Structural Aberrations in T-Even Bacteriophage V. Effects of Canavanine on the Maturation and Utilization of Specific Gene Products

Previous results have shown that when a T-even bacteriophage-infected cell was exposed to l-canavanine followed by an exposure to l-arginine, a monster phage particle, termed a lollipop, was formed. l-Canavanine was necessary for the induction event but l-arginine was required for the maturation of the particle. We now describe the effects of canavanine on the maturation of certain T4 proteins and their role in the induction of lollipops. The cleavage reactions of the head proteins P22, P23, P24, and IPIII are prevented by l-canavanine as shown by the accumulation of the precursor proteins and the failure of the cleaved products to appear. l-Canavanine also prevents the appearance of P12 (tailplate protein) and P20 (head protein) indicating that these proteins may undergo a proteolytic cleavage during normal assembly. The formation of P10 (tailplate protein) and P18 (tail sheath protein) is also affected by l-canavanine. The data suggest that P23 in conjunction with P20 plays a major role in the determination of the length of the phage head.     

Maturation of the head of bacteriophage T4. I. DNA packaging events

Pulse-chase experiments in wild-type and mutant phage-infected cells provide evidence that the following particles called prohead I, II and III are successive precursors to the mature heads. The prohead I particles contain predominantly the precursor protein P23 and possibly P22 (mol. wt 31,000) and IP III (mol. wt 24,000) and have an s value of about 400 S. Concomitantly with the cleavage of most of P23 (mol. wt 55,000) to P231 (mol. wt 45,000), they are rapidly converted into prohead II particles which sediment with about 350 S. The prohead II particles contain, in addition to P231, the major constituents of the viral shella—a core consisting of proteins P22 and IP III. In cell lysates, prohead I and prohead II particles contain no DNA in a DNase-resistant form and are not bound to the replicative DNA. We cannot, however, positively rule out the possibility that these particles may have contained some DNA while in the cells.The prohead II particles are in turn converted into particles which sediment with about 550 S after DNase treatment (prohead III). During this conversion about 50% of normal DNA complement becomes packaged in a DNase-resistant form, and roughly 50% of the core proteins P22 and IP III are cleaved. In lysates the prohead III particles are attached to the replicative DNA. The prohead III particle appears to be the immediate precursor of the full mature head (1100 S). Cleavage of protein P22 to small polypeptides and conversion of IP III IP III1 are completed at this time. No precursor proteins are found in the full heads. Studies with various mutant phage showed that the prohead II to III conversion is blocked by mutations in genes 16 and 17 and that the conversion of the prohead III particles to the mature heads is blocked by mutations in gene 49. Cleavage of the head proteins, however, occurs normally in these mutant-infected cells. We conclude that the cleavage of the major component of the viral shell, P23, into P231 precedes the DNA packaging event, whereas cleavage of the core proteins P22 and IP III appears to be intimately linked to the DNA packaging event. Models relating the cleavage processes to DNA encapsulation are discussed.     

Signals for site-specific cleavage of HSV DNA: maturation involves two separate cleavage events at sites distal to the recognition sequences

Mature Herpes Simplex Virus (HSV) genomes are cleaved from concatemeric precursors by a site-specific mechanism. These cleavage events are probably coupled to the encapsidation process. Sequences within the terminal repeat of HSV DNA are necessary for the cleavage and packaging reactions, and are also thought to be responsible for high frequency genome isomerization events. Here we present evidence to show that two viral DNA cleavage and packaging signals reside within a 250 bp subfragment of the terminal repeat, that the termini of mature viral DNA are generated by a process involving two separate DNA cleavages at sites distal to the cleavage signals, and that the sequences between these two cleavage sites are duplicated by the DNA maturation system.     

