Assembly of Bacillus subtilis phage phe29. 2. Mutants in the cistrons coding for the non-structural proteins.

The effect on phage morphogenesis of sus mutations in the cistrons coding for nonstructural proteins has been studied. Mutants in three cistrons analyzed that are involved in phage DNA synthesis, as well as in cistron 16 which codes for a late nonstructural protein, produce prolate capsids which are more rounded at the corners than complete phage heads and have an internal core; they contain the head proteins, the upper collar protein and protein p7, not present in mature phage particles. Mutants in cistron 7 do not produce capsids nor other phage-related structures; this result and the presence of p7 in phage capsids suggest an essential role in capsid assembly for this protein. The protein product of cistron 13 is probably needed for a stable DNA encapsulation since mutants in this cistron produce mainly DNA-free complete phage particles and only about 10% of uninfective DNA-containing complete phage. Cistron 15 codes for a late, partially dispensable, nonstructural protein which is present in the DNA-free capsids produced after infection with the delayed-lysis mutant sus14(1242), used as the wild-type control, or with mutants in cistrons 9, 11,12 and 13. Proteins p15 and p16 are probably involved in the encapsulation of viral DNA in a prohead.     

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

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

Gene Expression During the Development of Bacteriophage φ29 III. Analysis of Viral-Specific Protein Synthesis with Suppressible Mutants

Fifty-four suppressible mutants of bacteriophage phi29 have been isolated with a variety of mutagens and assigned to eight complementation groups. Viral-specific protein synthesis in UV light-irradiated, nonsuppressing Bacillus subtilis 60084 was analyzed with exponential acrylamide gels. Four additional phi29 proteins which were undetected on ordinary acrylamide gels are reported in this paper. Five phage phi29 proteins have been unambiguously assigned to specific cistrons. Two cistrons had pleiotropic effects on viral protein synthesis. Mutants in cistrons I or II were unable to synthesize DNA in nonsuppressing bacteria. Mutants in cistron I were unable to attach viral chromosomes to the host cell membrane, and the protein responsible for this function has been identified. The other viral protein playing a role in phage phi29 DNA synthesis is also identified and assigned to cistron II. Mutants in cistron II can attach viral chromosomes to membrane, but cannot synthesize DNA in nonsuppressing bacteria.     

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

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

Maturation of the head of bacteriophage T4. II. Head-related, aberrant tau-particles

Three somewhat different types of particle accumulate in cells infected with a phage carrying a mutation in gene 21 (in addition to the tubular variant (polyhead) of the head). The major type is the so-called τ-particle. These particles are very fragile, associated with the cell membrane, and have a sedimentation coefficient of about 420 S. They possess no DNA if isolated, and contain predominantly the precursor proteins P23, P24, P22 and the internal protein IP III, in addition to protein P20 and several proteins of unknown genetic origin.The remainder of the particles are partially or completely filled with DNA. The ratio of τ-particles to these partially or completely filled particles depends upon the particular mutant (in gene 21) phage used. In cells infected with a phage carrying the amber mutation (N90) in gene 21, about 10% of the precursor head protein P23 is cleaved to P231, and correspondingly about 10% of the particles are partially or completely filled with DNA. In cells infected with the temperature-sensitive mutant (N8) in gene 21, about 1% of the particles are fully or partially filled, and correspondingly about 1% of the P23 is cleaved to P231. In either case, the DNA-associated particles contain predominantly the cleaved proteins P231 and IP III1, and have none of the P22 and IP III found in τ-particles. This observation, and the correlation of the amount of partially or completely filled particles with the extent of the cleavage of P23 in the lysates, strongly suggest that cleavage of the head proteins is required for DNA packaging to occur.The τ-particles have properties similar to the so-called prohead I particles which we have isolated as intermediates in wild-type head assembly (preceding paper). However, temperature shift-down experiments, using several different phage carrying temperature-sensitive mutations in gene 21, indicate that the bulk of the τ-particles cannot be used for normal phage production.     

Order of assembly of the lower collar and the tail proteins of Bacillus subtilis bacteriophage phi 29.

