Introduction

The family Herpesviridae encompasses many human and animal pathogens that can cause a wide variety of medically and economically important diseases in a broad range of hosts. Of the more than 100 herpesviruses isolated to date, eight can cause significant disease in humans, especially in immunocompromised individuals. Understanding DNA packaging of herpesviruses has important practical implications because the process is a potential target for new antiviral strategies. In fact, resistance to...

Seeking the Mechanism

Dwight Anderson and Shelley Grimes Abstract The Bacillus subtilis bacteriophage 029 research team in Minneapolis has marveled at (and reveled in) the intricacies of 029 assembly for more than 30 years. Here we highlight the current state of knowledge of 029 DNA packaging. We describe the in vitro packaging system and focus on recent advances that address the mechanism of the packaging motor. Among advances, the head-tail connector has been visualized in proheads and the packaging motor resolved...

Discontinuous Headful Packaging

An interesting twist to the classical headful packaging model has been discovered during the large DNA cloning experiments in phage T4 and phage PI in vitro packaging.69'70 Both the cloning systems package linear headful size foreign DNA molecules cloned within a unique site of the vector followed by transduction of these molecules into E. colt. Surprisingly, a fraction of the clones showed very small, less than headful length, inserts. Subsequent experiments using only the 7-30 kb size vector...

Termination of DNA Packaging

After the headful DNA cleavage releases the capsid containing the newly packaged chromosome from the concatemer, the three head completion proteins (the products of genes 4, 10 and 26) add to the particle and stabilize the packaged DNA (Fig. 1). After the head completion proteins add to the stucture, 18 molecules of one more protein, called tailspike protein, add to the portal vertex. Tailspike protein is responsible for binding virions to the outer surface of cells to initate the next round of...

The Portal Vertex UL6

It has long been recognized that absence of a functional HSV-1 Ul6 gene precludes cleavage of concatemeric viral DNA.44'56'65'68,69 A high degree of homology exists between Ul6 homologs encoded by herpesvirus family members indicating that the encoded proteins likely play similar roles in the life cycles of all herpesviruses. pU ,6 is a component of procapsids, types A, B and C capsids, and virions.53'56 Thus, the capsid association of pUi,6 is unaffected by scaffold loss, capsid angularization...

ATP Binding Site I

The consensus sequence of the Walker-A nucleotide binding motif, (G A)XXXXGK(T S), is present in a large number of enzymes capable of nucleotide binding and or hydrolysis. Two Walker-A motifs have been identified in gpl7,9,18 the N -terminus proximal SRQLGKT16M67 (Walker-Ai) and a centrally locatedTAAVEGKS299-306 (Walker-An). As shown recendy, Walker-Ai is highly conserved among all the four T4-family (T4D, RB49, KVP40, KVP20) terminase sequences (Fig. 3) as well as in numerous other phage...

Assembly of T4 DNA Packaging Machine

Model for the assembly of a functional packaging machine. The small T4 terminase protein gp 16 exists as eight-subunit single or sixteen subunit double ring multimers in solution (see Fig. 2), whereas the large terminase protein gpl7 exists largely as a monomer. The stoichiometry of the terminase subunits in the holoenzyme is unknown. In the speculative model depicted above, gpl6 and gpl7 are shown as pentamers. The main point made is that gpl6 multimer interacts with gpl7 subunits...

Processivity of DNA Packaging Events during SPP1 Infection

After termination of the first packaging cycle initiated at pac (initiation cycle) a second packaging event is initiated at the nonencapsidated concatemer extremity created by the headful cleavage and additional cycles of encapsidation follow (processive headfuls) (Fig. 1). Processivity of SPP1 DNA packaging is high reaching an average of 5-6 headfuls per concatemer. Some of the packaging series can even yield 12 headfuls or more.8 The main factor that limits the series of encapsidation cycles...

Conclusions and Future Directions

Our current view of the ( )6 packaging and replication cascade is summarized in Figure 5. Even though considerable progress has been made in developing and analyzing the < )6 packaging system, our understanding of the packaging at mechanistic level is still relatively poor. The structural changes during the packaging process are still highly hypothetical. However, the estimations of the interior volumes of the intermediate particles gave some indications when the transitions might occur...