Processing of the L1 52/55k Protein by the Adenovirus Protease: a New Substrate and New Insights into Virion Maturation

Late in adenovirus assembly, the viral protease (AVP) becomes activated and cleaves multiple copies of three capsid and three core proteins. Proteolytic maturation is an absolute requirement to render the viral particle infectious. We show here that the L1 52/55k protein, which is present in empty capsids but not in mature virions and is required for genome packaging, is the seventh substrate for AVP. A new estimate on its copy number indicates that there are about 50 molecules of the L1 52/55k protein in the immature virus particle. Using a quasi-in vivo situation, i.e., the addition of recombinant AVP to mildly disrupted immature virus particles, we show that cleavage of L1 52/55k is DNA dependent, as is the cleavage of the other viral precursor proteins, and occurs at multiple sites, many not conforming to AVP consensus cleavage sites. Proteolytic processing of L1 52/55k disrupts its interactions with other capsid and core proteins, providing a mechanism for its removal during viral maturation. Our results support a model in which the role of L1 52/55k protein during assembly consists in tethering the viral core to the icosahedral shell and in which maturation proceeds simultaneously with packaging, before the viral particle is sealed.     

Bacteriophage p22 portal vertex formation in vivo

Bacteriophage with double-stranded, linear DNA genomes package DNA into pre-assembled icosahedral procapsids through a unique vertex. The packaging vertex contains an oligomeric ring of a portal protein that serves as a recognition site for the packaging enzymes, a conduit for DNA translocation, and the site of tail attachment. Previous studies have suggested that the portal protein of bacteriophage P22 is not essential for shell assembly; however, when assembled in the absence of functional portal protein, the assembled heads are not active in vitro packaging assays. In terms of head assembly, this raises an interesting question: how are portal vertices defined during morphogenesis if their incorporation is not a requirement for head assembly? To address this, the P22 portal gene was cloned into an inducible expression vector and transformed into the P22 host Salmonella typhimurium to allow control of the dosage of portal protein during infections. Using pulse-chase radiolabeling, it was determined that the portal protein is recruited into virion during head assembly. Surprisingly, over-expression of the portal protein during wild-type P22 infection caused a dramatic reduction in the yield of infectious virus. The cause of this reduction was traced to two potentially related phenomena. First, excess portal protein caused aberrant head assembly resulting in the formation of T=7 procapsid-like particles (PLPs) with twice the normal amount of portal protein. Second, maturation of the PLPs was blocked during DNA packaging resulting in the accumulation of empty PLPs within the host. In addition to PLPs with normal morphology, smaller heads (apparently T=4) and aberrant spirals were also produced. Interestingly, maturation of the small heads was relatively efficient resulting in the formation of small mature particles that were tailed and contained a head full of DNA. These data suggest that incorporation of portal vertices into heads occurs during growth of the coat lattice at decision points that dictate head assembly fidelity.     

Structure of T4 polyheads: II. A pathway of polyhead transformations as a model for T4 capsid maturation

We have studied the aberrant tubular polyheads of bacteriophages T4D and T2L as a model system for capsid maturation. Six different types of polyhead surface lattice morphology, and the corresponding protein compositions are reported and discussed. Using in vitro systems to induce transformations between particular polyhead types, we have deduced that the structural classes represent successive points in a transitional pathway. In the first step, coarse polyheads (analogous to the prohead τ-particle) are proteolytically cleaved by a phagecoded protease, a fragment of the gene 21 product. This cleavage of P23 to P23¹ induces a co-operative lattice transformation in the protein of the surface shell, to a conformation equivalent to that of T2L giant phage capsids. These polyheads (derived either from T4 or T2L lysates) can accept further T4-coded proteins. In doing so, they pass through intermediate structural states, eventually reaching an end point whose unit cell morphology is indistinguishable from that of the giant T4 capsids. At least one protein (called soc (Ishii & Yanagida, 1975)) is bound stoichiometrically to P23¹ in the end-state conformation. The simulation of several aspects of capsid maturation (cleavage of P23 to P23¹, stabilization, and lattice expansion) in the polyhead pathway suggest that it parallels the major events of phage T-even capsid maturation, decoupled from any involvement of DNA packaging.     

