High-throughput assay for temporal kinetic analysis of lytic coliphage activity

Plaque analysis allows the determination of phage titer and multiplicity of infection. Yet, this overnight assay provides only endpoint results, ignoring kinetic aspects. We introduce an alternative high-throughput and rapid method for kinetic analysis of lytic coliphage activity. Escherichia coli was infected with serial dilutions of MS2 coliphage, and bacterial growth was monitored using a multi-well plate reader providing within hours the equivalent data as obtained overnight. Additional information is yielded, including phage replication rate, progeny size per cycle, and viral propagation during bacterial growth. This method offers further insights into physicochemical mechanisms of lytic coliphage infection and temporal control. It also provides a virus–host interaction acumen.     

Rescue of abortive T7 gene 2 mutant phage infection by rifampin.

Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.     

Mechanism of replication of phichi174 single-stranded DNA. V. Dispersive and conservative transfer of parental DNA into progeny DNA

We have studied parent-to-progeny transfer of bacteriophage φX174 DNA during infection of Escherichia coli with isotopically-labeled, lysis-defective phage. After 60 minutes of infection at low multiplicities, 25 to 30% of the input viral DNA is transferred from the double-stranded replicative form into progeny phage; another 10 to 20% is transferred into the progeny single-stranded DNA pool. Thus, at times beyond the normal time of lysis, about 35 to 50% of the parental deoxyribonucleotides are found in progeny single-stranded DNA. Three quarters of the parental label found in the progeny phage is transferred by a dispersive process and one-quarter is transferred by a conservative, or non-dispersive, process such that the parental strand remains intact. At high multiplicities of infection the fraction of parental label transferred decreases.     

Manipulating or Superseding Host Recombination Functions: A Dilemma That Shapes Phage Evolvability

Phages, like many parasites, tend to have small genomes and may encode autonomous functions or manipulate those of their hosts''. Recombination functions are essential for phage replication and diversification. They are also nearly ubiquitous in bacteria. The E. coli genome encodes many copies of an octamer (Chi) motif that upon recognition by RecBCD favors repair of double strand breaks by homologous recombination. This might allow self from non-self discrimination because RecBCD degrades DNA lacking Chi. Bacteriophage Lambda, an E. coli parasite, lacks Chi motifs, but escapes degradation by inhibiting RecBCD and encoding its own autonomous recombination machinery. We found that only half of 275 lambdoid genomes encode recombinases, the remaining relying on the host''s machinery. Unexpectedly, we found that some lambdoid phages contain extremely high numbers of Chi motifs concentrated between the phage origin of replication and the packaging site. This suggests a tight association between replication, packaging and RecBCD-mediated recombination in these phages. Indeed, phages lacking recombinases strongly over-represent Chi motifs. Conversely, phages encoding recombinases and inhibiting host recombination machinery select for the absence of Chi motifs. Host and phage recombinases use different mechanisms and the latter are more tolerant to sequence divergence. Accordingly, we show that phages encoding their own recombination machinery have more mosaic genomes resulting from recent recombination events and have more diverse gene repertoires, i.e. larger pan genomes. We discuss the costs and benefits of superseding or manipulating host recombination functions and how this decision shapes phage genome structure and evolvability.     

Restoration by Chloramphenicol of Bacteriophage Production in Escherichia coli B Infected with a Ligase-Deficient Amber Mutant

The addition of chloramphenicol (CM) 5 min after infection of the nonpermissive host Escherichia coli B with the ligase-negative T4 amber, T4 AmH39X, allowed replication of parental deoxyribonucleic acid (DNA) and the production of high-molecular-weight progeny DNA, composed mostly of subunits with a D₂/D₁ of 0.6. When CM was removed after the accumulation of a large pool of this DNA, most of the infected bacteria were able to produce viable progeny phage, with an average yield of approximately 15 bacteriophage per bacterium. This phenomenon is called CM rescue of the ligase-negative T4 Am. CsCl and sucrose gradient analyses showed both the resulting phage and DNA extracted from them to be similar to the phage and DNA produced on the permissive host. The total transfer of the parental label to progeny phages was as high as 20%. In contrast, in bacteria not treated with CM or in bacteria to which CM was added after phage-coded nucleases had already been synthesized, both parental and progeny (newly synthesized) DNA was composed of very short fragments. Phage which are produced under conditions other than those of CM rescue are dead, light in CsCl, and contain only very short fragments of DNA. Parent-to-progeny transfer in this case is below 1%. When light radio-active parental DNA was used to infect heavy bacteria, DNA replicating in the CM rescue conditions assumed only a hybrid density. After removal of CM and maturation, the parental DNA was incorporated into progeny molecules in fragments constituting approximately 7 to 10% of its mass. This pattern of distribution is essentially what is observed in similar experiments in the permissive host. The role of ligase as an enzyme which compensates for the lethal action of phage-coded nuclease and which is stringently required for the repair of single-stranded nicks is emphasized. The possibility of specific sites for a unique cutting enzyme is discussed in connection with the hypothesis of a circularly permuted assembly of sets.     

