Search

Ubiquitous and abundant in ecosystems and microbiomes, gokushoviruses constitute a Microviridae subfamily, distantly related to bacteriophages ΦX174, α3, and G4. A high-resolution cryo-EM structure of gokushovirus ΦEC6098 was determined, and the atomic model was built de novo. Although gokushoviruses lack external scaffolding and spike proteins, which extensively interact with the ΦX174 capsid protein, the core of the ΦEC6098 coat protein (VP1) displayed a similar structure. There are, however, key differences. At each ΦEC6098 icosahedral 3-fold axis, a long insertion loop formed mushroom-like protrusions, which have been noted in lower-resolution gokushovirus structures. Hydrophobic interfaces at the bottom of these protrusions may confer stability to the capsid shell. In ΦX174, the N-terminus of the capsid protein resides directly atop the 3-fold axes of symmetry; however, the ΦEC6098 N-terminus stretched across the inner surface of the capsid shell, reaching nearly to the 5-fold axis of the neighboring pentamer. Thus, this extended N-terminus interconnected pentamers on the inside of the capsid shell, presumably promoting capsid assembly, a function performed by the ΦX174 external scaffolding protein. There were also key differences between the ΦX174-like DNA-binding J proteins and its ΦEC6098 homologue VP8. As seen with the J proteins, C-terminal VP8 residues were bound into a pocket within the major capsid protein; however, its N-terminal residues were disordered, likely due to flexibility. We show that the combined location and interaction of VP8’s C-terminus and a portion of VP1’s N-terminus are reminiscent of those seen with the ΦX174 and α3 J proteins. IMPORTANCE There is a dramatic structural and morphogenetic divide within the Microviridae. The well-studied ΦX174-like viruses have prominent spikes at their icosahedral vertices, which are absent in gokushoviruses. Instead, gokushovirus major coat proteins form extensive mushroom-like protrusions at the 3-fold axes of symmetry. In addition, gokushoviruses lack an external scaffolding protein, the more critical of the two ΦX174 assembly proteins, but retain an internal scaffolding protein. The ΦEC6098 virion suggests that key external scaffolding functions are likely performed by coat protein domains unique to gokushoviruses. Thus, within one family, different assembly paths have been taken, demonstrating how a two-scaffolding protein system can evolve into a one-scaffolding protein system, or vice versa.

Although microviruses do not possess a visible tail structure, one vertex rearranges after interacting with host lipopolysaccharides. Most examinations of host range, eclipse, and penetration were conducted before this “host-induced” unique vertex was discovered and before DNA sequencing became routine. Consequently, structure-function relationships dictating host range remain undefined. Biochemical and genetic analyses were conducted with two closely related microviruses, α3 and ST-1. Despite ∼90% amino acid identity, the natural host of α3 is Escherichia coli C, whereas ST-1 is a K-12-specific phage. Virions attached and eclipsed to both native and unsusceptible hosts; however, they breached only the native host’s cell wall. This suggests that unsusceptible host-phage interactions promote off-pathway reactions that can inactivate viruses without penetration. This phenomenon may have broader ecological implications. To determine which structural proteins conferred host range specificity, chimeric virions were generated by individually interchanging the coat, spike, or DNA pilot proteins. Interchanging the coat protein switched host range. However, host range expansion could be conferred by single point mutations in the coat protein. The expansion phenotype was recessive: genetically mutant progeny from coinfected cells did not display the phenotype. Thus, mutant isolation required populations generated in environments with low multiplicities of infection (MOI), a phenomenon that may have impacted past host range studies in both prokaryotic and eukaryotic systems. The resulting genetic and structural data were consistent enough that host range expansion could be predicted, broadening the classical definition of antireceptors to include interfaces between protein complexes within the capsid. IMPORTANCE To expand host range, viruses must interact with unsusceptible host cell surfaces, which could be detrimental. As observed in this study, virions were inactivated without genome penetration. This may be advantageous to potential new hosts, culling the viral population from which an expanded host range mutant could emerge. When identified, altered host range mutations were recessive. Accordingly, isolation required populations generated in low-MOI environments. However, in laboratory settings, viral propagation includes high-MOI conditions. Typically, infected cultures incubate until all cells produce progeny. Thus, coinfections dominate later replication cycles, masking recessive host range expansion phenotypes. This may have impacted similar studies with other viruses. Last, structural and genetic data could be used to predict site-directed mutant phenotypes, which may broaden the classic antireceptor definition to include interfaces between capsid complexes.

Two scaffolding proteins orchestrate ϕX174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature-sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned-gene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability. IMPORTANCE Genetic analyses have been instrumental in deciphering the temporal events of many biochemical pathways. However, pleiotropic effects can complicate analyses. Vis-à-vis virion morphogenesis, an improper protein-protein interaction within an early assembly intermediate can influence the efficiency of all subsequent reactions. Consequently, the flux of assembly intermediates cumulatively decreases as the pathway progresses. During morphogenesis, ϕX174 coat protein participates in at least four well-defined reactions, each one characterized by an interaction with a scaffolding or structural protein. In this study, genetic analyses, biochemical characterizations, and physiological assays, i.e., elevating the protein levels with which the coat protein interacts, were used to elucidate pleiotropic effects that may alter the flux of intermediates through a morphogenetic pathway.

