Early Synthesis of Membrane Protein After Bacteriophage T4 Infection

Proteins that associate with cellular membrane during the first 5 min after infection with bacteriophage T4 were examined. Several procedures, including electrophoretic separations in three sodium dodecyl sulfate polyacrylamide gel systems and inhibition of host protein synthesis by UV irradiation, were employed to distinguish host-specified proteins from those induced by T4. Residual host protein synthesis was found to account for much of the new protein in preparations of the total membrane and for almost all of the newly synthesized protein in the outer membrane. Preliminary evidence indicates that the synthesis of some host membrane proteins is shut off less rapidly than is host synthesis of soluble protein. One host-directed polypeptide of the outer membrane was unique in that its synthesis or incorporation into the membrane was preferentially inhibited by infection. Also, it was found that the detergent Sarkosyl solubilizes all early T4 membrane proteins; this observation provides the basis for a simple procedure for distinguishing phage proteins from host outer membrane proteins.     

Continued Synthesis of Bacterial DNA After Infection by Bacteriophage T4

Early in infection by bacteriophage T4, before replication has commenced, one can detect the presence of newly synthesized DNA which cosediments with parental phage DNA on sucrose gradients. As shown earlier (R. E. Murray and C. K. Mathews, 1969), some of this represents covalent attachment of new material to parental phage DNA molecules. However, as shown herein, most of it is bacterial DNA, which is synthesized after infection and presumably degraded to T4 DNA-sized pieces. The small amount of phage-specific DNA synthesis which occurs is apparently a repair process, for its extent is greatly increased if the phage are irradiated with ultraviolet light prior to infection. Analysis by means of pulse labeling with [(3)H]thymidine and DNA-DNA hybridization shows that host DNA synthesis continues at a significant rate (40 to 80% of the preinfection rate) as late as 10 min after infection at 37 C. Very early in infection this is primarily replicative synthesis, but later a repair process predominates. Presumably this represents attempted repair of damage being inflicted on host DNA by phage-coded nucleases.     

Unfolding of the Host Genome After Infection of Escherichia coli with Bacteriophage T4

The folded genome of Escherichia coli is converted to a slower-sedimenting form within 5 min after infection with bacteriophage T4 or T4nd28(den A)-amN82(44). Chloramphenicol sensitivity and response to UV-irradiation of the phage suggest participation of viral-induced functions.     

Membrane Interaction of the Portal Protein gp20 of Bacteriophage T4

Assembly of the bacteriophage T4 head structure occurs at the cytoplasmic face of the inner membrane of Escherichia coli with the formation of proheads. The proheads contain an internal scaffolding core that determines the size and the structure of the capsid. In a mutant where the major shell protein gp23 was compromised, core structures without a shell had been detected. Such core structures were also found in the mutant T4am20am23. Since the mutation in gene 20 is at the N terminus of gp20, it was assumed that these core structures assemble in the absence of gp20. However, sequencing showed that the mutation introduces a new ribosome binding site that leads to a restart at codon 15. Although the mutant protein gp20s lacks the very N-terminal sequence, we found that it still binds to the membrane of the host cell and can initiate prohead assembly. This explains its activity to allow the assembly of core structures and proheads at the membrane surface. With a cross-linking approach, we show here that gp20 and gp20s are escorted by the chaperones DnaK, trigger factor, and GroEL and dock on the membrane at the membrane protein YidC.     

Rescue of DNA Replication and Bacteriophage Production After Infection with T4 DNA Ligase Mutants

By preventing phage DNA synthesis during a critical period, conditions have been found under which DNA replication and phage production are rescued after infection with T4 DNA ligase mutants.     

Inhibition of T4 Bacteriophage Multiplication by Superinfecting Ghosts and the Development of Tolerance After Bacteriophage Infection

Simultaneous addition of T4 phage and ghosts to host cells prevents infective center formation. Cells which have been infected with phage for less than 2 min are also inhibited by superinfecting ghosts. After this time, a chloramphenicol-inhibitable reaction occurs which causes the phage-infected cells to become increasingly tolerant of added ghosts.     

Continuous Requirement for Host P10 Protein During Bacteriophage T4 Infection

The Escherichia coli 30S ribosomal protein P10, the product of the str gene, known to be involved only in the initiation of protein syntehsis, is required for all bacteriophage T4 protein synthesis.     

