Satellite bacteriophage P4: characterization of mutants in two essential genes

P4 is a helper-dependent bacteriophage which can use the late gene products of temperate phage P2 to encapsulate P4 DNA (Six, 1963; Six and Lindqvist, 1970; Gibbs, 1972; Six and Klug, 1973). P4 phage heads (d = 450 Å) contain only one-third the volume of P2 phage heads (d = 620 Å; Inman et al., 1971), and heads of the P4 size are not detected during a normal P2 infection (D. Walker, personal communication). Thus P4 directs the formation of 450 Å phage heads. P4 causes P2 prophage late genes to be expressed in the presence of immunity (Six and Klug, 1973), and without excision or replication of the prophage genome (Six and Lindqvist, 1971). In the absence of a helper, P4 DNA can replicate (Lindqvist and Six, 1971).We have isolated and characterized mutants in two essential P4 genes. P4 gene A mutants are unable to synthesize P4 DNA, but they retain the ability to transactivate² P2 prophage genes under nonpermissive conditions. Thus the A gene product may participate directly in the process of P4 DNA replication. P2-lysogenic, nonpermissive cells infected with P4 gene A mutants synthesize empty phage heads, 80% of which are intermediate in size between P4 heads and P2 heads. Heads of this intermediate size are also formed in small quantity during a normal P2 infection. The inability of P4 gene A mutants to synthesize P4-size heads may be due to a lack of replicating DNA or to lack of a size-directing protein.P4 gene B is defined by one temperature-sensitive mutation which is partially dominant to P4 wild type at 42 °. At the nonpermissive temperature this mutant can synthesize P4 DNA and cause a P2 prophage to be transcribed, but cannot cause the formation of head-like particles. Unlike P4 wild type, this mutant kills nonlysogenic cells at 42 °, and greatly depresses the synthesis of DNA, RNA, and protein.     

The bacteriophage f1 morphogenetic signal and the gene V protein/phage single-stranded DNA complex

The region of the bacteriophage f1 genome near the gene IV/intergenic region (IG) junction previously was shown to contain a sequence necessary for efficient packaging of single-stranded (SS) DNA into phage particles (the f1 “morphogenetic signal”) [G. P. Dotto, V. Enea, and N. D. Zinder (1981)Virology114, 463–473]. The DNA content of f1 phage/pBR322 chimeric plasmid-transformed, phage-infected bacteria has been investigated. The chimeric plasmids, constructed by Dotto et al. (1981), contained the f1 origins of DNA replication and, in some cases, also contained the “morphogenetic signal.” Chimeric plasmid SS DNA was detected in phage-infected bacteria harboring any one of these chimeric plasmids, and the majority of this SS DNA was found complexed to the phage gene V protein. Therefore, the “morphogenetic signal” is not required for the formation of the gene V protein/f1 SS DNA complex but instead must function at a later stage of filamentous phage morphogenesis.     

Substitution mutation in bacteriophage lambda with new specificity for late gene expression

The p4 mutation of phage λ was originally identified as a mutation leading to a small-plaque phenotype (Jacob and Wollman, 1954). Subsequent work has shown that the p4 mutation results in a net addition of DNA to the right arm of λ DNA (Weil et al., 1972; Herskowitz, 1971). We show here that p4 is a substitution mutation in which λ genes Q, S, and R are deleted and are replaced by analogous genes presumably from another phage.The qin mutations of Sato and Campbell (1970) appear very similar to the p4 mutation. The source of the substituted genes may be a hitherto unrecognized defective prophage present in certain strains of E. coli K12.Studies of late gene experession by phages carrying p4 or qin mutations give further evidence that late genes of λ are controlled by a diffusible factor (Q protein) which acts at an essential site in the right arm of λ DNA (the late gene promoter), and demonstrate the specificity of this interaction.     

Identification of a second protein (M2) encoded by RNA segment 7 of influenza virus

Recent analyses of the nucleotide sequences of RNA segment 7 of influenza virus from two strains of infuenza A virus (PR/8/34 and Udorn/72) have shown that in addition to the reading frame coding for the membrane protein (M₁) there is a second overlapping open reading frame that could code for 97 amino acids [Winter and Fields (1980). Nucleic Acids Res.8, 1965–1974;Allen et al. (1980). Virology107, 548–551;Lamb and Lai (1981). Virology112, 746–751]. We have identified such a protein (M₂) in infected cells and shown that it is synthesized from a separate mRNA. Hybrid-arrested translation experiments and the use of recombinant viruses of defined genome composition have demonstrated by both biochemical and genetic approaches that protein M₂ is coded for by RNA segment 7. Peptide mapping studies have shown that M₁ and M₂ are distinct proteins, and the peptide maps can be correlated with the nucleotide sequences coding for M₁ and M₂.     