Although ϕX174 DNA pilot protein H is monomeric during procapsid assembly, it forms an oligomeric tube on the host cell surface. Reminiscent of a double-stranded DNA phage tail in form and function, the H tube transports the single-stranded ϕX174 genome across the Escherichia coli cell wall. The 2.4-Å resolution H-tube crystal structure suggests functional and energetic mechanisms that may be common features of DNA transport through virally encoded conduits.

The øX174 DNA pilot protein H forms an oligomeric DNA-translocating tube during penetration. However, monomers are incorporated into 12 pentameric assembly intermediates, which become the capsid's icosahedral vertices. The protein's N terminus, a predicted transmembrane helix, is not represented in the crystal structure. To investigate its functions, a series of absolute and conditional lethal mutations were generated. The absolute lethal proteins, a deletion and a triple substitution, were efficiently incorporated into virus-like particles lacking infectivity. The conditional lethal mutants, bearing cold-sensitive ( cs ) and temperature-sensitive ( ts ) point mutations, were more amenable to further analyses. Viable particles containing the mutant protein can be generated at the permissive temperature and subsequently analyzed at the restrictive temperature. The characterized cs defect directly affected host cell attachment. In contrast, ts defects were manifested during morphogenesis. Particles synthesized at permissive temperature were indistinguishable from wild-type particles in their ability to recognize host cells and deliver DNA. One mutation conferred an atypical ts synthesis phenotype. Although the mutant protein was efficiently incorporated into virus-like particles at elevated temperature, the progeny appeared to be kinetically trapped in a temperature-independent, uninfectious state. Thus, substitutions in the N terminus can lead to H protein misincorporation, albeit at wild-type levels, and subsequently affect particle function. All mutants exhibited recessive phenotypes, i.e., rescued by the presence of the wild-type H protein. Thus, mixed H protein oligomers are functional during DNA delivery. Recessive and dominant phenotypes may temporally approximate H protein functions, occurring before or after oligomerization has gone to completion.

During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro . In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. IMPORTANCE Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo . We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.

It has been well established that chemical mutagenesis has adverse fitness effects in RNA viruses, often leading to population extinction. This is mainly a consequence of the high RNA virus spontaneous mutation rates, which situate them close to the extinction threshold. Single-stranded DNA viruses are the fastest-mutating DNA-based systems, with per-nucleotide mutation rates close to those of some RNA viruses, but chemical mutagenesis has been much less studied in this type of viruses. Here, we serially passaged bacteriophage ϕX174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU). We found that 5-FU was unable to trigger population extinction for the range of concentrations tested, but it negatively affected viral adaptability. The phage evolved partial drug resistance, and parallel nucleotide substitutions appearing in independently evolved lines were identified as candidate resistance mutations. Using site-directed mutagenesis, two single-nucleotide substitutions in the lysis protein E (T572C and A781G) were shown to be selectively advantageous in the presence of 5-FU. In RNA viruses, base analogue resistance is often mediated by changes in the viral polymerase, but this mechanism is not possible for ϕX174 and other single-stranded DNA viruses because they do not encode their own polymerase. In addition to increasing mutation rates, 5-FU produces a wide variety of cytotoxic effects at the levels of replication, transcription, and translation. We found that substitutions T572C and A781G lost their ability to confer 5-FU resistance after cells were supplemented with deoxythymidine, suggesting that their mechanism of action is at the DNA level. We hypothesize that regulation of lysis time may allow the virus to optimize progeny size in cells showing defects in DNA synthesis.

By acquiring resistance to an inhibitor, viruses can become dependent on that inhibitor for optimal fitness. However, inhibitors rarely, if ever, stimulate resistant strain fitness to values that equal or exceed the uninhibited wild-type level. This would require an adaptive mechanism that converts the inhibitor into a beneficial replication factor. Using a plasmid-encoded inhibitory external scaffolding protein that blocks φX174 assembly, we previously demonstrated that such mechanisms are possible. The resistant strain, referred to as the evolved strain, contains four mutations contributing to the resistance phenotype. Three mutations confer substitutions in the coat protein, whereas the fourth mutation alters the virus-encoded external scaffolding protein. To determine whether stimulation by the inhibitory protein coevolved with resistance or whether it was acquired after resistance was firmly established, the strain temporally preceding the previously characterized mutant, referred to as the intermediary strain, was isolated and characterized. The results of the analysis indicated that the mutation in the virus-encoded external scaffolding protein was primarily responsible for stimulating strain fitness. When the mutation was placed in a wild-type background, it did not confer resistance. The mutation was also placed in cis with the plasmid-encoded dominant lethal mutation. In this configuration, the stimulating mutation exhibited no activity, regardless of the genotype (wild type, evolved, or intermediary) of the infecting virus. Thus, along with the coat protein mutations, stimulation required two external scaffolding protein genes: the once inhibitory gene and the mutant gene acquired during evolution.

