Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: prohead restoration for DNA-gp3 packaging and assembly.

The DNA-protein complex DNA-gp3 of phi 29 is efficiently packaged into purified proheads with the aid of plasmid-derived gp16. The filled heads can be assembled to phage by addition of an extract providing the products for neck-tail assembly (Bjornsti et al., J. Virol. 50:766-772, 1984). However, purified proheads lost their competence to package DNA-gp3 upon storage for 2 months at 4 degrees C. Competence was restored by complementation with extracts of certain mutant-infected cells, and these experiments demonstrated that late proteins were not involved; restoration obtained with 4-8-14--infected cells was indistinguishable from that obtained with 7-8-14--infected cells. 2-8-14- and 3-8-14- extracts restored about one-third of the capacity to package exogenous DNA-gp3. A 1-8-14- extracts restored activity to package 20.6% of the DNA-gp3 added, but phage were not produced.     

Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage phi 29

The ATPase activity of the DNA packaging protein gp16 (gene product 16) of bacteriophage phi 29 was studied in the completely defined in-vitro assembly system. ATP was hydrolyzed to ADP and Pi in the packaging reaction that included purified proheads, DNA-gp3 and gp16. Approximately one molecule of ATP was used in the packaging of 2 base-pairs of phi 29 DNA, or 9 X 10(3) ATP molecules per virion. The hydrolysis of ATP by gp16 was both prohead and DNA-gp3 dependent. gp16 contained both the "A-type" and the "B-type" ATP-binding consensus sequences (Walker et al., 1982) and the predicted secondary structure for ATP binding. The A-type sequence of gp16 was "basic-hydrophobic region-G-X2-G-X-G-K-S-X7-hydrophobic", and similar sequences were found in the phage DNA packaging proteins gpA of lambda, gp19 of T7 and gp17 of T4. Having both the ATP-binding and potential magnesium-binding domains, all of these proteins probably function as ATPases and may have common prohead-binding capabilities. The phi 29 protein gp3, covalently bound to the DNA, may be analogous in function to proteins gpNul of lambda and gpl of phi 21 that bind the DNA.     

Prohead RNA of bacteriophage phi 29: size, stoichiometry and biological activity.

We previously demonstrated (Guo et al., 1987. Nucl. Acids Res. 15, 7081-7090) that purified proheads of bacteriophage phi 29 contain an RNA of 120 bases which is essential for DNA packaging. Here we report that this RNA exists primarily as a polymer of ca. 174 residues in phage-infected cells and that ca. 54 bases are cleaved from its 3'-terminus by adventitious nucleases during the purification of proheads. The long and short forms of the RNA had similar activity in in vitro DNA packaging and phage assembly. We report the sequence of the long form of the RNA and show that similar long and short forms can be isolated from the proheads of the phi 29 relatives phi 21, phi 15 and SF5. The concentration dependence in the reconstitution of RNA-free proheads suggests that one copy of the RNA is sufficient to restore DNA packaging activity to RNA-free proheads. However, quantitative measurements indicate that 5 to 6 copies of the RNA are present on proheads isolated from phage-infected cells.     

RNA dependence of the bacteriophage phi 29 DNA packaging ATPase.

The activity of the DNA packaging adenosine triphosphatase (ATPase) of the Bacillus subtilis bacteriophage phi 29 is dependent upon prohead RNA. The 174 nucleotide viral-encoded RNA is positioned on the head-tail connector at the portal vertex of the phi 29 precursor shell (prohead). Here, the RNA interacts with the ATP-binding gene 16 product (gp16) to constitute the DNA-packaging ATPase and initiate DNA packaging in vitro. Both the prohead connector (gene 10 product, gp10) and gp16 may utilize an RNA recognition motif characteristic of a number of RNA-associated proteins, and the binding of gp16 by proheads shields the prohead RNA from RNase A. The ATPase activity of gp16 is stimulated fourfold by RNA and tenfold by proheads with RNA. RNA is needed continuously for the gp16/RNA ATPase activity and is essential for the gp16/prohead ATPase activity. The prohead, with its connector, RNA and associated gp16 in an assembly-regulated configuration, hydrolyzes ATP and drives phi 29 DNA translocation.     