Sequential Packaging

< )6, like other dsRNA viruses with segmented genome, needs to have a mechanism to ensure that one copy of each of the individual genome segment will be packaged into the virus particle. Studies on < j> 6 have shed light to the mechanism how the tripartite genome may be incorporated into the capsid efficiendy and accurately. Apparently the polymerase complex has the ability to recognize the three viral single-stranded genomic precursor molecules in segment-specific manner. The...

KDa

Domain organization of the terminase gpNul and gpA subunits. Upper panel shows gpNul. wHTH indicates the winged helix-turn-helix motif. Lower panel shows gpA. In both panels, sites co-valently modified with 8-azido ATP are indicated with asterisks, and specific proteolysis sites are indicated with arrows, respectively. Details are presented in the text. the terminal cosN site however, cosQ alone is incapable of arresting DNA packaging. Rather, cosQ acts in concert with cosN and 12 to...

Selective Recognition and Cleavage of SPP1 DNA by the Terminase Complex gplgp2

Packaging of SPP1 DNA into procapsids is initiated by pac cleavage of the concatemeric DNA precursor (Fig. 1). This endonucleolytic cleavage is performed by the terminase enzyme. The terminase of SPP1 is a hetero-oligomer of small (gpl) and large (gp2) subunits. The ho-loenzyme possesses several activities including recognition, binding and cleavage of the DNA substrate, and ATP-hydrolysis. Gpl is an oligomer in solution with a native mass of about 200 kDa. Electron micrographs of negatively...

T3T7 DNA Packaging

During formation of a mature bacteriophage particle, a procapsid of protein packages the linear double-stranded DNA genome of the related bacteriophages, T3 and T7. Initiation of T3 T7 DNA packaging in vivo occurs near the genetic right end of a concatemer-associated genome. Initiation in vivo requires transcription near the initiation site and results in the formation of the genome's right end by cleavage of the concatemer. The initiation is co-operative among capsids that are packaging the...

O

Summary of allosteric interaction between the catalytic sites of terminase holoenzyme. The catalytic activity of the isolated gpA (red rectangle) and gpNul (blue sphere) subunits is shown at top. Assembly into the holoenzyme activates catalytic activity as indicated in step (ii). Interaction between the multiple catalytic sites of the holoenzyme is shown in the lower half of the figure. Details are described in the text. A color version of this figure is available online at http...

Components of the 6 RNA Packaging and Replication System

The < )6 in vitro RNA packaging and replication system contains isolated empty polymerase complexes (procapsids, PCs) and single-stranded copies of the viral genome segments in a defined reaction mixture (Fig. 3). The packaging or the replication is neither dependent on any viral encoded nonstructural protein, nor on any host components. Single-Stranded Genomic Precursor Molecules The virus specific plus-sense ssRNAs for packaging reaction can be obtained from an NC transcription reaction,23...

Concluding Remarks

The phenomenology of bacteriophage SPP1 DNA packaging is well known and some of the molecular mechanisms involved are among the best understood in tailed phages systems. These include the terminase recognition and cleavage of its target sequence pac, the structure and function of the portal protein, the mechanism of headful sensor, and connector assembly. Other aspects of the DNA packaging process were not yet studied in detail like the terminase-procapsid interaction, the properties and...

The j6 in Vitro ssRNA Packaging and Replication System

The ( > 6 in vitro ssRNA packaging and replication system (Fig. 3) allows to dissect the ssRNA binding, ssRNA packaging, combined ssRNA packaging and replication (i.e., minus-strand synthesis reaction), and combined packaging, replication, and transcription activities (i.e., plus-strand synthesis reaction) by modifying the reaction conditions.30'46'5052 The translocation of the ssRNA into the closed compartment requires energy in the form of hydrolysable nucleoside triphosphates, which can be...

