were produced, although most were empty. P6 was determined not to be a specificity factor, as the few filled particles seen in the P6-deficient infection contained only PRD1-specific DNA. The presence of P6 was not necessary for retention of DNA in the capsid once packaging had occurred, and P6-deficient DNA-containing particles were found to be stable and infectious, albeit not as infectious as wt PRD1 virions. A packaging model for bacteriophage PRD1, based on previous results and those obtained in this study, is presented.The encapsidation of viral double-stranded DNA (dsDNA) genomes in a protective spherical protein capsid presents a unique set of challenges. First, the genomes of icosahedral dsDNA viruses are enormous in length compared to the inner diameter and volume of the capsids they are to occupy. Packaging DNA to such a high density is energetically extremely unfavorable. Second, the DNA needs to be topologically organized so that when a suitable host cell is encountered, the genome can efficiently be translocated into the cell and a new round of infection can be initiated. Specificity is an additional challenge, because energy and material should not be wasted for nonproductive packaging of host DNA into the viral capsids.Icosahedral dsDNA viruses have developed ingenious machinery to perform such a complicated task. Encapsidation of the dsDNA genomes proceeds via formation of empty procapsids, devoid of DNA, that subsequently are packaged with DNA by a specific enzyme, called the terminase or packaging ATPase (for recent reviews, see references 24 and 46). This process requires a large amount of energy, usually provided in the form of ATP. It has been estimated that, for example, in the case of bacteriophage phi29, an average of 1 molecule of ATP per each 2 bp of DNA packaged is needed (38). Packaging occurs through a specific vertex of the capsid, which contains a ring-like portal structure, through which the DNA is threaded into the particle. However, packaging machineries are not quite as simple as this; many additional proteins, and in some cases even RNAs, are involved, with the details varying from one virus to another.The problem with studying these intricate apparatuses is their asymmetric location in the virion, which makes conventional structure determination methods that ...

Ternary Complex Formation on the Adenovirus Packaging Sequence by the IVa2 and L4 22-Kilodalton Proteins

“…For example, the molecular motor of bacteriophage T4 depends on complexes containing crucial accessory proteins and an active ATPase (29), and PRD1, which has many structural similarities with adenovirus (2), has a unique portal that possesses ATPase activity (20). Although no ATPase activity has yet been ascribed to the IVa2 protein, mutations in a putative Walker A motif of this protein, which is characteristic of AAAϩ ATPases (5), resulted in nonviable virus (27).…”

Probing protein interactions in the membrane-containing virus PRD1

“…Western blotting was performed by transferring the proteins from the SDS-PAGE gels onto PVDF membranes (Millipore), followed by visualization with peroxidaseconjugated secondary antibodies (Dako) and chemiluminescent detection using a Super Signal West Pico (Thermo) system. Specific rabbit antisera recognizing PRD1 proteins P2 (Grahn et al, 1999), P6 (Karhu et al, 2007), P3 (Rydman et al, 2001), P5 (Hänninen et al, 1997), P9, P22 (Strömsten et al, 2003), P15 (this study) and P31 (Rydman et al, 1999) as well as mAbs 7N41 (for P7 and P14), 11T25 (for P11) and 16A201 (for P16) (Hänninen et al, 1997) were used as primary antibodies for sequential labelling.…”

“…One of the fivefold vertices is different (special vertex), where the DNA-packaging machinery exists (Gowen et al, 2003;Strömsten et al, 2003). This special vertex complex consists of the integral membrane proteins P20 (5 kDa) and P22 (6 kDa) as well as packaging efficiency factor P6 (18 kDa) and packaging ATPase P9 (26 kDa) (Karhu et al, 2007;Strömsten et al, 2003Strömsten et al, , 2005. Although an in vitro DNA-packaging system is available (Ž iedaitė et al, 2009), the underlying molecular details of the DNA-packaging machinery and its structure remain to be solved.…”

“…In PRD1, the numerous suppressor-sensitive mutants have been invaluable in studying protein interactions and assigning functions to the corresponding proteins, e.g. revealing mechanisms for virus genome packaging (Karhu et al, 2007;Strömsten et al, 2005) or entry Grahn et al, 2002a, b). PRD1 is the first and the only virus with an internal membrane so far solved by X-ray crystallography at the atomic level (~4 Å resolution; Abrescia et al, 2004;Cockburn et al, 2004).…”

An icosahedral algal virus has a complex unique vertex decorated by a spike

“…Some icosahedral viruses, including tailed bacteriophages, HSV (15,23), mimivirus (12,16), and PRD1 (21,24), have a specialized pentameric vertex that is associated with genomic packaging and/or cell entry. Even the small icosahedral poliovirus has or develops a special pentameric vertex when delivering its genome to its host (25).…”