Structural organisation of the head-to-tail interface of a bacterial virus

In tailed icosahedral bacteriophages the connection between the 5-fold symmetric environment of the portal vertex in the capsid and the 6-fold symmetric phage tail is formed by a complex interface structure. The current study provides the detailed analysis of the assembly and structural organisation of such an interface within a phage having a long tail. The region of the interface assembled as part of the viral capsid (connector) was purified from DNA-filled capsids of the Bacillus subtilis bacteriophage SPP1. It is composed of oligomers of gp6, the SPP1 portal protein, of gp15, and of gp16. The SPP1 connector structure is formed by a mushroom-like portal protein whose cap faces the interior of the viral capsid in intact virions, an annular structure below the stem of the mushroom, and a second narrower annulus that is in direct contact with the helical tail extremity. The layered arrangement correlates to the stacking of gp6, gp15, and gp16 on top of the tail. The gp16 ring is exposed to the virion outside. During SPP1 morphogenesis, gp6 participates in the procapsid assembly reaction, an early step in the assembly pathway, while gp15 and gp16 bind to the capsid portal vertex after viral chromosome encapsidation. gp16 is processed during or after tail attachment to the connector region. The portal protein gp6 has 12-fold cyclical symmetry in the connector structure, whereas assembly-na?ve gp6 exhibits 13-fold symmetry. We propose that it is the interaction of gp6 with other viral morphogenetic proteins that drives its assembly into the 12-mer state.     

Bacillus subtilis mutants defective in bacteriophage phi 29 head assembly.

Virus assembly mutants of asporogenous Bacillus subtilis defective in bacteriophage phi 29 head assembly were detected by the use of antibodies that reacted strongly with the free dodecameric phi 29 portal vertex composed of gene product 10 (gp10) but weakly with the portal vertex assembled into proheads or phage. Phage adsorption and the synthesis of phage proteins, DNA-gene product 3, and prohead RNA were normal in these mutants, but prohead and phage production was greatly reduced. The assembly defect was transferred to competent B. subtilis by transformation and transduction. PBS1 transduction showed that the vam locus was linked to Tn917 located at 317 degrees on the B. subtilis chromosome.     

Initiation events in in-vitro packaging of bacteriophage phi 29 DNA-gp3

Initiation events in the packaging of bacteriophage phi 29 DNA-gp3 (DNA-gene product 3 complex) were studied in a completely defined in-vitro system that included purified proheads, DNA-gp3 and the DNA packaging protein gp16. In the sequential interactions, gp16 first bound to, and was modified by, the prohead. The prohead-gp16 complex then bound to DNA-gp3, resulting in a second modification of gp16 that permitted binding of ATP. DNA-gp3 aggregates were produced, and the hydrolysis of ATP accompanied DNA-gp3 packaging. Binding and hydrolysis of ATP by gp16 was both prohead- and DNA-gp3-dependent. Interruption of packaging by DNase I addition revealed filled heads but few particles containing partial lengths of DNA, suggesting that following a rate-limiting initiation, the translocation of DNA-gp3 into the prohead was much faster in the defined in-vitro system than in extracts.     

Analysis of gene function of bacteriophage phi 29 of Bacillus subtilis: identification of cistrons essential for viral assembly.

Restrictive infection of Bacillus subtilis by suppressor-sensitive (sus) mutants of phi 29 has been used to search for cistrons that function in viral assembly. The products of cistrons 7, 9, 10, and 16 are necessary for head morphogenesis. The neck upper collar protein P10 and the tail protein P9 must be present for DNA packaging to occur. The protein P7 must be present for phage-related particles to form. A prohead-like particle has been isolated during 16-restrictive infection. The particle is composed of the proteins Hd, P10, F, and P7. P16 must function for DNA-filled particles to accumulate. A DNA-containing particle produced in the absence of the cistron 11 product may be an intermediate in the phi 29 assembly pathway. The protein P13 interacts with P9 and P11 to form a stable DNA-filled particle. The products of cistrons 2 and 3 are essential for viral DNA synthesis, and in their absence virus-related particles are not detected.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: oriented and quantized in vitro packaging of DNA protein gp3.

The assembly of phage phi 29 occurs by a single pathway, and the DNA protein (DNA-gp3) of "packaging intermediates" can be obtained after DNase I interruption of in vitro complementation. A broad spectrum of DNA molecules of variable length was isolated from DNase I-treated proheads. Restriction endonuclease EcoRI digestion and electrophoretic analysis of these DNA molecules suggested that DNA-gp3 packaging was oriented with respect to the physical map and was a complex process. Proteinase K-treated exogenous DNA was not packaged. When exogenous DNA-gp3 was predigested with the restriction endonucleases BstEII. EcoRI, HpaI, and HpaII, the left-end fragments, ranging in size from 8 to 0.9 megadaltons, were selectively and efficiently packaged. During in vivo and in vitro assembly, DNA-gp3 is packaged into proheads, the "core-scaffolding" protein gp7 exits from the particles, and the DNA-filled heads assume the angular morphology of phage phi 29. The packaging of a 4.1-megadalton DNA-gp3 left-end fragment (one third of the genome) resulted in the exit of gp7 and the transition to angularity.     