Intranuclear Transport Proteins Ul17 Ul32

During infection with Herpes simplex virus 1, gene expression and DNA replication occur within globular intranuclear compartments termed replication compartments.99 At least four cleavage and packaging proteins, Ul6, Ul15, Ul32 and Ul33, accumulate in replication compartments at relatively early times after infection (6-8 hours).93'100102 Moreover, capsids have also been observed to colocalize completely within replication compartments at these times.101 These results have led to the suggestion...

Filling the Capsid with DNA

As in other tailed phages, the motor that drives P22 DNA into the capsid is thought to be composed of the portal protein and terminase components. Because P22 is a headful packaging phage, its head filling device must have two parts the force generating motor itself and a headful sensing device that controls the cleavage of DNA when the head is full. P22 heads contain DNA molecules that are on average 43,400 bp long, but their actual lengths vary from 42650 to 44150 bp.5 The headful nuclease...

Summary

P22 virion assembly is one of the prototypic virus nucleic acid packaging systems. Its terminase, portal, scaffold, coat and head completion proteins have little sequence similarity to the analogous proteins of other well-studied dsDNA virus types, yet a perfect parallel exists between these P22 general functions and those of their analogs in other systems. This relationship suggests that either the large dsDNA viruses (perhaps including herpesviruses, and even iridoviruses and adenoviruses 4...

Assembly of the SPP1 Procapsid the Proteinaceous DNA Container

The procapsid (or prohead) of SPP1 consists of four proteins the major capsid protein gpl3, the scaffold protein gpl 1, the portal protein gp6 and a minor component gp710 (Table 1). Gpl 3 forms the head shell of the SPP1 procapsid. The protein composition of the SPP1 procapsid suggests that 415 copies of gpl3 form a T 7 icosahedral head shell lattice.11 The Viral Genome Packaging Machines Genetics, Structure, and Mechanism, edited by Carlos Enrique Catalano. 2005 Eurekah.com and Kluwer Academic...

Biochemistry

Some Past Studies of the Optimization of in vitro Systems In vitro systems for bacteriophage DNA packaging provide the investigator with improved control of the conditions of DNA packaging. These systems also isolate the process of packaging from other cellular events. Finally, in vitro systems provide the investigator with the potential for single-particle observation of bacteriophage DNA packaging, i.e., observation of DNA packaging one packaging event at a time. One of the conditions tested...

Editor

Department of Pharmaceutical Chemistry The University of Colorado School of Pharmacy Denver, Colorado, U.S.A. Dwight Anderson Department of Oral Science and Department of Microbiology University of Minnesota Minneapolis, Minnesota, U.S.A. Chapter 7 and Immunology Cornell University Ithaca, New York, U.S.A. Chapter 9 Department of Biosciences and Institute of Biotechnology University of Helsinki Helsinki, Finland Chapter 8 Lindsay W. Black Department of Biochemistry and Molecular Biology...

Gp20 The Portal Protein

The T4 portal protein gp20 (61 kDa) was first determined to be directly connected to packaging by gene 20 cs mutations that blocked packaging initiation at low temperature. The prohead defect was reversed upon temperature shift or could be suppressed by specific gene 17 terminase mutations, thereby showing intimate association between terminase and portal proteins in packaging.31'32 As discussed above, a number of clustered cs mutations in the portal gene and their terminase suppressors have...

Components of the o29 DNA Packaging Motor

The DNA packaging motor consists of the prohead, which contains the dodecameric head-tail connector at its portal vertex, a multimer of prohead RNA (pRNA) that is attached to the connector, and a multimer of the ATPase gpl6 (gene product 16) that binds to pRNA (Table 1). The active motor also includes the packaging substrate DNA-gp3. Viral Genome Packaging Machines Genetics, Structure, and Mechanism, edited by Carlos Enrique Catalano. 2005 Eurekah.com and Kluwer Academic Plenum Publishers....

The Putative Terminase UL15 UL28 Ul33

It is logical to presume that herpesviruses encode a multisubunit functional homolog of bacteriophage terminases that specifically recognizes genomic ends within the DNA, links the DNA to the capsid, and mediates the packaging of DNA through the hydrolysis of ATP. Although the absence of an in vitro packaging system in HSV precludes definitive proof, a complex of the UlI 5 and Ul28 gene products is the most likely to fulfill the role of terminase at the present time. (The Ul28 protein was...