Synthesis of Messenger Ribonucleic Acid After Bacteriophage T1 Infection

Synthesis of host-specific and phage-specific messenger ribonucleic acid (mRNA) was studied in bacteria infected by unmodified (T1 . B) or modified [T1 . B(P1)] bacteriophage T1. In a "standard" infection of Escherichia coli B by T1 . B (no host-controlled modification involved), the rate and amount of T1 mRNA synthesis was intermediate between those values reported for infections by a virulent phage such as T4 or a temperate phage such as lambda. The initial rate of mRNA synthesis was slightly increased after T1 . B(P1) infection of E. coli B in comparison with T1 . B infection of the same host. Little or no phage mRNA synthesis could be detected in T1 . B infection of E. coli B(P1). Phage mRNA synthesis in T1 . B(P1)-infected E. coli B(P1) cells was approximately the same in amount as that seen in T1 . B(P1) infection of E. coli B. Synthesis of host-specific mRNA continued throughout the latent period in all infections studied. However, the enzyme beta-galactosidase could not be induced, except after T1 . B infection of E. coli B(P1). In an attempt to understand the apparent differences in mRNA synthesis after infection of E. coli B by phages T1 . B or T1 . B(P1), the effect of altered T1 deoxyribonucleic acid (DNA) methylation on mRNA synthesis was studied. Methyl-deficient T1 DNA, made in cells infected with ultraviolet-irradiated phage T3, inhibited (14)C-uridine incorporation more strongly than normal T1. One passage of methyl-deficient T1 through E. coli B restored uracil incorporation rates to those seen with ordinary T1. This suggests that methylation of T1 DNA can influence the rate of phage mRNA synthesis. However, attempts to relate the difference in mRNA synthesis seen between T1 . B and T1 . B(P1) in E. coli B to the activity of the P1 modification gene were not conclusive.     

Superinfection with Bacteriophage T4: Inverse Relationship Between Genetic Exclusion and Membrane Association of Deoxyribonucleic Acid of Secondary Bacteriophage

The majority of the deoxyribonucleic acid (DNA) of superinfecting T4 bacteriophage which is injected and not hydrolyzed does not attach to host cell membrane. Low levels of association of secondary phage DNA with membrane appear to be related to temporal genetic exclusion.     

Properties of Bacteriophage T4 Baseplate Protein Encoded by Gene 8

Gene product 8 (gp8, 344 amino acids per monomer) of bacteriophage T4 is one of the baseplate structural proteins. We constructed an expression vector of gp8 and developed a method for purification of recombinant protein. CD spectroscopy showed that gp8 is an / type structural protein. Its polypeptide chain consists of nearly 40% -structure and 15% -helix. These data agree with results of prediction of secondary structure based on the amino acid sequence of the protein. The sedimentation coefficient under standard conditions (S_20,w) is 4.6S. Analytical ultracentrifugation results demonstrated that gp8 in solution has two types of oligomers—dimer and tetramer. The tetramer of gp8 may be included in the wedge (1/6 of the baseplate), and the dimer may be an intermediate product of association.     

Degradation of Escherichia coli B Deoxyribonucleic Acid After Infection with Deoxyribonucleic Acid-Defective Amber Mutants of Bacteriophage T7

The degradation of bacterial deoxyribonucleic acid (DNA) was studied after infection of Escherichia coli B with DNA-negative amber mutants of bacteriophage T7. Degradation occurred in three stages. (i) Release of the DNA from a rapidly sedimenting cellular structure occurred between 5 and 6 min after infection. (ii) The DNA was cleaved endonucleolytically to fragments having a molecular weight of about 2 x 10(6) between 6 and 10 min after infection. (iii) These fragments of DNA were reduced to acid-soluble products between 7.5 and 15 min after infection. Stage 1 did not occur in the absence of the gene 1 product (ribonucleic acid polymerase sigma factor), stage 2 did not occur in the absence of the gene 3 product (phage T7-induced endonuclease), and stage 3 did not occur in the absence of the gene 6 product.     

Effect of Inhibition of Macromolecule Synthesis on the Association of Bacteriophage T4 DNA with Membrane

The "Mg(2+)-Sarkosyl crystals" (M band) technique distinguishes between membrane-bound and free intracellular DNA. This procedure was employed to investigate the nature of the reactions necessary to convert input T4 DNA to a rapidly sedimenting form. Energy poisoning inhibits this attachment reaction. Neither protein nor DNA synthesis appears to be required, but experiments with rifampin and extensively irradiated T4 suggest that RNA synthesis is involved. These results were confirmed by a second procedure for the determination of rapidly sedimenting DNA.     

Inhibition of Host Protein Synthesis During Infection of Escherichi coli by Bacteriophage T4: III. Inhibition by Ghosts

Deoxyribonucleic acid (DNA)-less T2 "ghosts" were prepared by osmotic shock and purified by KBr density gradient centrifugation. Escherichia coli B was treated with these ghosts in inorganic salts-glycerol medium to see which features of phage infection could be elicited by ghosts. At a multiplicity that was just sufficient to block induction of beta-galactosidase (EC 3.2.1.23), 89% of the bacteria were killed and the rates of ribonucleic acid (RNA) and DNA synthesis were about 10 to 15% of normal. However, protein synthesis was almost completely blocked but resumed after 30 min. During this period, it was possible to induce messenger RNA (mRNA) from the lactose operon, although this mRNA could not be translated into active beta-galactosidase. These results suggest to us that the viable cells surviving ghost infection synthesize nucleic acids at close to a normal rate but are temporarily blocked in protein synthesis. The continued formation of untranslated host mRNA mimics the pattern of bacterial synthesis just after whole-phage infection, and is consistent with the interpretation that the immediate block in the initiation of host translation by these viruses is due to their attachment.     