The ability for a virus to expand its host range is dependent upon a successful mode of viral entry. As such, the host range of the well-studied ΦX174 bacteriophage is dictated by the presence of a particular lipopolysaccharide (LPS) on the bacterial surface. The mutant ΦX174 strain JACS-K, unlike its ancestor, is capable of infecting both its native host Escherichia coli C and E. coli K-12, which does not have the necessary LPS. The conversion of an alanine to a very reactive threonine on its virion surface was found to be responsible for the strain's expanded host range.

Microviruses (canonical members are bacteriophages φX174, G4, and α3) are T=1 icosahedral virions with an assembly pathway mediated by two scaffolding proteins. The external scaffolding protein D plays a major role during morphogenesis, particularly in icosahedral shell formation. The results of previous studies, conducted with a cloned chimeric external scaffolding gene, suggest that the first α-helix acts as a substrate specificity domain, perhaps mediating the initial coat-external scaffolding protein interaction. However, the expression of a cloned gene could lead to protein concentrations higher than those found in typical infections. Moreover, its induction before infection could alter the timing of the protein's accumulation. Both of these factors could drive or facilitate reactions that may not occur under physiological conditions or before programmed cell lysis. In order to elucidate a more detailed mechanistic model, a chimeric external scaffolding gene was placed directly in the φX174 genome under wild-type transcriptional and translational control, and the chimeric virus, which was not viable on the level of plaque formation, was characterized. The results of the genetic and biochemical analyses indicate that α-helix 1 most likely mediates the nucleation reaction for the formation of the first assembly intermediate containing the external scaffolding protein. Mutants that can more efficiently use the chimeric scaffolding protein were isolated. These second-site mutations appear to act on a kinetic level, shortening the lag phase before virion production, perhaps lowering the critical concentration of the chimeric protein required for a nucleation reaction.

The φX174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven α-helices. The protein's core, α-helices 2 to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all Microviridae (canonical members are φX174, G4, and α3) external scaffolding proteins. On the other hand, the primary sequences of the first α-helices have diverged. The results of previous studies with α3/φX174 chimeric external scaffolding proteins suggest that α-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and φX174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. φX174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.

During the final stages of φX174 morphogenesis, there is an 8.5-Å radial collapse of coat proteins around the packaged genome, which is tethered to the capsid's inner surface by the DNA-binding protein. Two approaches were taken to determine whether protein-DNA interactions affect the properties of the mature virion and thus the final stages of morphogenesis. In the first approach, genome-capsid associations were altered with mutant DNA-binding proteins. The resulting particles differed from the wild-type virion in density, native gel migration, and host cell recognition. Differences in native gel migration were especially pronounced. However, no differences in protein stoichiometries were detected. An extragenic second-site suppressor of the mutant DNA-binding protein restores all assayed properties to near wild-type values. In the second approach, φX174 was packaged with foreign, single-stranded, covalently closed, circular DNA molecules identical in length to the φX174 genome. The resulting particles exhibited native gel migration rates that significantly differed from the wild type. The results of these experiments suggest that the structure of the genome and/or its association with the capsid's inner surface may perform a scaffolding-like function during the procapsid-to- virion transition.