The DNA Packaging Assay and Provisional Packaging Events

The packaging assay is based on protection of DNA in fdled heads from DNase treatment.28 Proheads, DNA-gp3 and gpl6 are generally mixed in a ratio of 2 1 12 to provide an estimated two-fold excess of proheads and gpl6, and the ternary complex forms during a 5 minute incubation at room temperature. ATP is added to initiate packaging, and incubation is continued for 10 minutes. DNase I is added to digest unpackaged DNA, and the protected packaged DNA is extracted and quantified by agarose gel...

Initiation of DNA Packaging

The first step in P22 (or any virus) DNA packaging is recognition of DNA to be packaged. Current evidence points strongly toward the P22 gene 3 protein being responsible for this recognition. P22 is a generalized transducing phage in that about 1-2 of the time it Figure 3. Processive packaging series. Concatemeric P22 DNA is represented by the thick horizontal line, and pac sites are represented by black circles. The vertical black line at thepac site indicates the location of series initiation...

Comparison to Tailed dsDNA Bacteriophages and Eukaryotic dsRNA Viruses

The packaging system of < )6 has certain similarities to the well-studied packaging systems of tailed dsDNA bacterial viruses. In both cases the genome is translocated into a preformed capsid (procapsid or prohead). The packaging is dependent on the hydrolysis of the high-energy bond in ATP (or any NTP in the case of ( )6). There are global conformational changes in procapsid during the maturation, and these can be induced by elevated temperature, change in pH or genome packaging.61,62 The...

ATPBinding Site II

A second Walker-A P-loop has been proposed in a number of terminases.18 In T4 gpl7, the second ATP-binding site is represented by the sequence TAAVEGKS299-306 with its apparent Walker-B motif close to the C-terminus GVSVAKSI.YMD468- 78.20 Unlike the striking conservation of Walker-Ai, the sequence conservation of Walker-An and its surrounding region is quite poor. Moreover, the gpl7s from phages KVP40 and KVP20 substitute isoleucine and valine respectively for the critical lysine of the...

Special Packaging Vertex

The structural approaches have indicated that ( > 6 PCs are symmetrical particles having one putative packaging NTPase (P4 hexamer) at each vertex. Earlier studies have also suggested that the packaging of more than one segment can take place simultaneously, and this was taken as evidence that there could be more than one entry portal in the PC.54 Recent biochemical and genetic analysis have, however, suggested that one of the 12 ver-texes in the < )6 PC may be physically and functionally...

Interactions of DNA Packaging with Other DNA Processes

Packaging in vivo must interact with numerous other DNA processes, suggesting that regulatory mechanisms governing these interactions are necessary. In fact, in several phages initiation of DNA packaging requires terminase interactions with other proteins, which help to control the process or facilitate it. For example, in phage X, the host coded proteins IHF and HU apparendy bend DNA to promote terminase binding.3 Phage T4 generally operates relatively independently of host components thus...

Building a Protective Shell for Viral DNA

Like other large dsDNA viruses, P22 assembles a protein procapsid and then inserts the DNA chromosome into this preformed container (Fig. 1). This strategy appears to be common to all dsDNA bacteriophages as well as the Herpesviridae. The genes required to build the procapsid and fill it with DNA map in a contiguous cluster on the P22 chromosome (Fig. 2). The four critical protein players in this process in P22 are analogous to those in other dsDNA viruses coat, scaffold, portal and terminase...

Model of the Packaging Mechanism

The most striking structural characteristic of the connector is the 36 long alpha helices in the central region, inclined roughly obliquely to the DNA helix, that give the connector the appearance of a compressible spring. The idea that the connector can expand and contract is the basis of a ratchet mechanism model5 (Fig. 7). There are three concentric symmetries of the active motor the 10-fold symmetry of DNA in the connector channel, the 12-fold symmetry of the connector, and the five-fold...