Transcription After Bacteriophage SPP1 Infection in Bacillus subtilis

The role of the host polymerase in Bacillus subtilis infected with phage SPP1 was studied in vivo with regard to production of phage-specific and host-specific ribonucleic acid (RNA) and to phage yield. Evidence is presented that the subunit(s) of B. subtilis RNA polymerase which is sensitive to rifampin and streptolydigin is necessary at all times during infection for phage production. The synthesis of phage RNA and the phage yield in strains resistant to either antibiotic were unaffected by the drug. Host RNA synthesis continued throughout infection; phage-specific RNA never accounted for more than 20% of pulse-labeled RNA at any time during infection.     

Multiple-Tailed T4 Bacteriophage.

Smith, Kendall O. (Baylor University College of Medicine, Houston, Tex.), and Melvin Trousdale. Multiple-tailed T4 bacteriophage. J. Bacteriol. 90:796-802. 1965.-T4 phage particles which appeared to have multiple-tails were observed. Experiments were designed to minimize the possibility that superimposed particles might account for this appearance. Double-tailed particles occurred at a frequency as high as 10%. Triple- and quadruple-tailed particles were extremely rare. All attempts to isolate pure lines of multiple-tailed phage have failed. Multiple-tailed phage particles were produced in highest frequency by Escherichia coli cells in the logarithmic growth phase which had been inoculated at a multiplicity of about 2.     

Localization and Importance of the Adenovirus E4orf4 Protein during Lytic Infection

The human adenovirus type 5 (Ad5) E4orf4 product has been studied extensively although in most cases as expressed from vectors in the absence of other viral products. Thus, relatively little is known about its role in the context of an adenovirus infection. Although considerable earlier work had indicated that the E4orf4 protein is not essential for replication, a recent study using dl359, an Ad5 mutant believed to produce a nonfunctional E4orf4 protein, suggested that E4orf4 is essential for virus growth in primary small-airway epithelial cells (C. O'Shea, et al., EMBO J. 24:1211-1221, 2005). Hence, to examine further the role of E4orf4 during virus infection, we generated for the first time a set of E4orf4 virus mutants in a common Ad5 genetic background. Such mutant viruses included those that express E4orf4 proteins containing various individual point mutations, those defective entirely in E4orf4 expression, and a mutant expressing wild-type E4orf4 fused to the green fluorescent protein. E4orf4 protein was found to localize primarily in nuclear structures shown to be viral replication centers, in nucleoli, and in perinuclear bodies. Importantly, E4orf4 was shown not to be essential for virus growth in either human tumor or primary cells, at least in tissue culture. Unlike E4orf4-null virus, mutant dl359 appeared to exhibit a gain-of-function phenotype that impairs virus growth. The dl359 E4orf4 protein, which contains a large in-frame internal deletion, clustered in aggregates enriched in Hsp70 and proteasome components. In addition, the late viral mRNAs produced by dl359 accumulated abnormally in a nuclear punctate pattern. Altogether, our results indicate that E4orf4 protein is not essential for virus growth in culture and that expression of the dl359 E4orf4 product interferes with viral replication, presumably through interactions with structures in the nucleus.     

Polymerization of Bacteriophage T4 Tail Sheath Protein Mutants Truncated at the C-Termini

Gene 18 of bacteriophage T4 encodes the contractile protein of the tail sheath. Previous work has shown that the full-length recombinant gene product (gp) 18 of 658 amino acid residues assembles in Escherichia coli cells into a long polysheath structure. However, the gp18 mutants truncated at the N-termini form insoluble aggregates similar to inclusion bodies. In this study, six plasmid vectors expressing the recombinant gp18 proteins truncated at the C-termini have been constructed. The CDelta58, CDelta129, CDelta152, C[g1]72, CDelta248, and CDelta287 proteins contain 600, 529, 506, 486, 410, and 371 residues of the full-length gp18 molecule, respectively. All the recombinant proteins were soluble and, except for the CDelta287 mutant, were assembled into polysheath-related structures. Electron microscopy of negatively stained purified proteins was performed and the resulting images were analyzed by computing their Fourier transforms. The CDelta58 and CDelta129 mutants, in addition to forming common contracted-type polysheath structures, assembled into thinner filaments that we called "noncontracted polysheaths" (NCP). The CDelta152, CDelta172, and CDelta248 proteins assembled into the NCP type only. Image processing showed that the NCP filaments significantly differ from both extended sheaths of T4 particle and polysheaths. The structure of the NCP filaments might correspond to the transitional helices postulated by Moody (J. Mol. Biol., 1973, 80, 613-636) that appeared during the process of tail contraction. Our results suggest that a short region at the C-terminus of the CDelta129 protein determines the contractile properties of the gp18 molecule. The shortest, the CDelta287 protein, does not assemble into regular structures, thus indicating that a sequence's stretch at the C-end of the CDelta248 mutant might be responsible for polymerization of gp18.