Intracellular visualization of precursor capsids in phage P22 mutant infected cells

Lenk, Elaine V.; Casjens, Sherwood; Weeks, J. Troy; King, Jonathan

doi:10.1016/0042-6822(75)90160-9

Cited by 75 publications

(46 citation statements)

References 29 publications

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“…Mutants in either protein gene prevent prohead formation. In cells infected with amber mutants in the coat protein gene no organized structures were observed, whereas small numbers of aberrant particles were present in cells infected with amber mutants in the scaffolding protein gene (King et al, 1973;Lenk et al, 1975;Earnshaw & King, 1978). Using proteins purified from proheads, the assembly process could be reproduced and studied in vitro.…”

Section: Discussionmentioning

confidence: 97%

Assembly of Herpes Simplex virus Type 1 Capsids Using a Panel of Recombinant Baculoviruses

Tatman

Preston

Nicholson

et al. 1994

Journal of General Virology

162

203

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Immature or B capsids of herpes simplex virus type 1 (HSV-1) are composed of seven proteins encoded by six viral genes. The proteins encoded by UL18 (VP23), UL19 (VP5), UL35 (VP26) and UL38 (VP19C) are components of the outer capsid shell whereas those specified by UL26 (VP21 and VP24) and UL26.5 (VP22a), are involved in scaffold formation. We have used a panel of recombinant baculoviruses, each expressing one of the capsid protein genes, to examine the requirements for capsid assembly. Coexpression of the six genes in insect cells resulted in the formation of capsids that were indistinguishable in appearance and protein composition from those made during HSV-1 infection of mammalian cells. This demonstrates that the proteins encoded by the known capsid genes contain all the structural information necessary for capsid assembly and that other virus-encoded proteins are not required for this process. Omission of single recombinant baculoviruses from this system allowed the role of individual HSV-1 proteins in capsid assembly to be determined. Thus, capsid assembly did not take place in the absence ofVP23, VP5 or VP19C, whereas lack of VP26 had no discernible effect on capsid formation. Capsids assembled in the absence of the UL26 gene products had a large-cored phenotype resembling that previously described for the HSV-1 mutant tsl201 which has a lesion in this gene. Some apparently intact capsid shells were also made in the absence of the major scaffolding protein, VP22a, whereas the omission of both UL26 and UL26.5 resulted in the appearance of large numbers of partial and deformed capsid shells.

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Section: Discussionmentioning

confidence: 97%

Assembly of Herpes Simplex virus Type 1 Capsids Using a Panel of Recombinant Baculoviruses

Tatman

Preston

Nicholson

et al. 1994

Journal of General Virology

162

203

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“…In agreement with the above results, approx-imately 20% of the total particles contained DNA, which means that if the particles are empty because of instability of the DNA, the exit of the DNA from the phage particle might take place within the cell or during the preparation of the thin sections. This resembles the case of mutants in cistrons 10 and 26 from phage P22 in which empty particles are accumulated due to instability of the DNA in the particle within the cell [26]. The analysis by polyacrylamide gel electrophoresis of the DNA-free particles produced after infection with sus mutants in cistron 13 showed that the faster sedimenting fractions of the peak contained mainly the phage structural proteins HPO, HP1, NP2 and HP3 and a small amount of proteins NP1, TP1 and NP3.…”

Section: Discussionmentioning

confidence: 98%

“…lOE), in a way similar to the proheads of phage P22 [26] and different from that which happens in the case of the Tau particles of phage T4, which are found attached to the cell membrane [27].…”

Section: Discussionmentioning

confidence: 99%

Assembly of Bacillus subtilis Phage Phi29. 2. Mutants in the Cistrons Coding for the Non-structural Proteins

et al. 1977

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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 pl5 and p16 are probably involved in the encapsulation of viral DNA in a prohead.It is now well established that DNA encapsulation in several bacteriophages takes place on a preformed head, named prohead, containing some electron-dense internal material which is released when the DNA is encapsulated [I -61.The results presented in the preceding paper suggest that in the Bacillussubtilis phage @29 a structure similar to a prohead is produced after infection with a delayed lysis mutant which behaves like wild-type phage, or with several mutants in cistrons coding for phage structural proteins. In this paper we present evidence on the accumulation of proheadlike structures after restrictive infection with sus mutants in several cistrons involved in phage DNA synthesis and in cistron 16, coding for a late, nonstructural protein (see Fig. I of the preceding paper).Sus mutants in cistron 7, also coding for a late, nonstructural protein, p7, do not produce any kind of phage-related structure suggesting a role for this protein in head morphogenesis. Protein p7 is present in all prohead-like structures and is absent from all DNA-containing heads, suggesting that this protein is released from the phage heads prior to or simultaneously with the encapsulation of DNA. The effect on phage morphogenesis of mutations in cistrons coding for other nonstructural proteins is also de6nzymes Lysozyme or mucopeptide N-acet ylmuramylhydrolase (EC 3 2 1 17), pancreatic ribonudease (EC 3 1 4 23), pancreatic deoxyribonuclease (EC 3 1 4 5) -scribed in this paper. A preliminary account of some of these results has already been given [7].Taking into account the results presented in this and in the preceding paper, a preliminary morphogenetic route for phage @29 assembly is proposed. MATERIALS AND METHODS Bacteria, Phage and MediaThe nonpermissive host, B. subtilis 110NA try--s...

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“…Electron microscopic searches, using visualization both by negative staining of infected cell extracts (24) and by making ultrathin sections of infected cells (36), have failed to reveal any kind of particles in 5-infected cells. Experiments designed to display structures in sucrose gradients of labeled 5-infected cell extracts (as described above for 8-extracts) have similarly failed to show any large aggregates of p8.…”

Section: Gene 8 Product Performs a Scaffolding Function For Coat Protmentioning

confidence: 99%

“…These particles lack the scaffolding protein (24); (g, h) aberrant structures from cells infected with a phage carrying an amber mutation in gene 8. A more detailed account of the in vivo morphology of the various head-related structures formed during P22 infection will be presented later (36).…”

mentioning

confidence: 99%

P22 morphogenesis I: Catalytic scaffolding protein in capsid assembly

1974

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About 250 molecules of the 42,000 molecular weight gene 8 product catalyze the polymerization of the major phage coat protein into a precursor shell temporarily containing both proteins. The resulting prohead appears to be a shell structure with the P8, or scaffolding protein, on the inside, and the coat protein on the outside. In concert with DNA condensation inside the shell, all 250 scaffolding molecules exit from the prohead, without proteolytic cleavage. These molecules then recycle and catalyze the formation of more proheads from newly synthesized coat protein. Such proteins, which catalyze assembly by temporarily associating with an intermediate stage, may represent a general mechanism of macromolecular assembly.

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Intracellular visualization of precursor capsids in phage P22 mutant infected cells

Cited by 75 publications

References 29 publications

Assembly of Herpes Simplex virus Type 1 Capsids Using a Panel of Recombinant Baculoviruses

Assembly of Herpes Simplex virus Type 1 Capsids Using a Panel of Recombinant Baculoviruses

Assembly of Bacillus subtilis Phage Phi29. 2. Mutants in the Cistrons Coding for the Non-structural Proteins

P22 morphogenesis I: Catalytic scaffolding protein in capsid assembly

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