DNA Packaging in Bacteriophage T4

Black Introduction Double-stranded (ds) DNA packaging in phage T4 and other icosahedral viruses is a fascinating biological problem. During packaging, a complex, metabolically active, concatemeric DNA is translocated into an empty prohead in an ATP-driven process and condensed as a highly ordered structure of near crystalline density.13 dsDNA packaging serves as an excellent model system to understand fundamental biological mechanisms such as the reversible...

G

Gpl6 40-44, 46,47, 49, 50, 52, 53,68, 69, 90, 97, 102-104, 106-109, 113, 114 gpl7 40, 41, 43-48, 51-53, 55,69 gpl7s 46 gpl8 64,65,67 gpl 9 62, 64, 65,67, 68,73 gp20 40,41,46,48,49,53, 54 gp23 40, 41 gp24 40, 41 gpA 5, 6, 8-11, 14-20, 22-24, 26-30, 32, 52 gpD protein 30 gpFI 6, 24-28, 31, 33 gpNul 5, 6, 8-14, 16-20, 22, 23, 26, 28, 33 H Headful packaging 50, 54, 89, 97, 99, 125 Helicase 8, 14-18, 23, 28, 29, 45, 63, 131 Herpesvirus 1, 3-5, 29, 40, 41, 44, 86, 99, 135-137,140,142-145 HSV DNA...

Model of DNA Cleavage and Packaging

A model for the maturation of capsids in herpesvirus-infected cells that is consistent with the current literature is depicted in Figures 2 and 4. The procapsid containing unprocessed scaffold proteins is depicted as the precursor to the angular type A, B, and C capsids found within cells infected with wild type viruses (Fig. 4). As described above, procapsids contain the unprocessed versions of the pUi26 and pUi26.5 gene products. It is believed that immature capsids are delivered via pUi,17...

Mutations That Change the Sequential Packaging

Mutations in PI (Argi4 to Gly) and P4 (Ser25o to Gin) affect the sequential packaging cascade so that s is not packaged or is packaged poorly without considerable effect on m and 1 packaging. This phenotype of the PI capsid mutant has been taken as an evidence that PI contains the primary recognition sites for genomic ssRNA segments.43 Interestingly, the inability to package s by the P4 NTPase mutant particles appears only after the particles have been frozen and thawed. Fresh P4 mutant...

References

Genetic exchange in Salmonella. J Bacteriol 1952 64 679-699. 2. Ebel-Tsipis J, Botstein D, Fox MS. Generalized transduction by phage P22 in Salmonella typhimurium. I. Molecular origin of transducing DNA. J Mol Biol 1972 71 433-448. 3. Poteete AR. Bacteriophage P22. In Calendar R, ed. The Bacteriophages. New York Plenum Press, 1988 647-682. 4. Susskind MM, Botstein D. Molecular genetics of bacteriophage P22. Microbiol Rev 1978 42 385-413. 5. Casjens S, Hayden M. Analysis...

Virion Completion

Subsequent to separation of the DNA-filled capsid from the concatemer, the minor capsid proteins gpW and gpFII are sequentially added to the portal (Fig. 11). Presumably, gpW and gpFII are required to prevent DNA loss from the filled capsid139 and to provide an attachment site for tail addition, respectively.145 A high-resolution 3D structure of gpW (68 residues) has recendy been solved by NMR146 The solution structure represents a novel fold, consisting of two CC-helixes and a two-stranded...

Ja

Fluorescence microscopy of a single T7 100S+ DNA molecule in the presence ofT7 procapsids. Fluorescence microscopy was performed of the following mixture (i) Alexa 488-stained (green fluorescence seeref. 114) particles from a procapsid fraction of a Nycodenz gradient, (ii)T7 100S+ DNA that was stained with 1 mg ml ethidium bromide (orange fluorescence), and (iii) 1.1 low-gelling agarose (SeaPrep, Biowhittaker, Rockland, ME). The buffer was 0.039 M NaCl, 0.77 mMTris-Cl, pH 7.4,0.077 mM...