The Gene Product of Human Cytomegalovirus Open Reading Frame UL56 Binds the pac Motif and Has Specific Nuclease Activity

Using the cis-acting human cytomegalovirus (HCMV) packaging elements (pac 1 and pac 2) as DNA probes, specific DNA-protein complexes were detected by electrophoretic mobility shift assay (EMSA) in both HCMV-infected cell nuclear extracts and recombinant baculovirus-infected cell extracts containing the HCMV p130 (pUL56) protein. DNA-binding proteins, which were common in uninfected and infected cell extracts, were also detected. Mutational analysis showed that only the AT-rich core sequences in these cis-acting motifs, 5′-TAAAAA-3′ (pac 1) and 5′-TTTTAT-3′ (pac 2), were required for specific DNA-protein complex formation. The specificity of the DNA-protein complexes was confirmed by EMSA competition. Furthermore, a specific endonuclease activity was found to be associated with lysates of baculovirus-infected cells expressing recombinant p130 (rp130). This nuclease activity was time dependent, related to the amount of rp130 in the assay, and ATP independent. Nuclease activity remained associated with rp130 after partial purification by sucrose gradient centrifugation, suggesting that this activity is a property of HCMV p130. We propose a possible involvement of p130 in HCMV DNA packaging.Human cytomegalovirus (HCMV), one of eight human herpesviruses, can cause serious illness in neonates as well as in immunocompromised adults (2). For example, transplant and AIDS patients may develop life-threatening diseases as a consequence of primary infection or reactivation of latent infection. Present therapeutic approaches are limited, and new strategies that may result from a better understanding of the molecular events involved in viral maturation are needed.The HCMV virion consists of an envelope, an amorphous tegument, and an icosahedral nucleocapsid, which is assembled in the nuclei of infected cells. The precise molecular events of HCMV capsid assembly and subsequent DNA packaging are not well understood. It is generally accepted that viral DNA is packaged into a procapsid consisting of major capsid protein (UL86), minor capsid protein (UL85), minor capsid protein-binding protein (UL46), smallest capsid protein (UL47/48), assembly protein (UL80.5), and proteinase precursor protein (UL80a) (8). The assembly protein is removed during DNA insertion. It is unclear how the concatenated viral DNA contacts empty capsids and is cleaved and packaged into the capsid.Recent studies with herpes simplex virus type 1 (HSV-1) mutants that were temperature sensitive suggest that cleavage of the concatenated DNA does not occur in the absence of packaging (1). One possible model would be the involvement of cleavage packaging protein(s) which could facilitate incorporation of DNA into the procapsid by attaching to a specific motif within the viral genome. With HSV-1, the UL36 gene product (ICP1) and a smaller protein (possibly encoded by UL37) are part of a complex that recognizes the HSV-specific a sequence and are required for cleavage and packaging of viral DNA from concatemers (6, 7). In addition, the HSV-1 ICP 18.5 (UL28) gene product and the pseudorabies virus (PrV) homolog (16) were also reported to play an important role in DNA packaging (1, 14). Addison et al. (1) demonstrated that empty capsids were observed under conditions nonpermissive for the expression of the HSV-1 ICP 18.5 gene product. The HSV-1 ICP 18.5 mutants failed to cleave concatenated viral DNA in noncomplementing cells, suggesting that cleavage and packaging require ICP 18.5. Similar results were reported by Mettenleiter et al. (14) for PrV mutant protein. These observations suggest that the HSV-1 UL36, UL37, and UL28 gene products are involved in cleavage and packaging of concatenated viral DNA.In a recent study, we identified and partially characterized the gene product of HCMV UL56 (4). The HCMV UL56 gene product of 130 kDa is the homolog of the HSV-1 UL28 gene product. It is therefore postulated that UL56 possesses properties comparable to those of HSV-1 UL28, implying an involvement in cleavage and packaging of DNA. The HCMV genomic a sequence is a short sequence located at both termini of the genome and repeated in an inverted orientation at the L-S junction. The a sequence plays a key role in replication as a cis-acting signal for cleavage and packaging of progeny viral DNA and circularization of the viral genome. The HCMV a sequence contains two conserved motifs, pac 1 and pac 2, which are required for cleavage and packaging of the viral DNA (18). Both sequence motifs are located on one side of the cleavage site. The pac 1 and pac 2 motifs have an AT-rich core flanked by a GC-rich sequence. During the initial step of viral DNA packaging, a capsid-associated protein may bind to the pac sequences and may be involved in cleavage of the viral DNA concatemer.In this study, electrophoretic mobility shift assays (EMSAs) were performed with DNA probes spanning the region of these cis-acting elements. These studies demonstrate that specific proteins from HCMV-infected nuclear extracts or baculovirus-UL56-infected cell extracts bind to the pac motifs. Using affinity-purified monospecific antibodies, we show that p130 is present in specific DNA-protein complexes containing the pac motifs of the viral genome. Furthermore, evidence is presented for a sequence-specific endonuclease activity of recombinant HCMV p130, using circular plasmid DNA bearing the a sequence as a substrate.     