The visualization of a circular DNA-protein complex from adenovirions

A DNA-protein complex was extracted from adenovirus type 5 virions with 4 M guanidine hydrochloride, and was purified by sucrose gradient velocity centrifugation. The isolated DNA-protein complex contained about 0.8% of the total protein of intact virions. Electron microscopic visualization of the DNA-protein complexes revealed circular DNA containing one protein entity as well as linear molecules with protein knobs at both molecular ends. The location of the protein knobs makes it probable that these knobs indicate the presence of a terminal protein discovered by 4., 5., which is able to keep linear adenovirus DNA molecules in a circular state.     

New att mutants of phage λ

Phage λ inserts its chromosome into that of its host Escherichia coli by recombination at specific attachment sites (atts). New deletion and point mutants affecting att were isolated. The point mutants map as if they are in a region common to the atts and could not be distinguished from att mutants described earlier (Shulman and Gottesman, 1973).Effects of the mutations on the physiology of insertion and excision are presented.     

Participation of the s gene product of phage T4 in the establishment of resistance to T4 ghosts

T-even phage-infected cells of Escherichia coli have been shown to become resistant to phage ghosts (Duckworth, 1971; Vallée et al., 1972, Okamoto, 1973). The present data show that a mutation in the s gene of T4 phage leads to a failure to establish the resistance. If cells are infected with a temperature-sensitive s mutant (T4tss) at lower temperatures and then transferred to a high temperature in the presence of chloramphenicol, the once established resistance is destroyed depending on the extent of heat treatment. These results indicate that the s gene product (protein) is likely to be directly involved in the establishment of the resistance, and is needed throughout infection for the maintenance of the resistant state.If the cells that were infected with T4tss at a low temperature and then challenged with ghosts, are transferred to a high temperature, the inhibitory action of the ghosts begins to exert itself. This suggests that the phage-induced resistance does not appear to be a result of inactivation of the adsorbed ghosts.Comparison of a mutant T4s2 with T4imm2, another mutant which fails to confer resistance to ghosts on the host cells (Vallée and Cornett, 1972) reveals that cells infected with either of these phages lack resistance to a comparative degree. Infection with a double mutant, T4s2-imm2, renders cells much more sensitive to ghosts than cells infected with individual parental phage. These facts imply that both s- and imm-gene products are required for the establishment of the resistance and that these two products probably act “cooperatively” rather than “additively.”     

Inhibition of vesicular stomatitis virus-defective interfering particle synthesis by Shope fibroma virus.

Undiluted passage of vesicular stomatitis virus (VSV) in a line of African green monkey kidney cells results in a cyclic synthesis of standard infectious VSV. This pattern of virus yield is due to the cyclic production of defective interfering (DI) particles [Perrault, J., and Holland, J. J. (1972). Virology50, 148–158; Palma, B. L., and Huang, A. S. (1974). J. Infect. Dis.129, 403–410; Holland, J. J., Villareal, L. P., and Briendl, M. (1976). J. Virol.17, 805–813]. When such cells were infected with Shope fibroma virus (ShFV) prior to serial undiluted passage of VSV, the normal cyclic yield of VSV was altered. Using two criteria for the determination of DI particles, i.e., the interference assay and the detection of physical particles by gradient technique, it was shown that ShFV exerted its effect by inhibiting the synthesis of VSV-DI particles. It is suggested that ShFV affects both the induction and the amplification of DI particles in this system. Experiments also indicate that the ShFV-mediated inhibition of VSV-DI particle synthesis is probably not due to poxvirus-induced inhibition of cellular macromolecular biosynthesis.     

Tryptic and chymotryptic methionine peptide analysis of the in Vitro translation products specified by the transforming region of adenovirus type 2

Early region 1 (El) cytoplasmic RNAs of adenovirus type 2 were translated in vitro. Structural relationships between these proteins were then established by tryptic and chymotryptic digestion and two-dimensional peptide mapping. This analysis also allowed a direct comparison of these proteins with polypeptides isolated from early infected cell extracts and previously assigned to E1 by indirect means (M., Green, W. S. M. Wold, K. H. Brackmann, and M. A. Cartas, 1979, Virology 97, 275–286). E1a (0–4.5%) RNA encodes five proteins in vitro: 35K, 41K, 47K and 53K at early times, and a 28K protein at later times in infection. These five proteins are all highly related by peptide mapping analysis, and are also related to a group of four proteins isolated from cell extracts prepared early in infection. E1b (4.5–11.0%) early RNA encodes 52K and one or more 15K proteins in vitro. The 52K protein shares tryptic peptides with a 53K immunoprecipitated T antigen. A 15K protein shares some peptides with the 52K protein and with several proteins isolated from infected cells. At intermediate to late times, E1b RNA encodes a 12K protein that corresponds to the virion structural polypeptide IX. These data also permit the correlation of E1 proteins synthesized in vitro with the polypeptides predicted by E1 sequence analysis.     