Structure of Capsids

The capsid of the mature bacteriophage T3 T7 particle has the following components (a) Most mass is associated with an icosahedral outer shell (T 7) that is made of gp 10 (Fig. 3a).7779 (b) One 5-fold symmetric vertex (of twelve 5-fold symmetric vertices) of the outer shell has a 12-fold symmetrical ring attached.7780 This ring is made of gp8 and is called either the connector or the portal the connector has been purified in the case of both T380 and T7.81 All connectors have an axial hole...

Genetics Genome Sequence

Complete nucleotide sequences have been determined for both theT7 genome10 and the T3 genome.11 These two bacteriophages appear to be representatives of a much larger family of bacteriophages. For example, both Vibrio parahaemolyticus bacteriophage VpV26225 and Roseophage SIOl26 have distant homology to T7, seen primarily in the nucleotide sequence and genetic organization of genes encoding proteins that participate in morphogenesis. These studies found no T3 T7-like RNA polymerase in either...

Cooperative gpNulgpA Binding Interactions

Model for cooperative assembly of gpA (red rectangle) and gpNul (blue rectangle) at cos. A symmetric gpA dimer binds to cosN. Binding of gpA to the cosNL half-site relies strictly on intrinsic binding interactions. Binding of gpA to the cosNR half-site is supported by cooperative interactions with gpNu 1 and IHF (purple lobes) assembled at cosB. A color version of this figure is available online at http www.Eurekah.com. mutations into the cosNR half-site are masked by supportive...

Bacteriophage SPP1 DNA Packaging

Anja Dr ge and Paulo Tavares Introduction SPP1 is a virulent double-stranded DNA (dsDNA) phage that infects the Gram-positive bacterium Bacillus subtilis strain 168. SPP1 belongs to the Siphoviridae family. The virion is composed of an icosahedral, isometric capsid (-60 nm diameter) and a long, flexible, noncontractile tail.1 The phage head encloses the mature SPP1 chromosome, which is a linear double-stranded DNA molecule of -45.9 kbp size.2,3 Replication of the SPP1 genome produces DNA...

Unit

Viral Genome Packaging Machines Genetics, Structure, and Mechanism Carlos Enrique Catalano, Pharm.D., Ph.D. Department of Pharmaceutical Chemistry The University of Colorado School of Pharmacy Denver, Colorado, U.S.A. Kluwer Academic Plenum Publishers New York, New York U.S.A. Viral Genome Packaging Machines Genetics, Structure, and Mechanism Landes Bioscience Eurekah.com Kluwer Academic Plenum Publishers Copyright 2005 Eurekah.com and Kluwer Academic Plenum Publishers No part of this book may...

Conclusions

Phage T4 fundamentally employs the same mechanisms as the other well characterized dsDNA bacteriophages for packaging DNA. However, it also presents some unique challenges. For instance, the mechanism of DNA substrate recognition and regulation of concatemer maturation and cutting represents one such problem. This is complicated by the fact that the T4 packaging substrate is an endless concatemer with multiple branches that are associated with protein complexes. T4 may exploit multiple modes of...

Termination of Packaging

Genome packaging is terminated when the packaging motor encounters the next downstream cos in the concatemer. When terminase encounters this site, translocation stops and symmetric nicks are introduced into the coWsubsite (Fig. 11) this terminal cos cleavage reaction is less dependent on cosB, but requires that the wQand 12 subsites be present for efficient termination (described in detail below). The strand separation activity of terminase separates the cohesive ends, releasing the DNA-filled...

Procapsid to Core Transition

Cryo-electron microscopy based image reconstructions have revealed a considerably different conformation for the empty polymerase complex (depicted in Fig. 5A) compared to that of the virion-derived dsRNA-filled core particle (depicted in Fig. 5F) despite the same protein composition. The empty particle is smaller (-46 nm versus -50 nm) and more angular. It also appeared to have inwards oriented cups at each of the five-fold facets instead of the round appearance of the core particle.12 The...

R o D

GroELS j * V gpB > gpB* 11 - qpC 11 gpC+gpE.> pX1,pX2 Figure 7. Procapsid assembly and processing. Details are described in the text. proteins are presumably required for proper folding of gpB, but the role of gpNu3 in assembly of the ring is not clear. The phage-encoded gpC protein next adds to the preconnector to yield a 30S initiator structure this reaction may also involve gpNu3.95 Neither the stoichiometry nor the structure of gpC in the initiator complex is known. The initiator serves...