Characterization of Empty adenovirus particles assembled in the absence of a functional adenovirus IVa2 protein

The molecular mechanism for packaging of the adenovirus (Ad) genome into the capsid is likely similar to that of DNA bacteriophages and herpesviruses-the insertion of viral DNA through a portal structure into a preformed prohead driven by an ATP-hydrolyzing molecular machine. It is speculated that the IVa2 protein of adenovirus is the ATPase providing the power stroke of the packaging machinery. Purified IVa2 binds ATP in vitro and, along with a second Ad protein, the L4 22-kilodalton protein (L4-22K), binds specifically to sequences in the Ad genome that are essential for packaging. The efficiency of binding of these proteins in vitro was correlated with the efficiency of packaging in vivo. By utilizing a virus unable to express IVa2, pm8002, it was reported that IVa2 plays a role in assembly of the empty virion. We wanted to address the question of whether the ATP binding, and hence the putative ATPase activity, of IVa2 was required for its role in virus assembly. Our results show that ATPase activity was not required for the assembly of empty virus particles. In addition, we present evidence that particles were assembled in the absence of IVa2 by using two viruses null for IVa2-a deletion mutant virus, ΔIVa2, and the previously described mutant virus, pm8002. Empty virus particles produced by these IVa2 mutant viruses did not contain detectable viral DNA. We conclude that the major role of IVa2 is in viral DNA packaging. A characterization of the empty particles obtained from the IVa2 mutant viruses compared to wild-type empty particles is presented.     

Initiation of bacteriophage P22 DNA packaging series. Analysis of a mutant that alters the DNA target specificity of the packaging apparatus

Bacteriophage P22 is thought to package its double-stranded DNA chromosome from concatemeric replicating DNA in a "processive" sequential fashion. According to this model, during the initial packaging event in such a series the packaging apparatus recognizes a nucleotide sequence, called pac, on the DNA, and then condenses DNA within the coat protein shell unidirectionally from that point. DNA ends are generated near the pac site before or during the condensation reaction. The opposite end of the mature chromosome is created by a cut made in the DNA after a complete chromosome is condensed within the phage head. Subsequent packaging events on that concatemeric DNA begin at the end generated by the headful cut of the previous event and proceed in the same direction as the previous event. We report here the identification of a consensus nucleotide sequence for the pac site, and present evidence that supports the idea that the gene 3 protein is a central participant in this recognition event. In addition, we tentatively locate the portion of the gene 3 protein that contacts the pac site during the initiation of packaging.     

Recognition and cleavage of the bacteriophage P1 packaging site (pac). I. Differential processing of the cleaved ends in vivo

The packaging of bacteriophage P1 DNA into viral capsids is initiated at a specific DNA site called pac. During packaging, that site is cleaved and at least one of the resulting ends is encapsidated into a P1 virion. We show here that pac is located on a 620 base-pair fragment of P1 DNA (EcoRI-20). When that fragment is inserted into the chromosome of cells that are then infected with P1, packaging of host DNA into phage particles is initiated at pac and proceeds down the chromosome, unidirectionally, for about five to ten P1 "headfuls" (about 5 X 10(5) to 10 X 10(5) bases of DNA). Using an assay for pac cleavage that does not depend on DNA packaging, we have identified a set of five amber mutations that are mapped adjacent to pac, and that define a gene (gene 9) essential for pac cleavage. Amber mutations that are located in genes necessary for viral capsid formation (genes 4, 8 and 23), or in a gene necessary for "late" protein synthesis (gene 10), do not affect pac cleavage. The latter result suggests that the synthesis of the pac cleavage protein is not regulated co-ordinately with other phage morphogenesis proteins. The products of pac cleavage were analyzed using two different DNA substrates. In one case, a single copy of pac was placed in the chromosome of P1-sensitive cells. When those cells were infected with P1, we could detect the cleavage of as much as 70% of the pac-containing DNA. The pac end destined to be packaged in the virion was detected five to 20 times more efficiently than was the other end. Since this result is obtained whether or not the infecting P1 phage can encapsidate the cut pac site, the differential detection of pac ends is not simply a consequence of one end being packaged and the other not. In a second case, pac was located in cells on a small (5 X 10(3) bases) multicopy plasmid. When those cells were infected with P1, neither pac end was detected efficiently after P1 infection, unless the cells carried a recBCD- mutation. In recBCD- cells, the results with plasmid-pac substrates were similar to those obtained with chromosomally integrated pac substrates. We interpret these results to mean that, following pac cleavage, the end destined to be packaged is protected from cellular nucleases while the other end is degraded by the action of at least two nucleases, one of which is the product of the host recBCD gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of Gag mutation and processing on retroviral dimeric RNA maturation