Viral determinants of polarized assembly for the murine leukemia virus

Retrovirus transmission via direct cell-cell contact is more efficient than diffusion through the extracellular milieu. This is believed to be due to the ability of viruses to efficiently coordinate several steps of the retroviral life cycle at cell-cell contact sites (D. C. Johnson et al., J. Virol. 76:1-8, 2002; D. M. Phillips, AIDS 8:719-731, 1994; Q. Sattenau, Nat. Rev. Microbiol. 6:815-826, 2008). Using the murine leukemia virus (MLV) as a model retrovirus, we have previously shown that interaction between viral envelope (Env) and receptor directs viral assembly to cell-cell contact sites to promote efficient viral spreading (J. Jin et al., PLoS Biol. 7:e1000163, 2009). In addressing the underlying mechanism, we observed that Env cytoplasmic tail directs this contact-induced polarized assembly. We present here the viral determinants in the Env cytoplasmic tail and Gag that are important in this process. A tyrosine residue within the cytoplasmic tail of Env was identified, which directs polarized assembly. MLV matrix-mediated membrane targeting is required for Gag recruitment to sites of cell-cell contact. Our results suggest that MLV polarized assembly is mediated by a direct or indirect interaction between both domains, thereby coupling Gag recruitment and virus assembly to Env accumulation at the cell-cell interface. In contrast, HIV Gag that assembles outside of cell-cell interfaces can subsequently be drawn into contact zones mediated by MLV Env and receptor, a finding that is consistent with the previously observed lateral movement of HIV into the virological synapse (W. Hubner et al., Science 323:1743-1747, 2009; D. Rudnicka et al., J. Virol. 83:6234-6246, 2009). As such, we observed two distinct modes of virus cell-to-cell transmission that involve either polarized or nonpolarized assembly, but both result in virus transmission.     

Role of the amino-terminal domain of bacteriophage phi 29 connector in DNA binding and packaging.

The connector of bacteriophage phi 29 is required for prohead assembly, binds DNA, and drives DNA packaging into viral proheads. Limited proteolysis of the connector protein with endoproteinase Glu-C from Staphylococcus aureus V8 and chymotrypsin showed that a domain of the NH2-terminal region is involved in DNA binding and in the subsequent packaging into preformed proheads, but not in prohead assembly. Mutants in specific amino acids of the NH2-terminal domain, obtained by directed mutagenesis techniques, showed that the Ala1-Arg2-Lys3-Arg4 region of the connector is absolutely necessary for DNA packaging into the proheads as well as for efficient DNA binding.     

Defining molecular and domain boundaries in the bacteriophage phi29 DNA packaging motor

Cryo-electron microscopy (cryo-EM) studies of the bacteriophage phi29 DNA packaging motor have delineated the relative positions and molecular boundaries of the 12-fold symmetric head-tail connector, the 5-fold symmetric prohead RNA (pRNA), the ATPase that provides the energy for packaging, and the procapsid. Reconstructions, assuming 5-fold symmetry, were determined for proheads with 174-base, 120-base, and 71-base pRNA; proheads lacking pRNA; proheads with ATPase bound; and proheads in which the packaging motor was missing the connector. These structures are consistent with pRNA and ATPase forming a pentameric motor component around the unique vertex of proheads. They suggest an assembly pathway for the packaging motor and a mechanism for DNA translocation into empty proheads.     

Sequential interactions of structural proteins in phage phi 29 procapsid assembly.