Viral assembly is an ideal system in which to investigate the transient recognition and interplay between proteins. During morphogenesis, scaffolding proteins temporarily associate with structural proteins, stimulating conformational changes that promote assembly and inhibit off-pathway reactions. Microviridae morphogenesis is dependent on two scaffolding proteins, an internal and an external species. The external scaffolding protein is the most conserved protein within the Microviridae, whose canonical members are fX174, G4, and a3. However, despite 70% homology on the amino acid level, overexpression of a foreign Microviridae external scaffolding protein is a potent cross-species inhibitor of morphogenesis. Mutants that are resistant to the expression of a foreign scaffolding protein cannot be obtained via one mutational step. To define the requirements for and constraints on scaffolding protein interactions, chimeric external scaffolding proteins have been constructed and analyzed for effects on in vivo assembly. The results of these experiments suggest that at least two cross-species inhibitory domains exist within these proteins; one domain most likely blocks procapsid formation, and the other allows procapsid assembly but blocks DNA packaging. A mutation conferring resistance to the expression of a chimeric protein (chiD r ) that inhibits DNA packaging was isolated. The mutation maps to gene A, which encodes a protein essential for packaging. The chiD r mutation confers resistance only to a chimeric D protein; the mutant is still inhibited by the expression of foreign D proteins. The results presented here demonstrate how closely related proteins could be developed into antiviral agents that specifically target virion morphogenesis. The proper assembly of viral proteins and nucleic acids into a biologically active virion involves numerous and diverse macromolecular interactions. While structural proteins must correctly interact with other structural proteins, proper morphogenesis is equally dependent on interactions between structural and scaffolding proteins. Viral scaffolding proteins are transiently associated with morphogenetic intermediates but are not found in the mature viral particle (10, 12, 17, 22). These proteins promote the efficiency and fidelity of particle formation by ensuring proper interactions between viral proteins, promoting the nucleation of assembly, and aiding in the formation of a precisely sized capsid (13, 16, 19). In the Microviridae, assembly is dependent on two scaffolding proteins, an internal and an external species. The assembly pathway has been extensively characterized (8), and the atomic structures of both the fX174 virion and a morphogenetic intermediate containing a full complement of scaffolding proteins have been solved (2, 3, 14, 15). Therefore, the results of genetic and biochemical analyses can be interpreted within a structural context. The Microviridae assembly pathway is illustrated in Fig. 1. The first detectable morphogenetic intermediates are the 9S and 6S particles, respective pentamers of the viral coat and spike proteins. In a reaction mediated by the internal scaffolding (or B) protein, these intermediates associate, forming the 12S particle. Twelve 12S particles are then organized into the procapsid by 240 copies of the external scaffolding (or D) protein. The preinitiation complex, consisting of one copy of viral proteins A and C, replicative-form (RF) DNA, and the host cell Rep protein, associates with the procapsid, probably along a twofold axis of symmetry (5), forming the 50S complex. Single-stranded genomic DNA is then concurrently synthesized and packaged. The internal scaffolding protein is extruded during this reaction, yielding the infectious provirion, which may represent the end of the intracellular assembly pathway. The external scaffolding protein may dissociate upon cell lysis. External scaffolding proteins are not unique to the Microviridae. The coliphage P4 Sid protein and the triplex proteins of the Herpesviridae perform analogous functions (13, 23). Although the Microviridae morphogenetic pathway has been well characterized, the mechanism in which viral scaffolding proteins enable nascent intermediates to reach a biologically active form remains somewhat obscure. Here we report that the expression of closely related external scaffolding proteins inhibits morphogenesis. Using chimeric proteins, we have defined the existence of two inhibitory domains. These results have implications for the use of closely related proteins in the development of antiviral analogs specific to viral morphogenesis.

C-terminal, aromatic amino acids in the ϕX174 internal scaffolding protein B mediate conformational switches in the viral coat protein. These switches direct the coat protein through early assembly. In addition to the aromatic amino acids, two acidic residues, D111 and E113, form salt bridges with basic, coat protein side chains. Although salt bridge formation did not appear to be critical for assembly, the substitution of an aromatic amino acid for D111 produced a lethal phenotype. This side chain is uniquely oriented toward the center of the coat-scaffolding binding pocket, which is heavily dominated by aromatic ring-ring interactions. Thus, the D111Y substitution may restructure pocket contacts. Previously characterized B − mutants blocked assembly before procapsid formation. However, the D111Y mutant produced an assembled particle, which contained the structural and external scaffolding proteins but lacked protein B and DNA. A suppressor within the external scaffolding protein, which mediates the later stages of particle morphogenesis, restored viability. The unique formation of a postprocapsid particle and the novel suppressor may be indicative of a novel B protein function. However, genetic data suggest that the particle represents the delayed manifestation of an early assembly error. This seemingly late-acting defect was rescued by previously characterized suppressors of early, preprocapsid, B − assembly mutations, which act on the level of coat protein flexibility. Likewise, the newly isolated suppressor in the external scaffolding protein also exhibited a global suppressing phenotype. Thus, the off-pathway product isolated from infected cells may not accurately reflect the temporal nature of the initial defect.

Viruses often evolve resistance to antiviral agents. While resistant strains are able to replicate in the presence of the agent, they generally exhibit lower fitness than the wild-type strain in the absence of the inhibitor. In some cases, resistant strains become dependent on the antiviral agent. However, the agent rarely, if ever, elevates dependent strain fitness above the uninhibited wild-type level. This would require an adaptive mechanism to convert the antiviral agent into a beneficial growth factor. Using an inhibitory scaffolding protein that specifically blocks φX174 capsid assembly, we demonstrate that such mechanisms are possible. To obtain the quintuple-mutant resistant strain, the wild-type virus was propagated for approximately 150 viral life cycles in the presence of increasing concentrations of the inhibitory protein. The expression of the inhibitory protein elevated the strain's fitness significantly above the uninhibited wild-type level. Thus, selecting for resistance coselected for dependency, which was characterized and found to operate on the level of capsid nucleation. To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly.