Genetic and Immunological Studies of Bacteriophage T4 Thymidylate Synthetase

Thymidylate synthetase, which appears after infection of Escherichia coli with bacteriophage T4, has been partially purified. The phage enzyme is immunologically distinct from the host enzyme and has a molecular weight of 50,000 in comparison to 68,000 for the host enzyme. A system has been developed to characterize T4 td mutants previously known to have impaired expression of phage thymidylate synthetase. For this system, an E. coli host lacking thymidylate synthetase was isolated. Known genetic suppressors were transduced into this host. The resulting isogenic hosts were infected with phage T4 td mutants. The specific activities and amounts of cross-reacting material induced by several different types of phage mutants under conditions of suppression or non-suppression have been examined. The results show that the phage carries the structural gene specifying the thymidylate synthetase which appears after phage infection, and that the combination of plaque morphology, enzyme activity assays, and an assay for immunologically cross-reacting material provides a means for identifying true amber mutants of the phage gene.     

The mechanism of replication of phi X174 single-stranded DNA. II. The role of viral proteins

We have studied the replication of φX174 DNA in Escherichia coli infected with various amber mutants (cistrons I to VII) of φX. Previous research showing that some of these mutants are able to form replicative form (RF) DNA but are unable to produce net amounts of viral progeny single-stranded DNA has been confirmed and extended. Evidence is presented that a defect in any one of four viral cistrons prevents the asymmetric replication of the RF to produce progeny viral DNA. At least four virus-coded proteins, three of which are part of the mature virion, must be present before single-stranded DNA synthesis can even be initiated; the possibility that single-stranded DNA is made and then degraded or converted to RF is eliminated. Mutants in one cistron (II) do permit the asymmetric replication of RF at late times, but the displaced viral strand is incorporated into a defective particle and subsequently may be partially degraded. Both RFI (superhelix) and RFII are present in roughly comparable amounts throughout the normal latent period in infections with wild-type phage or any of the phage mutants.     

ppGpp-Dependent Negative Control of DNA Replication of Shiga Toxin-Converting Bacteriophages in Escherichia coli

The pathogenicity of enterohemorrhagic Escherichia coli (EHEC) strains depends on the production of Shiga toxins that are encoded on lambdoid prophages. Effective production of these toxins requires prophage induction and subsequent phage replication. Previous reports indicated that lytic development of Shiga toxin-converting bacteriophages is inhibited in amino acid-starved bacteria. However, those studies demonstrated that inhibition of both phage-derived plasmid replication and production of progeny virions occurred during the stringent as well as the relaxed response to amino acid starvation, i.e., in the presence as well as the absence of high levels of ppGpp, an alarmone of the stringent response. Therefore, we asked whether ppGpp influences DNA replication and lytic development of Shiga toxin-converting bacteriophages. Lytic development of 5 such bacteriophages was tested in an E. coli wild-type strain and an isogenic mutant that does not produce ppGpp (ppGpp⁰). In the absence of ppGpp, production of progeny phages was significantly (in the range of an order of magnitude) more efficient than in wild-type cells. Such effects were observed in infected bacteria as well as after prophage induction. All tested bacteriophages formed considerably larger plaques on lawns formed by ppGpp⁰ bacteria than on those formed by wild-type E. coli. The efficiency of synthesis of phage DNA and relative amount of lambdoid plasmid DNA were increased in cells devoid of ppGpp relative to bacteria containing a basal level of this nucleotide. We conclude that ppGpp negatively influences the lytic development of Shiga toxin-converting bacteriophages and that phage DNA replication efficiency is limited by the stringent control alarmone.     