After their release from host cells, most retroviral particles undergo a maturation process, which includes viral protein cleavage, core condensation, and increased stability of the viral RNA dimer. Inactivating the viral protease prevents protein cleavage; the resulting virions lack condensed cores and contain fragile RNA dimers. Therefore, protein cleavage is linked to virion morphological change and increased stability of the RNA dimer. However, it is unclear whether protein cleavage is sufficient for mediating virus RNA maturation. We have observed a novel phenotype in a murine leukemia virus capsid mutant, which has normal virion production, viral protein cleavage, and RNA packaging. However, this mutant also has immature virion morphology and contains a fragile RNA dimer, which is reminiscent of protease-deficient mutants. To our knowledge, this mutant provides the first evidence that Gag cleavage alone is not sufficient to promote RNA dimer maturation. To extend our study further, we examined a well-defined human immunodeficiency virus type 1 (HIV-1) Gag mutant that lacks a functional PTAP motif and produces immature virions without major defects in viral protein cleavage. We found that the viral RNA dimer in the PTAP mutant is more fragile and unstable compared with those from wild-type HIV-1. Based on the results of experiments using two different Gag mutants from two distinct retroviruses, we conclude that Gag cleavage is not sufficient for promoting RNA dimer maturation, and we propose that there is a link between the maturation of virion morphology and the viral RNA dimer.     

Evidence that a phage T4 DNA packaging enzyme is a processed form of the major capsid gene product

A phage T4 DNA packaging enzyme appears to arise as a processed form of the major T4 capsid structural protein gp23. The enzyme activity and antigen are missing from all head gene mutants that block the morphogenetic proteolytic processing reactions of the head proteins in vivo. The enzyme antigen can be formed in vitro by T4 (gp21) specific processing of gp23 containing extracts. Enzyme antigen is found in active processed proheads but not in full heads. The enzyme and the major capsid protein show immunological cross-reactivity, produce common peptides upon proteolysis, and share an assembly-conformation-dependent ATP binding site. The packaging enzyme and the mature capsid protein (gp23*) both appear to arise from processing of gp23, the former as a minor product of a specific gp23 structure in the prohead, acting in DNA packaging as a DNA-dependent ATPase, and a headful-dependent terminase.     

The DNA maturation domain of gpA, the DNA packaging motor protein of bacteriophage lambda, contains an ATPase site associated with endonuclease activity

Terminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in Escherichia coli. Biochemical characterization of gpA-DeltaN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A "P-loop" sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme, DNA maturation and DNA packaging, are discussed.     

Analysis in vivo of the bacteriophage P22 headful nuclease

Bacteriophage P22 packages its double-stranded DNA chromosomes from concatemeric replicating DNA in a processive, sequential fashion. According to this model, during the initial packaging event in such a series the packaging apparatus recognizes a nucleotide sequence, called pac, on the DNA, and then condenses DNA within the coat protein shell unidirectionally (rightward) from that point. DNA ends are generated near the pac site before or during the condensation reaction. The right end of the mature chromosome is created by a cut made in the DNA by the "headful nuclease" after a complete chromosome is condensed within the phage head. Subsequent packaging events on that concatemeric DNA begin at the end generated by the headful cut of the previous event and proceed in the same direction as the previous event. We report here accurate measurements of the P22 chromosome length (43,400( +/- 750) base-pairs, where the uncertainty is the range in observed lengths), genome length (41,830( +/- 315) base-pairs, where the uncertainty represents the accuracy with which the length is known), the terminal redundancy (1600( +/- 750) base-pairs or 3.8( +/- 1.8)%, where the uncertainty is the observed range) and the imprecision in the headful measuring device ( +/- 750 base-pairs or +/- 1.7%). In addition, we present evidence for a weak nucleotide sequence specificity in the headful nuclease. These findings lend further support to, and extend our understanding of, the sequential series model of P22 DNA packaging.     