Proper virion assembly requires numerous macromolecular interactions proceeding along an ordered morphogenetic pathway. While structural proteins are incorporated into the final product, morphogenesis is equally dependent upon scaffolding proteins, which are not included in the mature virion. During morphogenesis, these proteins mediate conformational switches to ensure the efficiency and fidelity of assembly (13). Although many large DNA viruses rely on a single internal scaffolding protein, the small microviruses (canonical species φX174, G4, and α3) and the satellite P4-like viruses require both internal and external scaffolding proteins (10, 14, 21). Although the internal scaffolding proteins in these dual systems are absolutely essential, several lines of evidence suggest that the external scaffolding proteins may be more critical. Successive targeted genetic selections have led to the isolation of a φX174 multiple mutant that can assemble without its internal scaffolding protein in vivo (5). Moreover, P4 procapsid-like particles can be assembled in vitro using only the capsid and external scaffolding proteins (26). The morphogenetic roles of the φX174 internal and external scaffolding proteins are illustrated in Fig. ​Fig.1A.1A. The first identifiable assembly intermediates are pentamers of the viral coat F and major spike G proteins, the 9S and 6S particles, respectively, which can form in the absence of both scaffolding proteins (23). Five internal scaffolding B proteins bind to the underside of the 9S particle, yielding the 9S* intermediate. This interaction induces a conformational change that allows 9S*-6S particle associations, forming the 12S* intermediate. This particle also includes the DNA pilot protein H (10, 16). Twenty external scaffolding D proteins, most likely in the form of five tetramers or ten asymmetric dimers (18), interact with the 12S* particle to form the short-lived 18S intermediate (4). D-D contacts mediate the construction of the procapsid (108S), an immature virus particle, presumably by allowing 18S pentamers to interact across twofold axes of symmetry (6, 8, 18). FIG. 1. (A) The φX174 morphogenetic pathway. (B) The four external scaffolding subunits (D1 [purple], D2 [blue], D3 [brown], and D4 [green]), coat protein (gray), and internal scaffolding protein (peach) in the asymmetric unit. (C) The four external scaffolding ... In the atomic structure of the viral procapsid (6, 8), there are four structurally distinct external scaffolding proteins (D1 to D4), arranged as dimers of dimers (D1D2, D3D4), per viral coat protein (Fig. 1B and C). To achieve this unique arrangement, one monomer in each asymmetric dimer, D1 and D3, must be bent 30°; this occurs at glycine residue 61 in α-helix 3 (Fig. ​(Fig.1C).1C). This kink is also present in the crystal structure of the assembly naive dimer DADB (18). A mutation at this site may allow the protein to fold into only one of these two conformations. Although the mutant protein would retain the ability to interact with other D and structural proteins, the product of these interactions may inhibit lattice formation and subsequent virus assembly. The putative dominant lethal phenotypes of some glycine 61 substitutions have been reported (3). However, the molecular mechanism of inhibition could not be characterized due to difficulties associated with propagating the mutant strains. To determine the mechanism of inhibition and circumvent the problems associated with propagating dominant lethal viral mutants, genetically engineered D genes were constructed, and the proteins were expressed in wild-type φX174-infected cells. This alternate approach also allowed mutants resistant to the expression of the inhibitory proteins to be isolated, elucidating possible resistance mechanisms that may be anticipated for antiviral therapies that target virus-specific assembly processes. The results of this analysis suggest that the presence of the wild-type external scaffolding protein is required for the mutant inhibitory D protein to enter the morphogenetic pathway. This may lead to premature D protein interactions with structural proteins or off-pathway reactions involving later intermediates. The location of the resistance mutations within the procapsid crystal structure suggest a mechanism in which inhibitory D protein subunits are excluded from occupying particular positions atop 12S* pentamers.

Linear viral DNA present in cytoplasmic extracts of cells newly infected with human immunodeficiency virus type 1 can be induced to form 1-LTR and 2-LTR circles by incubation of the extracts in the presence of added nucleoside triphosphates. No circular DNA forms are detected when extracts are incubated in the absence of added nucleoside triphosphates. Restriction enzyme analysis and polymerase chain reaction analysis with selected primers, as well as DNA sequence analysis of the polymerase chain reaction products, show that most of the 2-LTR circles are the result of autointegration reactions, while 1-LTR circles result from recombination between the long terminal repeats on the linear viral DNA. In addition, a small amount of simple 2-LTR circles, formed by end-to-end joining of the linear viral DNA, is formed in vitro. Integration of the linear viral DNA into heterologous DNA competes effectively with the formation of 2-LTR circles by autointegration. However, concentrations of target DNA which completely block autointegration have no effect on the formation of 1-LTR circles or simple 2-LTR circles. Factors present in extracts of uninfected cells can mediate the formation of 1-LTR circles and simple 2-LTR circles from purified deproteinated linear viral DNA, indicating that viral proteins are not necessary for the formation of these two types of circular viral DNA. These experiments demonstrate that all the transformations of linear viral DNA which occur in the nuclei of cells infected with human immunodeficiency virus type 1 can be reproduced in vitro.

Oligodeoxyribonucleotide mutagenesis has been used to produce a G----A mutation at nucleotide 557 of the phi X174 genome. This changes the ribosome-binding sequence GAGG of gene E to GAAG without affecting the amino acid, glutamine, encoded by the overlapping gene D. The phi X174rb(E)557 mutant does not lyse infected Escherichia coli C and therefore results in the accumulation of a large number intracellular mature phage particles. Thus, the mutation inactivates production of the gene E lytic product, presumably by blocking translation of gene E, without affecting other phage functions.