Direct Inhibition of Cellular Fatty Acid Synthase Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses

Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a fatty acid utilized for synthesis of more complex fatty acids, plasma membrane structure, and post-translational palmitoylation of host and viral proteins. We have developed a potent inhibitor of FASN (TVB-3166) that reduces the production of respiratory syncytial virus (RSV) progeny in vitro from infected human lung epithelial cells (A549) and in vivo from mice challenged intranasally with RSV. Addition of TVB-3166 to the culture medium of RSV-infected A549 cells reduces viral spread without inducing cytopathic effects. The antiviral effect of the FASN inhibitor is a direct consequence of reducing de novo palmitate synthesis; similar doses are required for both antiviral activity and inhibition of palmitate production, and the addition of exogenous palmitate to TVB-3166-treated cells restores RSV production. TVB-3166 has minimal effect on RSV entry but significantly reduces viral RNA replication, protein levels, viral particle formation and infectivity of released viral particles. TVB-3166 substantially impacts viral replication, reducing production of infectious progeny 250-fold. In vivo, oral administration of TVB-3166 to RSV-A (Long)-infected BALB/c mice on normal chow, starting either on the day of infection or one day post-infection, reduces RSV lung titers 21-fold and 9-fold respectively. Further, TVB-3166 also inhibits the production of RSV B, human parainfluenza 3 (PIV3), and human rhinovirus 16 (HRV16) progeny from A549, HEp2 and HeLa cells respectively. Thus, inhibition of FASN and palmitate synthesis by TVB-3166 significantly reduces RSV progeny both in vitro and in vivo and has broad-spectrum activity against other respiratory viruses. FASN inhibition may alter the composition of regions of the host cell membrane where RSV assembly or replication occurs, or change the membrane composition of RSV progeny particles, decreasing their infectivity.     

On the lack of host-cell reactivation of UV-irradiated phage T5 II. Further characterization of the repair inhibition exerted by T5 infection

Experiments reported in the preceding paper [4] had shown that host-cell reactivation (HCR) of UV-irradiated phage T1 in excision-repair proficient Escherichia coli cells is inhibited by superinfection with phage T5. Theoretical considerations have led to predictions concerning the dependence of repair inhibition on the multiplicity of superinfecting T5 phage and on the UV fluence to which they were exposed. These predictions have been supported by experimental results described in this paper. The fluence dependence permitted calculation of the relative UV sensitivity of the gene function responsible for repair inhibition; it was found to be about 2.3% that of the plaque-forming ability of phage T5.The T5-inhibitable step in excision repair occurs early in the infective cycle of T1. Furthermore, experiments involving the presence of 400 μg/ml chloramphenicol showed that HCR inhibition of T1 is caused by a protein produced after the FST segment of T5 (i.e. the first 8% of the T5 genome) has entered the host cell. A previously described minor T1 recovery process, occuring in both excision-repair-proficient and -deficient host cells, is inhibited by T5 infection due to a different substance, which is most likely associated with the “second-step-transfer” region of T5 DNA (involving the remainder of the genome). Superinfection with T4ν₁ phage resulted in HCR inhibition of T1, resembling that observed after T5 superinfection. The discussion of these results suggests that inhibition of the bacterial excision repair system by T5 or T4 infection occurs at the level of UV-endonucleolytic incision, and that lack of HCR both in T-even phages and in T5 can be explained in the same manner.     

Effect of Prophage W on the Propagation of Bacteriophages T2 and T4

Studies have been undertaken to determine whether the temperate phage ω present in Escherichia coli strain W is responsible for the inability of this strain to act as a host for T2 and T4. E. coli WS, cured of phage ω, was sensitive to T2 and T4. Lysogenation of E. coli C and WS with phage ω resulted in loss of ability to plate T2 and T4. However, E. coli K-12 lysogens still served as hosts for the T -even phage. Two of three WS lysogens studied resembled strain W at the biochemical level. They converted about 30% of infecting T2 deoxyribonucleic acid (DNA) to acid-soluble fragments and limited macromolecular synthesis to a few minutes after infection. The third lysogen did not degrade phage DNA, and nucleic acid and protein synthesis continued for some time, although no phage production occurred. It is concluded that phage ω plays a role in the restriction of virulent phage but that it is not the only factor involved. Since acid solubilization was not observed in all cases of phage ω-mediated restriction of T -even phage, a hypothesis for the restriction has been proposed which is based on an alteration in the cell envelope after lysogenation with phage ω.     