Extracts obtained after restrictive infection of Bacillus subtilis with mutants in cistron 11 of bacteriophage phi 29 are complemented in vitro by extract donors of the lower collar protein (p11). Purified 11- heads, containing the major capsid protein (p8), the fiber protein (p8.5), the upper collar protein (p10), and the virus DNA, can be also complemented in vitro to produce infective virus. This result suggests that 11- heads are intermediates in phage phi 29 morphogenesis. The order of assembly of the lower collar protein p11 and the tail protein p9 was determined in vitro in two complementation steps. The results obtained indicate that the lower collar protein is assembled before the tail protein.     

Morphogenesis of Bacteriophage φ80: Identification of the Cistron 13 Product

An in vitro complementation reaction leading to the assembly of bacteriophage phi80 tails from component proteins is described. Tail assembly occurs when a lysate of any mutant in cistron 13 is mixed with a second lysate of a mutant in any of the other cistrons involved in tail formation. Lysates of mutants that are blocked in tail formation contain phage heads that can unite with free tails to form infective particles. The rate of the complementation reaction shows little dependence upon temperature, suggesting that the assembly depends largely upon the kinetic encounter of the interacting components. The tail component missing in cistron 13 mutant lysates was purified approximately 55-fold and shown to be, at least in part, a protein having a molecular weight of approximately 22,000. This protein was also released from highly purified infective phi80 particles after osmotic shock followed by heattreatment, suggesting that it most probably is an integral structural protein of the phage tail. Lysates of mutants of bacteriophage lambda that are defective in tail formation were shown to contain a tail component identical with or similar to the phi80 cistron 13 product.     

Length and shape variants of the bacteriophage T4 head: mutations in the scaffolding core genes 68 and 22.

The shape and size of the bacteriophage T4 head are dependent on genes that determine the scaffolding core and the shell of the prohead. Mutants of the shell proteins affect mainly the head length. Two recently identified genes (genes 67 and 68) and one already known gene (gene 22), whose products are scaffold constituents, have been investigated. Different types of mutants were shown to strongly influence the proportion of aberrantly shaped particles. By model building, these shape variants could be represented as polyhedral bodies derived from icosahedra, through outgrowths along different polyhedral axes. The normal, prolate particle is obtained by elongation along a fivefold axis. The mutations of the three core genes (genes 67, 68, and 22) affect the width mainly by lateral outgrowths of the prolate particle, although small and large isometric particles are also found. Many of the aberrant particles are multitailed, suggesting a correlation between tail attachment sites and shape.     

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.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage p22. I. Genes, proteins, structures and DNA maturation

The functions of ten known late genes are required for the intracellular assembly of infectious particles of the temperate Salmonella phage P22. The defective phenotypes of mutants in these genes have been characterized with respect to DNA metabolism and the appearance of phage-related structures in lysates of infected cells. In addition, proteins specified by eight of the ten late genes were identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis; all but two are found in the mature phage particle. We do not find cleavage of these proteins during morphogenesis.The mutants fall into two classes with respect to DNA maturation; cells infected with mutants of genes 5, 8, 1, 2 and 3 accumulate DNA as a rapidly sedimenting complex containing strands longer than mature phage length. 5^? and 8^? lysates contain few phage-related structures. Gene 5 specifies the major head structural protein; gene 8 specifies the major protein found in infected lysates but not in mature particles. 1^?, 2^? and 3^? lysates accumulate a single distinctive class of particle (“proheads”), which are spherical and not full of DNA, but which contain some internal material. Gene 1 protein is in the mature particle, gene 2 protein is not.Cells infected with mutants of the remaining five genes (10, 26, 16, 20 and 9) accumulate mature length DNA. 10^? and 26^? lysates accumulate empty phage heads, but examination of freshly lysed cells shows that many were initially full heads. These heads can be converted to viable phage by in vitro complementation in concentrated extracts. 16^? and 20^? lysates accumulate phage particles that appear normal but are non-infectious, and which cannot be rescued in vitro.From the mutant phenotypes we conclude that an intact prohead structure is required to mature the virus DNA (i.e. to cut the overlength DNA concatemer to the mature length). Apparently this cutting occurs as part of the encapsulation event.     

Overproduction and purification of the connector protein of Bacillus subtilis phage phi 29.