Gene K of phi X 174, which overlaps genes A, B, and C, was found to be nonessential, although possibly beneficial for the growth of the phage. Viable mutants of gene K made less than 4% of the normal amount of K protein as judged by quantitative fluorography of sodium dodecyl sulfate-polyacrylamide gels; compared with the wild-type phi X, K mutants had an identical latent period but a two- to threefold reduction in burst size.

The gene products of the lethal lysis genes S and E of the bacteriophages lambda and phiX174, respectively, were shown to be associated primarily with inner membrane material by isopycnic sucrose gradient centrifugation of lysates of infected cells. A small amount of each polypeptide appeared to be in the outer membrane fraction.

The phi X174 lysis gene E was placed under control of the lac promoter by cloning into the multicopy plasmid pBH20. Other phi X174 gene sequences were removed by nuclease digestion. Expression of gene E was shown to be necessary and sufficient to produce lysis phenomena exhibited by infection with intact phage. Lysis, its inhibition by MgSO4 and spermine, its progression through a spheroplasting stage, and its dependence on an early chloramphenicol-sensitive step were reproduced in clones induced for expression of the E gene product. Escherichia coli clones carrying the E gene not under lac control, and clones under lac control but only minimally induced for gene E expression, exhibited morphological aberrations consistent with the view that the mechanism by which gene E mediates cell lysis is related to host cell division processes.

The A* gene of bacteriophage phi X174 has been cloned into the inducible expression vector pCQV2 under conditions allowing its lethal action to be controlled by the lambda cI857 repressor. Upon induction of expression, DNA synthesis in Escherichia coli carrying the recombinant plasmid is severely inhibited; however, these same cells permit beta-galactosidase induction at a rate similar to that observed in control cells at the inducing (for A*) temperature. Cells in which A* is expressed form filaments and produce more RecA protein, indicating at least a partial induction of the SOS response; however, there is no evidence of damage to the bacterial chromosome. It appears that the A* protein has as one function the inhibition of cell division and DNA replication but not transcription or protein synthesis during phage infection.

An M13 phage deletion mutant, M13 delta E101, developed as a vector for selecting DNA sequences that direct DNA strand initiation on a single-stranded template, has been used for cloning restriction enzyme digests of phi X174 replicative-form DNA. Initiation determinants, detected on the basis of clear-plaque formation by the chimeric phage, were found only in restriction fragments containing the unique effector site in phi X174 DNA for the Escherichia coli protein n' dATPase (ATPase). Furthermore, these sequences were functional only when cloned in the orientation in which the phi X174 viral strand was joined to the M13 viral strand. A 181-nucleotide viral strand fragment containing this initiation determinant confers a phi X174-type complementary-strand replication mechanism on M13 chimeras. The chimeric phage is converted to the parental replicative form in vivo by a mechanism resistant to rifampin, a specific inhibitor of the normal RNA polymerase-dependent mechanism of M13. In vitro, the chimeric single-stranded DNA promotes the assembly of a functional multiprotein priming complex, or primosome, identical to that utilized by intact phi X174 viral strand DNA. Chimeric phage containing the sequence complementary to the 181-nucleotide viral strand sequence shows no initiation capability, either in vivo or in vitro.

Phi X174-infected minicells, produced by Escherichia coli PC2251, synthesized 11 phi X174-encoded polypeptides. The infecting single-stranded viral genome was converted to a double-stranded, closed circular, replicative form (replicative form I). Little, if any, replicative form I replication took place, and synthesis of progeny single-stranded molecules could not be detected.

The gene A protein cleaves phi X174 single-stranded DNA (ssDNA). The cleavage appears to be stoichiometric, whereby a gene A protein molecule breaks a phosphodiester bond and binds to the 5' end. The enzyme introduces mostly a single break in a circular ssDNA molecule. However, at high enzyme-to-DNA ratios, more than one break in the DNA could be observed. The cleavage of the ssDNA by gene A protein renders the DNA sensitive to the action of terminal transferase to incorporate [alpha -32P]ATP. Thus, the 3'OH end is free. All attempts to label the 5' end by T4-induced polynucleotide kinase and [gamma-32P]ATP failed. The formation of a gene A-ssDNA complex was demonstrated directly by using 3H-labeled gene A protein and 32P-labeled ssDNA in the reaction. Such a complex is resistant to treatments with 0.2 M NaOH, banding in CsCl, and boiling in 2.5% sodium dodecyl sulfate. Only treatment with a nuclease released the bound protein. Also, after cleaving [32P]ssDNA by gene A protein, followed by either DNase I or micrococcal nuclease digestion, a fraction of the 32P label remained resistant to nuclease treatment and comigrated with gene A protein on polyacrylamide gels.