A Forward-Genetic Screen and Dynamic Analysis of Lambda Phage Host-Dependencies Reveals an Extensive Interaction Network and a New Anti-Viral Strategy

Latently infecting viruses are an important class of virus that plays a key role in viral evolution and human health. Here we report a genome-scale forward-genetics screen for host-dependencies of the latently-infecting bacteriophage lambda. This screen identified 57 Escherichia coli (E. coli) genes—over half of which have not been previously associated with infection—that when knocked out inhibited lambda phage''s ability to replicate. Our results demonstrate a highly integrated network between lambda and its host, in striking contrast to the results from a similar screen using the lytic-only infecting T7 virus. We then measured the growth of E. coli under normal and infected conditions, using wild-type and knockout strains deficient in one of the identified host genes, and found that genes from the same pathway often exhibited similar growth dynamics. This observation, combined with further computational and experimental analysis, led us to identify a previously unannotated gene, yneJ, as a novel regulator of lamB gene expression. A surprising result of this work was the identification of two highly conserved pathways involved in tRNA thiolation—one pathway is required for efficient lambda replication, while the other has anti-viral properties inhibiting lambda replication. Based on our data, it appears that 2-thiouridine modification of tRNA^Glu, tRNA^Gln, and tRNA^Lys is particularly important for the efficient production of infectious lambda phage particles.     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates II. Folate Metabolism and Polyglutamate Cleavage Activity of Uninfected and Infected Escherichia coli Cells and Bacteriophage Particles

We investigated the role of the T4D bacteriophage gene 28 product in folate metabolism in infected Escherichia coli cells by using antifolate drugs and a newly devised assay for folyl polyglutamate cleavage activity. Preincubation of host E. coli cells with various sulfa drugs inhibited phage production by decreasing the burst size when the phage particles produced an altered gene 28 product (i.e., after infection under permissive conditions with T4D 28^ts or T4D am28). In addition, we found that another folate analog, pyrimethamine, also inhibited T4D 28^ts production and T4D 28am production, but this analog did not inhibit wild-type T4D production. A temperature-resistant revertant of T4D 28^ts was not sensitive to either sulfa drugs or pyrimethamine. We developed an assay to measure the enzymatic cleavage of folyl polyglutamates. The high-molecular-weight folyl polyglutamate substrate was isolated from E. coli B cells infected with T4D am28 in the presence of labeled glutamic acid and was characterized as a folate compound containing 12 to 14 labeled glutamate residues. Extracts of uninfected bacteria liberated glutamate residues from this substrate with a pH optimum of 8.4 to 8.5. Extracts of bacteriophage T4D-infected E. coli B cells exhibited an additional new folyl polyglutamate cleavage activity with a pH optimum of about 6.4 to 6.5, which was clearly distinguished from the preexisting activity in the uninfected host cells. This new activity was induced in E. coli B cells by infection with wild-type T4D and T4D amber mutants 29⁻, 26⁻, 27⁻, 51⁻, and 10⁻, but it was not induced under nonpermissive conditions by T4D am28 or by T4D 28^ts. Mutations in gene 28 affected the properties of the induced cleavage enzyme. Wild-type T4D-induced cleavage activity was not inhibited by pyrimethamine, whereas the T4D 28^ts activity induced at a permissive temperature was inhibited by this folate analog. Folyl polyglutamate cleavage activity characteristic of the activity induced in host cells by wild-type T4D or by T4D gene 28 mutants was also found in highly purified preparations of these phage ghost particles. The T4D-induced cleavage activity could be inhibited by antiserum prepared against highly purified phage baseplates. We concluded that T4D infection induced the formation of a new folyl polyglutamate cleavage enzyme and that this enzyme was coded for by T4D gene 28. Furthermore, since this gene product was a baseplate tail plug component which had both its antigenic sites and its catalytic sites exposed on the phage particle, it was apparent that this enzyme formed part of the distal surface of the phage baseplate central tail plug.     

In vivo methylation of replicating bacteriophage phi chi174 DNA

The pattern of DNA methylation during the infection of Escherichia coli C cells with bacteriophage φX174, has been studied. In vivo methylated DNA was isolated and analyzed using the following techniques: velocity sedimentation through neutral and alkaline sucrose gradients, isopycnic analysis, chromatography on benzoylated DBAE-cellulose columns and specific enzymatic digestion. All these analytical methods indicated that the DNA molecules that are methylated during the process of phage φX DNA replication are the replicating intermediates composed of a circular complementary strand and a viral strand larger than one genome length. It is concluded that methylation occurs on the nascent DNA strand of the replicating intermediates involved in the synthesis of progeny single-stranded DNA.     