A phi 29 DNA fragment containing genes 10 and 11, coding for the connector protein and the lower collar protein, respectively, has been cloned in the pBR322 derivative plasmid pKC30 under the control of the PL promoter of phage lambda. Two polypeptides with the electrophoretic mobility of proteins p10 and p11 were labelled with 35S-methionine after heat induction. The proteins were characterized as p10 and p11 by radioimmunoassay and they represented about 10% and 7%, respectively, of the total E. coli protein after 4 hours of induction. These proteins represent less than 1% of the B. subtilis protein in phi 29-infected cells. Protein p10 has been highly purified from the E. coli cells carrying the recombinant plasmid. Antibodies raised against the purified protein p10 reacted with the connector protein produced in phi 29-infected B. subtilis.     

Amber mutants in gene 67 of phage T4. Effects on formation and shape determination of the head

Two amber mutations in gene 67 of bacteriophage T4 were constructed by oligonucleotide-directed mutagenesis and the resulting mutated genes were recombined back into the phage genome and their phenotype was studied. The 67amK1 mutation is close to the amino terminus of the gene, and phage carrying this mutation are unable to form plaques on suppressor-negative hosts. A second mutation, 67amK2, which lies in the middle of the gene, three codons N-terminal to a proteolytic cleavage site, produces a small number of viable phage particles. In suppressor-negative hosts, both mutants produce polyheads and proheads. 67amK1 assembles only few proheads that have a disorganized core structure, as judged from thin sections of infected cells. The proheads and the mature phages of both mutants are mainly isometric rather than having the usual prolate shape. Depending on the 67 mutant and the host, between 20% and 73% of the particles that are produced are isometric, and 1 to 10% are two-tailed biprolate particles. 67amK2 phages grown on a supD suppressor strain that inserts serine in place of the wild-type leucine do not contain gp67* derived from gene product 67 (gp67) by proteolytic cleavage. This demonstrates the importance of the correct amino acid at this position in the protein. Other abnormalities in these 67amK2 phages are the presence of uncleaved scaffolding core proteins (IPIII and gp68), indicating a structural alteration in the prohead scaffold, resulting in only partial cleavage. In wild-type phages these proteins are found in the head only in the cleaved form. With double-mutants of 67 with mutations in the major shell protein gp23 no naked scaffolding cores were found, confirming the necessity of gp67 for the assembly or persistence of a "normal" core.     

φX-174 Bacteriophage Structural Mutants Which Affect Deoxyribonucleic Acid Synthesis

Seven cistrons in X-174 were identified and one in particular was studied intensively: cistron A, which is assigned a protein in the mature phage. Amber mutants in this cistron synthesize a new deoxyribonucleic acid (DNA) form in addition to circular phage DNA upon infection of the restrictive host. This DNA is linear, non-infectious, and single-stranded; it is formed from the phage strand of replicative form X-174 DNA. These mutants produce two different defective particles in the restrictive host. One particle contains circular phage DNA but is not infectious; the other contains the new DNA form and is similar to the 70S particles found in wild-type phage lysates. The mutant A gene product acts independently of normal A protein upon mixed infection of the restrictive host with an A mutant and a mutant from any other cistron or wild type.     

The lysis function of RNA bacteriophage Qbeta is mediated by the maturation (A2) protein

Complete or partial cDNA sequences of the RNA bacteriophage Qbeta were cloned in plasmids under the control of the lambdaP(L) promoter to allow regulated expression in Escherichia coli harbouring the gene for the temperature-sensitive lambdaCI857 repressor. Induction of the complete Qbeta sequence leads to a 100-fold increase in phage production, accompanied by cell lysis. Induction of the 5'-terminal sequence containing the intact maturation protein (A2) cistron also causes cell lysis. Alterations of the A2 cistron, leading to proteins either devoid of approximately 20% of the C-terminal region or of six internal amino acids, abolish the lysis function. Expression of other cistrons in addition to the A2 cistron does not enhance host lysis. Thus, in Qbeta, the A2 protein, in addition to its functions as maturation protein, appears to trigger cell lysis. This contrasts with the situation in the distantly related group I RNA phages such as f2 and MS2 where a small lysis polypeptide is coded for by a region overlapping the end of the coat gene and the beginning of the replicase gene.     

Head morphologies in bacteriophage T4 head and internal protein mutant infections.