Replacement of virtually all the cytosine residues with 5-methylcytosine residues in the complementary strand of the replicative form (RF) of phi X174 DNA caused a 300- to 500-fold loss in its transfecting activity. Similar results were obtained with analogously methylated M13 RF. Transfection experiments with phi X RF hemimethylated in only part of the molecule, as assessed by analysis with restriction endonucleases, indicated that gene A of phi X, which needs to be nicked at a specific site by the gene A protein for RF replication, was not the main target for this inhibition by DNA methylation. We propose that the loss of transfecting activity was due to hemimethylation of the phi X RF interfering with the processively catalyzed movement of the replication fork.

An algorithm to locate transcription terminators (V. Brendel and E. N. Trifonov, Nucleic Acids Res. 12:4411-4427, 1984) was applied to the genomes of bacteriophages phi x174 and G4. The proposed sites are similarly located in phiX and G4 and fit with transcript lengths previously observed in vivo and in vitro.

The bacteriophage phi X174 strain ins6 constructed previously was used to investigate the maximum genome size that could be packaged into the icosahedral phage without concomitant loss of phage viability. The J-F intercistronic region of ins6, which already contains an insert of 117 base pairs with a unique PvuII site, was enlarged further by insertion of HaeIII restriction fragments of the plasmid pBR322 into that PvuII site. By using a biochemical approach for the site-specific mutagenesis as well as selection of mutant genomes, a series of mutants was isolated with genomes of up to 5,730 nucleotides, 6.4% larger than that of the wild-type DNA. Phages with genomes larger than 5,550 nucleotides were highly unstable and were rapidly outgrown by spontaneously occurring deletion mutants. The data predict that genomes of at least 6,090 nucleotides could be constructed and, most likely, packaged, but the resulting phages would not grow well. We speculate that the volume of the phage capsid is not the limiting factor of genome size or is not the only limiting factor.

Different-size species of phi X174-specific mRNA's decayed exponentially, with half-lives ranging from 4.5 to 11 min.

The J-F intercistronic region of the genomes of bacteriophages phi X174 and G4 is transcribed but not translated. It contains the potential for a perfectly base-paired hairpin structure, which has been proposed to act as a terminator of transcription or as a regulatory region for mRNA turnover (or both). We measured phage-specific protein synthesis in mutants with modifications of the hairpin sequence and found a relative decrease in the expression of the upstream gene D as compared to the downstream genes F, G, and H in all mutants. The mutations also appeared to affect the efficiency of the gene F ribosome-binding site. These data strongly support the regulatory significance of the J-F intercistronic region and the putative hairpin structure therein.

Site-directed mutagenesis has been used to produce a T----A change at nucleotide 70 of phi X174 genome. This generates an am codon, TAG, in the gene K reading frame without affecting the amino acid, leucine, encoded by the overlapping gene A. The gene K mutant produces small plaques on su- hosts. It has an identical latent period, but a more reduced burst size than that of the wild-type phi X174. The reduced burst size in the gene K mutant suggests that the gene K protein, although not essential, has a role in increasing infectivity by increasing the burst size three- to sixfold.

The suppression of polarity by UV irradiation was similar to the suppression by rho mutants. This was demonstrated for a polar nonsense mutant of phage phi X174. Treatment of the host for 30 min with 100 micrograms of the radiomimetic drug mitomycin C per ml was about as effective as 550 J of UV irradiation per m2 in relieving polarity. The shape of the UV survival curves for rho mutants could be linked to a proposed mechanism of UV relief of polarity. Host cell reactivation of phage lambda and W-reactivation of phage G4 were unaffected by rho mutations. UV suppression of polarity is independent of the Hcr- and RecA- phenotypes. An explanation for the UV sensitivity of rho mutants is provided, and several ways are considered in which UV irradiation may deplete cellular rho activity and thereby cause UV relief of polarity. We propose a novel theory that relates the UV inactivation of normal repair-proficient cells to a decrease in rho activity.

Analysis of the bacteriophage phi X174 eclipse period in terms of Arrhenius kinetic theory suggests the following hypothesis: mutants should exist with two concomitant physiological characteristics as their phenotype. These are an eclipse rate lower than that of the wild type at permissive temperatures for plaque formation and an eclipse rate too low at lower temperatures to permit plaque development. Thus, enrichment of a mutagenized virus population for mutants that fail to eclipse during a short period at permissive temperatures should yield eclipse mutants with the cold-sensitive (cs; nonpermissive temperature, 25 degrees C), and not the temperature-sensitive (ts; nonpermissive temperature, 42 degrees C), plaque phenotype. In several trials, the frequency of the cs phenotype in the population increased from less than 0.2% to between 2 and 4% after the enrichment step, whereas the frequency of the ts phenotype remained unchanged (less than 0.2%). Moreover, 80% of these cs mutants have eclipse rates that are 3- to 40-fold lower than that of the wild type at both 37 degrees C and 25 degrees C. The successful application of the Arrhenius theory to phi X eclipse may provide insights into the molecular mechanism whereby the phi X174 genome is delivered into the host cell. Since the eclipse kinetics of other nonenveloped viruses are similar to those of phi X174, kinetic theory may be broadly applicable in the selection and characterization of viral eclipse mutants.