Host restriction of emerging high-pathogenic bunyaviruses via MOV10 by targeting viral nucleoprotein and blocking ribonucleoprotein assembly

Bunyavirus ribonucleoprotein (RNP) that is assembled by polymerized nucleoproteins (N) coating a viral RNA and associating with a viral polymerase can be both the RNA synthesis machinery and the structural core of virions. Bunyaviral N and RNP thus could be assailable targets for host antiviral defense; however, it remains unclear which and how host factors target N/RNP to restrict bunyaviral infection. By mass spectrometry and protein-interaction analyses, we here show that host protein MOV10 targets the N proteins encoded by a group of emerging high-pathogenic representatives of bunyaviruses including severe fever with thrombocytopenia syndrome virus (SFTSV), one of the most dangerous pathogens listed by World Health Organization, in RNA-independent manner. MOV10 that was further shown to be induced specifically by SFTSV and related bunyaviruses in turn inhibits the bunyaviral replication in infected cells in series of loss/gain-of-function assays. Moreover, animal infection experiments with MOV10 knockdown corroborated the role of MOV10 in restricting SFTSV infection and pathogenicity in vivo. Minigenome assays and additional functional and mechanistic investigations demonstrate that the anti-bunyavirus activity of MOV10 is likely achieved by direct impact on viral RNP machinery but independent of its helicase activity and the cellular interferon pathway. Indeed, by its N-terminus, MOV10 binds to a protruding N-arm domain of N consisting of only 34 amino acids but proving important for N function and blocks N polymerization, N-RNA binding, and N-polymerase interaction, disabling RNP assembly. This study not only advances the understanding of bunyaviral replication and host restriction mechanisms but also presents novel paradigms for both direct antiviral action of MOV10 and host targeting of viral RNP machinery.     

Escherichia coli Mutants Permissive for T4 Bacteriophage with Deletion in e Gene (Phage Lysozyme)

Escherichia coli mutants have been isolated that are permissive for the infection by T4 phage with deletion in the cistron for the phage lysozyme, the e gene. Some, but not all, of these mutants are simultaneously permissive for the infection by T4 phage defective in the t gene, the product of which has also been implicated in the release of progeny phages. Most of these mutants shared the following properties: temperature sensitivity in growth and cell division, increased sensitivity towards a number of unrelated antibiotics and colicins, and increased sensitivity towards anionic detergents (sodium dodecyl sulfate and sodium deoxycholate). The possible biochemical basis for these phenotypes is discussed.     

Inhibition of aldolase A blocks biogenesis of ATP and attenuates Japanese encephalitis virus production

Viral replication depends on host proteins to supply energy and replication accessories for the sufficient production of viral progeny. In this study, we identified fructose-bisphosphate aldolase A as a binding partner of Japanese encephalitis virus (JEV) untranslated regions (UTRs) on the antigenome via RNA affinity capture and mass spectrometry. Direct interaction of aldolase A with JEV RNAs was confirmed by gel mobility shift assay and colocalization with active replication of double-stranded RNA in JEV-infected cells. Infection of JEV caused an increase in aldolase A expression of up to 33%. Knocking down aldolase A reduced viral translation, genome replication, and viral production significantly. Furthermore, JEV infection consumed 50% of cellular ATP, and the ATP level decreased by 70% in the aldolase A-knockdown cells. Overexpression of aldolase A in aldolase A-knockdown cells increased ATP levels significantly. Taken together, these results indicate that JEV replication requires aldolase A and consumes ATP. This is the first report of direct involvement of a host metabolic enzyme, aldolase A protein, in JEV replication.     

The dependence of viral RNA replication on co-opted host factors

Positive-sense RNA ((+)RNA) viruses such as hepatitis C virus exploit host cells by subverting host proteins, remodelling subcellular membranes, co-opting and modulating protein and ribonucleoprotein complexes, and altering cellular metabolic pathways during infection. To facilitate RNA replication, (+)RNA viruses interact with numerous host molecules through protein-protein, RNA-protein and protein-lipid interactions. These interactions lead to the formation of viral replication complexes, which produce new viral RNA progeny in host cells. This Review presents the recent progress that has been made in understanding the role of co-opted host proteins and membranes during (+)RNA virus replication, and discusses common themes employed by different viruses.