Structure and inherent properties of the bacteriophage lambda head shell. VI. DNA-packaging-defective mutants in the major capsid protein

Some amino acid substitutions in the major capsid protein (gene E product) of lambda phage are found to cause a defect in DNA packaging. These substitutions permit initiation of DNA packaging and expansion of the prohead. However, cleavage of the concatemer DNA at the cos site takes place only to a very small extent, and the capsid eventually becomes empty. Interestingly, the mutations are suppressed by a decrease of the DNA length between the cos sites by 8000 to 10,000 bases. These properties are similar to those of amber mutants in gene D, which codes for the capsid outer-surface protein. Studies on the E missense.D amber double mutant show that the E protein and the D protein contribute additively to the stabilization of the condensed form of the DNA molecule in phage heads.     

Coliphage P1 morphogenesis: analysis of mutants by electron microscopy.

We used electron microscopy and serum blocking power tests to determine the phenotypes of 47 phage P1 amber mutants that have defects in particle morphogenesis. Eleven mutants showed head defects, 30 showed tail defects, and 6 had a defect in particle maturation (which could be either in the head or in the tail). Consideration of previous complementation test results, genetic and physical positions of the mutations, and phenotypes of the mutants allowed assignment of most of the 47 mutations to genes. Thus, a minimum of 12 tail genes, 4 head genes, and 1 particle maturation gene are now known for P1. Of the 12 tail genes, 1 (gene 19, located within the invertible C loop) codes for tail fibers, 6 (genes 3, 5, 16, 20, 21, and 26) code for baseplate components (although one of these genes could code for the tail tube), 1 (gene 22) codes for the sheath, 1 (gene 6) affects tail length, 2 (genes 7 and 25) are involved in tail stability, and 1 (gene 24) either codes for a baseplate component or is involved in tail stability. Of the four head genes, gene 9 codes for a protein required for DNA packaging. The function of head gene 4 is unclear. Head gene 8 probably codes for a minor head protein, whereas head gene 23 could code for either a minor head protein or the major head protein. Excluding the particle maturation gene (gene 1), the 12 tail genes are clustered in three regions of the P1 physical genome. The four head genes are at four separate locations. However, some P1 head genes have not yet been detected and could be located in two regions (for which there are no known genes) adjacent to genes 4 and 8. The P1 morphogenetic gene clusters are interrupted by many genes that are expressed in the prophage.     

Assembly and Maturation of the Bacteriophage Lambda Procapsid: gpC Is the Viral Protease

Viral capsids are robust structures designed to protect the genome from environmental insults and deliver it to the host cell. The developmental pathway for complex double-stranded DNA viruses is generally conserved in the prokaryotic and eukaryotic groups and includes a genome packaging step where viral DNA is inserted into a pre-formed procapsid shell. The procapsids self-assemble from monomeric precursors to afford a mature icosahedron that contains a single “portal” structure at a unique vertex; the portal serves as the hole through which DNA enters the procapsid during particle assembly and exits during infection. Bacteriophage λ has served as an ideal model system to study the development of the large double-stranded DNA viruses. Within this context, the λ procapsid assembly pathway has been reported to be uniquely complex involving protein cross-linking and proteolytic maturation events. In this work, we identify and characterize the protease responsible for λ procapsid maturation and present a structural model for a procapsid-bound protease dimer. The procapsid protease possesses autoproteolytic activity, it is required for degradation of the internal “scaffold” protein required for procapsid self-assembly, and it is responsible for proteolysis of the portal complex. Our data demonstrate that these proteolytic maturation events are not required for procapsid assembly or for DNA packaging into the structure, but that proteolysis is essential to late steps in particle assembly and/or in subsequent infection of a host cell. The data suggest that the λ-like proteases and the herpesvirus-like proteases define two distinct viral protease folds that exhibit little sequence or structural homology but that provide identical functions in virus development. The data further indicate that procapsid assembly and maturation are strongly conserved in the prokaryotic and eukaryotic virus groups.     

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.