A previous report (Hayashi et al., Proc. Natl. Acad. Sci. U.S.A. 73:3519-3523, 1976) indicated that in vivo bacteriophage phi X174 mRNA's terminate after genes J, F, G, and H. However, termination at these sites is not stringent. To determine whether termination of phi X174 transcription depends on rho factor activity, we introduced a temperature-sensitive rho mutation (nitA) into a phi X174-sensitive host cell line and determined termination sites in wild-type and nitA cells. We found that (i) normal phi X174 terminators were recognized in phi X174-infected nitA cells, (ii) the rho mutation relieved polar effects caused by nonsense mutations in the phage genome or by chloramphenicol treatment of the host cells, and (iii) polarity was not caused by premature termination of transcription at the site of the polar mutation. RNA synthesis continued beyond the site to the first rho-sensitive site.

Three simian virus (SV40)-phi X174 recombinant genomes were isolated from single BSC-1 monkey cells cotransfected with SV40 and phi X174 RF1 DNAs. The individual cell progenies were amplified, cloned, and mapped by a combination of restriction endonuclease and heteroduplex analyses. In each case, the 600 to 1,000 base pairs of phi X174 DNA (derived from different regions of the phi X174 genome) were present as single inserts, located in either the early or late SV40 regions; the deletion of SV40 DNA was greater than the size of the insert; and the remaining portions of the hybrid genome were indistinguishable from wild-type SV40 DNA, as judged by both mapping and biological tests. Hence, apart from the deletion which accommodates the phi X174 DNA insert, no other rearrangements of SV40 DNA were detected. The restriction map of a SV40-phi X174 recombinant DNA isolate before molecular cloning was indistinguishable from those of two separate cloned derivatives of that isolate, indicating that the species cloned was the major amplifiable recombinant structure generated by a single recombinant-producing cell. The relative simplicity of the SV40-phi X174 recombinant DNA examined is consistent with the notion that most recombinant-producing BSC-1 cells support single recombination events generating only one amplifiable recombinant structure.

The insertion of a particular phi X DNA sequence in the plasmid pACYC177 strongly decreased the capacity of Escherichia coli cells containing such a plasmid to propagate bacteriophage phi X174. The smallest DNA sequence tested that showed the effect was the HindII fragment R4. This fragment does not code for a complete protein. It contains the sequence specifying the C-terminal part of the gene H protein and the N-terminal part of the gene A protein, as well as the noncoding region between these genes. Analysis of cells that contain plasmids with the "reduction sequence" showed that (i) the adsorption of the phages to the host cells is normal, (ii) in a single infection cycle much less phage is formed, (iii) only 10% of the infecting viral single-stranded DNA is converted to double-stranded replicative-form DNA, and (iv) less progeny replicative form DNA is synthesized. The reduction process is phi X174 specific, since the growth of the related G4 and St-1 phages was not affected in these cells. The effect of the recombinant plasmids on infecting phage DNA shows similarity to the process of superinfection exclusion.

A hairpin-like secondary structure in the intercistronic region between genes J and F of bacteriophages, phi X174 and G4 has been postulated to act as a transcription termination signal. We analyzed the in vivo transcripts of both phages and mutants derived from them with modifications of this hairpin structure. The phi X174 mutants appeared to fall into two groups with respect to the stability of two mRNA species. Class 1 mutants showed an mRNA profile very similar to the parental strain, whereas class 2 mutants lacked two major mRNA species normally terminated near the J-F region. The G4 mutants behaved like class 2 mutants of phi X174. Analysis of the stability of phi X174 mRNA revealed that messages specific for the genes upstream of the hairpin turn over more rapidly in class 2 mutants than in class 1 mutants. In class 1 mutants, the mRNA decay rates are similar but not identical to those of the wild-type strain. These data suggest a role for the nucleotide sequence within the J-F intercistronic region in mRNA degradation. They further imply that transcription termination occurs downstream from this site.

The phi X174 A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated end. To determine the nature of the covalent linkage and the amino acid involved, we used the A protein to cleave DNA synthesized in vitro with [alpha-32P]dATP to form the complex of A protein covalently linked to single-stranded DNA. The complex was then digested with DNase I, and the 32P-labeled A protein was isolated by electrophoresis on polyacrylamide gels. The isolated complex was treated extensively with trypsin, and the released peptide-oligonucleotide complexes were incubated with formic acid and diphenylamine (Burton reaction). The Burton reaction caused a transfer of the labeled phosphate from dAMP to the peptide. The labeled phosphopeptides were isolated and hydrolyzed, revealing a linkage of the phosphate to a tyrosine. These results indicate that the A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated terminus by a tyrosyl-dAMP phosphodiester bond.