Escherichia coli infected with phage T4 mutants defective in synthesis of the three major internal proteins found in the phage head, IPI-, IPII-, IPIII-, or IP degrees (lacking all three) were examined in the electron microscope for head formation. Infection with IPI- or IPII- does not appear to induce increased aberrant head formation, whereas IPII- or IP degrees infections result in production of polyheads and viable phage. Multiple mutants of the early head formation genes 20, 21, 22, 23, 24, 31, 40 and IP degrees were constructed. Combination with IP degrees increases polyhead formation when head formation is not blocked at a more defective stage but results in a qualitative shift to lump formation in association with gene 22 mutants. Thin-sectioning studies show morphologically similar cores in amber 21 and 21am IP degrees tau particles. These morphological observations, genetic evidence for interaction between ts mutants in gene 22 and the IP mutants, and analysis of the protein composition of tau particles further support the idea that p22 and the internal proteins form an unstable assembly core necessary for an early stage of head formation (M. K. Showe and L. W. Black, 1973).     

On the internal structure of bacteriophage lambda

The structure of bacteriophage lambda has been studied by electron microscopy of negatively stained particles. The phage particles will eject their DNA if they are heated or dialyzed against a chelating agent. The ghost particles, so formed, have a channel running down their tails. Since the channel is not visible in normal particles, the channel may be filled with part of the DNA molecule. Up to 30% of the ghosts contain round objects about half the internal diameter of the head. The round objects, called "cores," have the same buoyant density as the coat protein. The core may be a protein spool about which the phage DNA is wound.     

Construction and mutagenesis of an artificial bicistronic tick-borne encephalitis virus genome reveals an essential function of the second transmembrane region of protein e in flavivirus assembly

Flaviviruses have a monopartite positive-stranded RNA genome, which serves as the sole mRNA for protein translation. Cap-dependent translation produces a polyprotein precursor that is co- and posttranslationally processed by proteases to yield the final protein products. In this study, using tick-borne encephalitis virus (TBEV), we constructed an artificial bicistronic flavivirus genome (TBEV-bc) in which the capsid protein and the nonstructural proteins were still encoded in the cap cistron but the coding region for the surface proteins prM and E was moved to a separate translation unit under the control of an internal ribosome entry site element inserted into the 3' noncoding region. Mutant TBEV-bc was shown to produce particles that packaged the bicistronic RNA genome and were infectious for BHK-21 cells and mice. Compared to wild-type controls, however, TBEV-bc was less efficient in both RNA replication and infectious particle formation. We took advantage of the separate expression of the E protein in this system to investigate the role in viral assembly of the second transmembrane region of protein E (E-TM2), a second copy of which was retained in the cap cistron to fulfill its other role as an internal signal sequence in the polyprotein. Deletion analysis and replacement of the entire TBEV E-TM2 region with its counterpart from another flavivirus revealed that this element, apart from its role as a signal sequence, is important for virion formation.     

Cysteine biosynthesis in Salmonella typhimurium: the presence of ATP-sulfurylase and APS-kinase in various cysteine-requiring mutants.

Enzymatic tests were performed on a series of cysteine-requiring mutants for the presence of the sulfate activating enzymes. ATP-sulfurylase (sulfate adenylyltransferase EC 2.7.7.4) and APS-kinase (adenylylsulfate kinase EC 2.7.1.25). The enzymatic products adenosine 5'-[35S]sulfatophosphate and adenosine 3'-phosphate 5'-[35S]sulfatophosphate were identified by paper electrophoresis and measured quantitatively without elution from the paper. Cys mutants mapping in cistrons, A, H, I, J, G, and Ea contain both enzymes. Mutation in the D cistron leads to the loss of ATP-sulfurylase. Mutants mapping in the C cistron lack APS-kinase. Ba, Bb, and Bc mutants lack both enzymes. The control of the synthesis of these enzymes by cysteine was examined. Both enzymes are missing when cells are grown on cysteine.     

Structure of the head-tail connector of bacteriophage φ29

The head-to-tail connecting region of bacteriophage φ29 has been studied by isolating neck-tail complexes from disrupted phage. These complexes can be isolated with appendages (from wild-type phage) or without appendages (from phage mutant sus12). Treatment of the neck-tail complex without appendages with urea or guanidinium hydrochloride releases the tail protein (p9) from the neck complex (proteins p10 and p11). Electron micrographs of φ29 necks show that they are composed of two collars and a thin axial tube. There is an internal hole along the longitudinal axis, from the upper collar to the thin tube.Image-processing analysis of electron micrographs of two-dimensional crystals of necks shows that the neck of phage φ29 consists of 12 external units and an internal area of apparent 6-fold symmetry, with a hole in the centre.