The mechanism of viral capsid assembly is an intriguing problem because of its fundamental importance to research on synthetic viral particle vaccines, gene delivery systems, antiviral drugs, chimeric viruses displaying antigens or ligands, and the study of macromolecular interactions. The genes coding for the scaffolding (gp7), capsid (gp8), and portal vertex (gp10) proteins of the procapsid of bacteriophage phi 29 of Bacillus subtilis were expressed in Escherichia coli individually or in combination to study the mechanism of phi 29 procapsid assembly. When expressed alone, gp7 existed as a soluble monomer, gp8 aggregated into inclusion bodies, and gp10 formed the portal vertex. Circular dichroisin spectrum analysis indicated that gp7 is mainly composed of alpha helices. When two of the proteins were coexpressed, gp7 and gp8 assembled into procapsid-like particles with variable sizes and shapes, gp7 and gp10 formed unstable complexes, and gp8 and gp10 did not interact. These results suggested that gp7 served as a bridge for gp8 and gp10. When gp7, gp8, and gp10 were coexpressed, active procapsids were produced. Complementation of extracts containing one or two structural components could not produce active procapsids, indicating that no stable intermediates were formed. A dimeric gp7 concatemer promoted the solubility of gp8 but was inactive in the assembly of procapsid or procapsid-like particles. Mutation at the C terminus of gp7 prevented it from interacting with gp8, indicating that this part of gp7 may be important for interaction with gp8. Coexpression of the portal protein (gp20) of phage T4 with phi 29 gp7 and gp8 revealed the lack of interaction between T4 gp20 and phi 29 gp7 and/or gp8. Perturbing the ratio of the three structural proteins by duplicating one or another gene did not reduce the yield of potentially infectious particles. Changing of the order of gene arrangement in plasmids did not affect the formation of active procapsids significantly. These results indicate that phi 29 procapsid assembly deviated from the single-assembly pathway and that coexistence of all three components with a threshold concentration was required for procapsid assembly. The trimolecular interaction was so rapid that no true intermediates could be isolated. This finding is in accord with the result of capsid assembly obtained by the equilibrium model proposed by A. Zlotnick (J. Mol. Biol. 241:59-67, 1994).     

Multifunctional roles of a bacteriophage phi 29 morphogenetic factor in assembly and infection

Low copy number proteins within macromolecular complexes, such as viruses, can be critical to biological function while comprising a minimal mass fraction of the complex. The Bacillus subtilis double-stranded DNA bacteriophage phi 29 gene 13 product (gp13), previously undetected in the virion, was identified and localized to the distal tip of the tail knob. Western blots and immuno-electron microscopy detected a few copies of gp13 in phi 29, DNA-free particles, purified tails, and defective particles produced in suppressor-sensitive (sus) mutant sus13(330) infections. Particles assembled in the absence of intact gp13 (sus13(342) and sus13(330)) had the gross morphology of phi 29 but were not infectious. gp13 has predicted structural homology and sequence similarity to the M23 metalloprotease LytM. Poised at the tip of the phi 29 tail knob, gp13 may serve as a plug to help restrain the highly pressurized packaged genome. Also, in this position, gp13 may be the first virion protein to contact the cell wall in infection, acting as a pilot protein to depolymerize the cell wall. gp13 may facilitate juxtaposition of the tail knob onto the cytoplasmic membrane and the triggering of genome injection.     

Molecular evolution of human immunodeficiency virus env in humans and monkeys: similar patterns occur during natural disease progression or rapid virus passage

Neonatal rhesus macaque 95-3 was inoculated with nonpassaged simian-human immunodeficiency virus strain SHIV-vpu(+), which encodes env of the laboratory-adapted human immunodeficiency virus (HIV) strain IIIB and is considered nonpathogenic. CD4(+) T-cell counts dropped to <200 cells/microl within 4.6 years, and monkey 95-3 died with opportunistic infections 5.9 years postinoculation. Transfer of blood from 95-3 to two naive adult macaques resulted in high peak viral loads and rapid, persistent T-cell depletion. Progeny virus evolved in 95-3 despite high SHIV-vpu(+) neutralizing antibody titers and still used CXCR4 but, in contrast to parental SHIV-vpu(+), productively infected macrophages and resisted neutralization. Sequence analysis revealed three new potential glycosylation sites in gp120; another two were lost. Strikingly similar mutations were detected in a laboratory worker who progressed to AIDS after accidental HIV-IIIB infection (T. Beaumont et al., J. Virol. 75:2246-2252, 2001), thus supporting the SHIV-vpu(+)/rhesus macaque system as a relevant model. Similar mutations were also described after rapid passage of chimeric viruses encoding IIIB env in rhesus and pig-tailed macaques (M. Cayabyab et al., J. Virol. 73:976-984, 1999; Z. Q. Liu et al., Virology 260:295-307, 1999; S. V. Narayan et al., Virology 256:54-63, 1999; R. Raghavan et al., Brain Pathol. 7:851-861, 1997; E. B. Stephens et al., Virology 231:313-321, 1997). Thus, HIV-IIIB env evolved similarly in three different species; this selection occurred in chronically infected individuals during disease progression as well as after rapid virus passage. We postulate that evolutionary pressure led to the outgrowth of more aggressive viral variants in all three species.     