Encapsidation of the Segmented Double Stranded RNA Genome of Bacteriophage

Pirttimaa and Dennis H. Bamford Abstract Bacteriophage < ))6 has a segmented double-stranded RNA genome that is incorporated into a preformed capsid during viral assembly. The three viral genomic segments are packaged as single-stranded precursors, which are later replicated into the mature double-stranded genome inside the capsid by the viral polymerase. The packaging efficiency of < j> 6 is high virtually all particles released from < )6-infected cells are...

Bacteriophage Lambda Terminase and the Mechanism of Viral DNA Packaging

Michael Feiss and Carlos Enrique Catalano Abstract The developmental pathways of many double-stranded DNA (dsDNA) viruses, both prokaryotic and eukaryotic, are remarkably similar. In viruses as diverse as bacteriophage X and the herpesviruses, DNA replication proceeds through a rolling circle mechanism where the circular genome serves as a template for the synthesis of linear concatemers multiple genomes in length. Concurrendy, viral gene expression produces structural proteins, which...

Ongoing Analysis of o29 DNA Packaging The Path to Enlightenment

The paradigm of analysis of any cell biological process at the molecular level, attributed to Tom Pollard and published in a review of the meeting Proteins as Machines in reference 38, is reproduced in Figure 10. The approach is to identify all components, describe all reaction intermediates, determine the rates of all transitions, and determine component structures at atomic resolution. Although arduous, all parts of the approach are vital to a complete understanding of the biological process....

T

Schematic diagram of HSV-1 DNA. Unique long (Ul) and unique short (Us) components are flanked by inverted repeats (boxes). The a sequences at genomic ends contain sequences necessary for packaging DNA. They are reiterated at the internal L S junction in opposite orientation (designated by a'). Detail of the a sequence is also shown. The lengths of the a sequence components are indicated in bp. containing a DR1, Pac2, Pac 1, and at least some repeated units, are sufficient for the...

Activities Associated with gpl7 DNA Packaging

Packaging efficiencies measured at 10 wild type (> 108 ml infected bacteria) can be obtained using purified terminase proteins.12'25 Gpl6 enhances gpl7-dependent packaging activity by about 100-fold at low concentrations of gpl7.8 DNA packaging in vitro normally requires both gpl6 and gpl7, but gpl7 alone may suffice at high concentrations.11'12 Endogenous phage T4 concatemeric DNA that accumulates in packaging defective phage infections is packaged at about 100-fold greater efficiency than...

Components of the Phage T4 DNA Packaging Machine

Gpl6 is an 18 kDa protein.8,9 It is the small subunit of the terminase holoenzyme. Gpl6 is dispensable for in vitro DNA packaging although packaging efficiencies are low in its absence. It is essential in vivo since 16am mutations are lethal, although microscopy reveals that, in double am mutants of gene 16, packaging begins late after infection and proceeds slowly and incompletely.10 Gpl6 appears to play an important role in DNA recognition and in modulating the functions of the terminase...

Viral Genome Packaging Machines

Doughnut Packaging Packaging Structure

A virus particle is a marvel of nature, designed to replicate with a minimal genetic repertoire. All viruses are, of course, obligate intracellular parasites and require an appropriate host in which to develop and multiply. They have evolved a variety of strategies to infect a host cell and to usurp the cellular machinery to manufacture the components required to construct a virion. These precursors are then assembled into an infectious virus particle within the cell. Virus assembly is a...

Capsid Maturation

Herpesvirus Assembly

During the herpesvirus cleavage packaging reaction, DNA is inserted into preformed capsids. Cleaved viral genomes are not detected in cells infected with viruses that fail to assemble capsids, suggesting that capsids contain essential parts of the cleavage packaging machinery.32 There are four morphologic types of capsids that can be distinguished by electron microscopic examination of thin sections of herpesvirus-infected cells, and they are designated procapsids and capsid types A, B, and...