Replication of spleen focus-forming friend virus in fibroblasts from C57BL mice that are genetically resistant to spleen focus formation

Induction of erythroleukemia and spleen focus formation by the defective spleen focus-forming component of Friend virus (SFFV) is governed by a genetic locus of the mouse termed Fv-2 (F. Lilly, J. Nat. Cancer Inst.45, 163–169, 1970). Subsequently, the Fv-2 gene was reported to act by directly controlling SFFV, but not helper virus replication in the animal (R. A. Steeves, R. J. Eckner, M. Bennett, E. A. Mirand, and P. J. Trudel, J. Nat. Cancer Inst.44, 1209–1217, 1971; R. A. Steeves, F. Lilly, G. Steinheider, and K. Blank, In “Differentiation of Normal and Neoplastic Hemopoietic Cells” (B. Clarkson, P. A. Marks, and J. Tills, eds.), pp. 591–600. Cold Spring Harbor Conference on Cell Proliferation, Cold Spring Harbor, N. Y., 1978) or in cultured cells (N. M. Teich and T. M. Dexter, In “Oncogenic Viruses and Host Cell Genes” (Y. Ikawa and T. Odaka, eds.), pp. 263–276. Academic Press, New York, 1979). To determine how Fv-2 controls SFFV-induced erythroleukemia, we have examined its effect on SFFV replication in fibroblasts of resistant C57B1 mice infected with an ecotropic SFFV-Friend leukemia virus pseudotype. It was found that virus produced by C57B1 cells had spleen focus-forming activity in susceptible mice. RNA isolated from virus produced by C57Bl cells was indistinguishable in electrophoretic mobility and RNase T,-resistant oligonucleotides from authentic SFFV RNA described previously. We conclude that the Fv-2 gene does not inhibit SFFV replication in fibroblasts of Fv-2-resistant C57BI mice. Based on previous work of others and on our results, we suggest that Fv-2 acts either by controlling in hematopoietic cells susceptibility to helper virus, or by controlling a target necessary for transformation. Loss of SFFV from SFFV-helper virus stocks passaged in resistant animals would be a consequence of selection against, rather than direct inhibition of, SFFV. In susceptible animals, rapidly proliferating, SFFV-transformed cells would maintain a high ratio of SFFV to helper virus. Lack of transformed cells in resistant animals, and the consequent lack of selection for SFFV, would favor helper virus over SFFV.     

The properties and origin of the proteins in the SV40 nucleoprotein complex

Infection of African green monkey kidney cells with simian virus 40 (SV40) results in the formation of an SV40 specific DNA-protein complex that sediments at about 50 S (Green et al., 1971; White and Eason, 1971). This nucleoprotein complex has been fixed with formaldehyde and the DNA to protein ratio was estimated from its buoyant density in CsCl equilibrium density gradients. Most of the complex (>95%) had a buoyant density of 1.47 g/cc while a minor fraction (<5%) had a density of 1.67 g/cc. This corresponded to a protein: DNA ratio of 0.82–0.95 for the major species and 0.04–0.06 for the minor component. The effect of ionic strength on the nucleoprotein complex was studied. The results indicated that conformational changes in the complex can be detected below 0.6 M NaCl, while above this ionic strength the protein to DNA ratio decreases. One or two viral coat proteins, determined by SV40 temperature-sensitive mutants tsB8 and tsB11, were found to be associated with the 50 S nucleoprotein complex. Evidence for a third tightly bound protein is also presented.     

Prophage deletion mapping of bacteriophage Mu-1

Bacteriophage Mu integrates at a very large number of sites which appear to be randomly distributed over the whole Escherichia coli chromosome (Taylor, 1963) and even within a single gene, lacZ (Bukhari and Zipser, 1972; Daniell et al., 1972). In order to determine whether integration also occurs at many sites on the Mu chromosome, deletion mapping of the Mu prophage was done in seven independently isolated lysogens in which the Mu prophages were located in leu, trp, or lys. The deletion strains were isolated as strains which had simultaneously become defective for phage production and for the function of a gene close to the prophage. Mapping of the prophage in these deletion strains was done by marker rescue using Mu amber mutants from eighteen complementation groups. Since the prophage map obtained is the same in all seven lysogens, it is clear that the integration of Mu does not occur randomly over the Mu chromosome. For the three prophages in leu the deletions entering the prophage from the arabinose operon remove different ends of the prophage in the different lysogens. This indicates that the prophage may be oriented in either direction in a given operon.     