RNA-mediated specificity of DNA packaging into hybrid lambda/phi 29 proheads.

A small RNA (pRNA, 174 nt) is known to be essential for DNA packaging in bacteriophage phi 29. However, in an in vitro DNA packaging system based on hybrid lambda/phi 29 proheads (made up of head proteins from phage lambda and connectors from phage phi 29), the specificity of DNA packaging is lost, and different RNA molecules fulfil the requirements for DNA packaging, albeit with less efficiency than phi 29 pRNA. Competition assays with RNAs from different sources have shown that phi 29 connectors bind preferentially pRNA. An increase in the efficiency of phi 29 DNA packaging into hybrid proheads induced by phi 29 pRNA is observed because, when phi 29 pRNA is incubated with hybrid proheads, phi 29 DNA is packaged more efficiently than other DNAs of similar length. Furthermore, when hybrid proheads carrying phi 29 pRNA are incubated with a mixture of DNAs from different sources, phi 29 DNA is selectively packaged, thus indicating that phi 29 pRNA determines the specificity of DNA packaging.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: preliminary isolation and characterization of intermediate particles of the assembly pathway.

Three classes of particles have been identified in restrictive phi 29 suppressor-sensitive (sus) mutant infections of Bacillus subtilis, including DNA-containing heads or phage, prohead, and empty heads. Pulse-chase labeling experiments indicate that the prohead, the first particle assembled in 14-infected cells, is converted to DNA-filled heads and phi 29. In addition to the proteins Hd, P10, and F found in mature phi 29, the prohead contains a "core" protein P7 that exits as the prohead matures and appears to recycle during subsequent rounds of prohead assembly. Prohead-like structures accumulate in UV-irradiated cells and are present in restrictive infections with sus mutants of cistrons 9 and 16. Empty heads are observed only when infection results in the formation of DNA-containing particles; this and other evidence indicates that the empty heads are probably not true intermediates. Phage phi 29 assembly apparently occurs by a single pathway in which neck and tail components interact to stabilize the completed DNA-containing head.     

DNA packaging motor assembly intermediate of bacteriophage phi29

Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here, the bacteriophage phi29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the phi29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration, and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent on the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone; therefore, the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway.     

Role of an internal and two 3'-terminal RNA elements in assembly of tombusvirus replicase

Plus-strand RNA virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. The replicase of Cucumber necrosis virus (CNV), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. In addition, the assembly of CNV replicase is stimulated in the presence of plus-stranded viral RNA (Z. Panaviene et al., J. Virol. 78:8254-8263, 2004). To define cis-acting viral RNA sequences that stimulate replicase assembly, we performed a systematic deletion approach with a model tombusvirus replicon RNA in Saccharomyces cerevisiae, which also coexpressed p33 and p92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations from yeast revealed critical roles for three RNA elements in CNV replicase assembly: the internal p33 recognition element (p33RE), the replication silencer element (RSE), and the 3'-terminal minus-strand initiation promoter (gPR). Deletion or mutagenesis of these elements reduced the activity of the CNV replicase to a minimal level. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structures among these elements may also play a role in replicase assembly. Altogether, the role of multiple RNA elements in tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection during replication.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: DNA-gp3 intermediate in in vivo and in vitro assembly

The assembly of phage phi 29 occurs by a single pathway, and DNA-protein (DNA-gp3) has been shown to be an intermediate on the assembly pathway by a highly efficient in vitro complementation. At 30 degrees C, about one-half of the viral DNA synthesized was assembled into mature phage, and the absolute plating efficiency of phi 29 approached unity. DNA packaging at 45 degrees C was comparable to that at 30 degrees C, but the burst size was reduced by one-third. When cells infected with mutant ts3(132) at 30 degrees C to permit DNA synthesis were shifted to 45 degrees C before phage assembly, DNA synthesis ceased and no phage were produced. However, a variable amount of DNA packaging occurred. Superinfection by wild-type phage reinitiated ts3(132) DNA synthesis at 45 degrees C, and if native gp3 was covalently linked to this DNA during superinfection replication, it was effectively packaged and assembled. Treatment of the DNA-gp3 complex with trypsin prevented in vitro maturation of phi 29, although substantial DNA packaging occurred. A functional gp3 linked to the 5' termini of phi 29 DNA is a requirement for effective phage assembly in vivo and in vitro.