Neurohistochemical changes in the liver of guinea pigs following ligation of the common bile duct

The common bile duct of guinea pigs was ligated without injuring the nerves entering the liver. The innervation of the liver was studied for catecholamines and acetylcholinesterase with the methods of Falck et al. (1962) and Coupland and Holmes (1957), respectively, on the first, second, fourth, and fourteenth postoperative days. The results obtained were compared with the innervation pattern observed in the liver of control animals. Four days after the application of the ligature the liver of control animals. Four days after the application of the ligature monoaminergic and acetylcholinesterase-positive innervation could only be observed in the hilus of the hepatic lobes. On the fourteenth day the histology of the liver indicated cirrhosis, the nerves, however, become demonstrable in a rearranged form within the expanded connective tissue areas. Views on the origin and plasticity of the reorganizing nerves are discussed.     

Role of T4 phage-directed protein in the establishment of resistance to T4 ghosts

It has been reported that after infection of Escherichia coli with T4 phage, the cells become resistant to the action of T4 ghosts (Duckworth, 1971; Vallée and Cornett, 1972). The process of acquiring the resistance has been analyzed by following the rate of ${}^3$H-uridine incorporation. Within 5 min after T4 infection, the resistance reached a maximum level, which is 500–1000-fold higher than that of uninfected cells. This resistance is destroyed by a large number of ghosts with a greatly reduced efficiency. The addition of chloramphenicol in the course of development immediately blocks the increase of the resistance. Kinetic analyses show that the conversion from the sensitive to the resistant state takes place “gradually” in each cell depending on the amount of protein synthesized, rather than in all-or-none fashion. The resistance is specific to T-even phage ghosts.The resistance to ghosts in T4-infected cells is discussed in relation to the “immunity” phenomenon in the colicin system.     

Bacteriophage T3 DNA binding protein interaction with DNA

Single-stranded DNA binding protein (DBP), coded by T3 phage, is essential for concatemer formation (H. Fujisawa, M. Yamagishi, H. Matsuo-Kato, and T. Minagawa, 1980, Virology, 105, 480–489.) T3 DBP was purified to homogeneity and found to exist as a dimer. The structure of the T3 DBP-fd DNA complex appears as a condensed and beaded ring structure by electron microscopy. As judged by sucrose gradient centrifugation, DBP binds to single-stranded, but not to double-stranded DNA; at saturation, one protein monomer is bound per every 100 nucleotides. The T3 DBP has a strong ability to catalyze renaturation of DNA even in the absence of Mg. The results suggest that the T3 DBP acts in concatemer formation by stimulating pairing of the single-stranded redundant termini of T3 DNA.     

Establishment and analysis of a system which allows assembly and disassembly of alphavirus core-like particles under physiological conditions in vitro

Core-like (CL) particles which closely resemble alphavirus cores in size, shape, and relative amount of nucleic acid and protein have been assembled in vitro from Sindbis (SIN) virus core (C) protein and single-stranded nucleic acids in buffer containing 1 M urea [G. Wengler, U. Boege, G. Wengler, H. Bischoff, and K. Wahn (1982)Virology118, 401–410]. We have now analyzed the interaction of SIN virus C protein and nucleic acids in vitro under conditions designed to resemble those present in the cell during core assembly. In buffer containing 100 mM K-acetate, 1.7 mM Mg-acetate, pH 7.4, CL particles are efficiently assembled from all single-stranded nucleic acids analyzed, and even heparin and polyvinylsulfate are incorporated into such particles. A reticulocyte lysate translates SIN virus-specific mRNA into C protein under these ionic conditions. Interactions of C protein with nucleic acids and ribosomes in a reticulocyte lysate have also been analyzed. The following conclusions can be drawn from these analyses: (1) In accordance with earlier findings [N. Glanville and I. Ulmanen (1976) Biochem. Biophys. Res. Commun.71, 393–399] the C protein translated in vitro efficiently binds to ribosomes. (2) Exogenously added C protein binds to the large subunit of the ribosomes in the lysate. (3) CL particles can be assembled in the lysate from exogenous added 42 S genome RNA and exogenous added C protein if both components are present at sufficiently high concentrations. (4) The C protein translated from viral mRNA in the lysate is transferred from the ribosomes into preassembled CL particles containing 42 S RNA in the lysate. (5) If only small amounts of CL particles are added into a lysate these particles disaggregate and core protein molecules are transferred from the particles to the large subunit of the ribosomes. The results on the assembly of CL particles in vitro allow the formulation of some hypotheses concerning the assembly and disassembly of core particles in vivo.