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We have isolated and characterized several hundred mutants of bacteriophage λ that are defective in prophage excision. Almost all resemble classical int and xis mutants in complementation pattern and ability to integrate. One int mutant is exceptional in that it is defective in excision but proficient in integration. The mutant sites are located in a region that begins within 50 base-pairs to the right of the attachment site and extends about 1350 base-pairs rightward, int is approximately 1240 base-pairs and xis 110 base-pairs long.

We have established that in the Becker strain of pseudorabies virus (PRV), the glycoprotein gIII gene is not essential for growth in cell culture. This was accomplished by construction and analysis of viral mutants containing two defined deletion mutations affecting the gIII gene. These mutations were first constructed in vitro and introduced into Escherichia coli expression plasmids to verify structure and protein production. Each mutation was then crossed onto PRV by cotransfection of plasmid DNA and parental viral DNA by using gIII-specific monoclonal antibodies as selective and screening reagents. One resultant virus strain, PRV-2, contained an in-frame deletion of a 402-base-pair (bp) SacI fragment contained within the gIII gene. Another virus strain, PRV-10, contained a deletion of a 1,480-bp XhoI fragment removing 230 bp of the upstream, putative transcriptional control sequences and 87% of the gIII coding sequence. The deletion mutants were compared with parental virus by analysis of virion DNA, gIII specific RNA, and proteins reacting with gIII specific antibodies. Upon infection of PK15 cells, the deletion mutants did not produce any proteins that reacted with two gIII specific monoclonal antibodies. However, two species of truncated glycosylated proteins were observed in PRV-2 infected cells that reacted with antiserum raised against bacterially produced gIII protein. PRV-10 produced no detectable gIII-specific RNA or protein. PRV-10 could be propagated without difficulty in tissue culture. Virus particles lacking gIII were indistinguishable from parental PRV virus particles by analysis of infected-cell thin sections in the electron microscope. We therefore conclude that expression of the gIII gene was not absolutely essential for PRV growth in tissue culture.

A simple, efficient method for introducing recombinant DNA into a herpesvirus vector and retrieving it at a later time has been developed. By using the Cre-lox site-specific recombination system of coliphage P1, DNA can be readily inserted in vitro into a pseudorabies virus (PRV) vector containing the lox recombination site. The vector PRV42 contains a lox site within the nonessential gIII gene, which encodes a virion envelope glycoprotein. Incubation in vitro of PRV42 DNA with Cre protein and a circular plasmid containing a lox site generates approximately 5% recombinant molecules having the plasmid integrated into the PRV genome at the lox site. Transfection of the reaction mixture into cultured cells allows recovery of the infectious recombinant virus, which is readily identified by a nondestructive "black-plaque assay" using a gIII-specific monoclonal antibody. PRV42 plaques stain black when treated with the gIII monoclonal antibody and a peroxidase-linked second anti-antibody because the lox site placed within the gIII gene of PRV42 does not destroy the gIII epitope. However, Cre-mediated integration of heterologous DNA at the lox site disrupts the gIII epitope so that the resulting recombinant virus produces white plaques. The recombinant virus is infectious, stable, and grows as well as the parental PRV42 vector. The inserted plasmid can be efficiently excised (greater than 50%) from viral DNA by Cre and recovered by transformation of Escherichia coli.

The DNA of actinophage φEC was characterized by physical and enzymatic methods. In the electron microscope, the DNA appeared as a linear, double-stranded molecule with an average chain length of 43.2 ± 1.7 kb. Further studies suggested that the DNA carried cohesive ends. The DNA was analyzed by digestion with 32 restriction endonucleases. The enzymes BamHI, HpaI, PvuII, XbaI and XhoI were used to construct a map of the φEC genome.

Using partially purified mouse liver 23S mRNA known to be associated with the Ah locus and 3-methylcholanthrene-induced cytochrome P 1 -450, we synthesized double-stranded cDNA by the successive action of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) and the Klenow A fragment of Escherichia coli DNA polymerase I. The double-stranded cDNA was inserted into pBR322 plasmid DNA by Pst I cleavage and homopolymeric “tailing” and cloned in E. coli LE392. Clone 46 hybridized with [ 32 P]cDNA made from 23S mRNA from “ Ah -responsive” C57BL/6N mice but did not hybridize with similarly prepared [ 32 P]cDNA from “ Ah -nonresponsive” DBA/2N mice. Clone 30 was positive, and clone 7 was negative, with both C57BL/6N and DBA/2N [ 32 P]cDNA probes; these two clones were therefore used as “positive” and “negative” control clones, respectively. By translation-arrest experiments, clone 46 DNA and clone 30 DNA were shown to be associated with anti- P 1 -450- and anti-albumin-precipitable material, respectively. By agarose gel electrophoresis of Pst I digests, the clone 46 DNA insert was shown to be 1100 base pairs in total length and to contain one internal Pst I site. The cDNA made from total mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mice hybridized to the two fragments of Pst I-digested DNA from clone 46, whereas similarly prepared cDNA from 3-methylcholanthrene-treated DBA/2N and control C57BL/6N and DBA/2N mice did not. Of 11 restriction endonucleases used, two ( Pst I and Xba I) had sites within the clone 46 DNA insert. After hybridization of clone 46 32 P-labeled nick-translated DNA to Eco RI fragments from A/HeJ mouse genomic DNA and fractionation by RPC-5 chromatography and gel electrophoresis, only one positive band (3-4 kilobase pairs appeared. These data demonstrate conclusively that pBR322 clone 46 DNA is associated with mRNA controlled by the murine Ah locus, presumably the structural gene encoding 3-methylcholanthrene-induced P 1 -450.

When coliphage Lambda replicates its DNA, it commandeers portions of the host replication system for its own use. How phage and host functions interact and are regulated present interesting problems in the DNA replication program of phage Lambda.

We describe a simple technique for isolating deletion mutants of phage lambda and use it to dissect a cloned fragment of foreign DNA. The technique is based on our previous finding that the normally essential product of lambda head gene D is dispensible for phage growth if the DNA content of the phage is less than 82% that of lambda wild-type (Sternberg and Weisberg, 1977). A significant fraction of the few phage that form plaques when a D amber mutant is plated on a nonsuppressing host contains deletions that reduce the phage chromosome size to less than 82% that of wild-type. It is possible to isolate deletions ranging in size from less than 1.5 kb to 14 kb (3 to 27% of wild-type lambda), and the size range can be restricted by an appropriate choice of the DNA content of the starting phage. This method, unlike the older EDTA or heat resistance methods, permits the scoring of deletions because of the absence of phenotypic variants. We investigated the effect of several host and phage mutations on deletion frequency and type and have determined that a host polA mutation increases the frequency of deletions about 30-50-fold without changing the type of deletions. A host mutD mutation or thymine deprivation increases deletion frequency about 10-fold. In contrast, a host ligts mutation has no effect on the frequency of deletions. We have also determined that the size of the smallest lambda chromosome packageable in a plaque-forming phage particle is 72-73% that of lambda wild-type.

We have identified and characterized a 3.0 kilobase (kb) mRNA containing coding sequences of the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene. The synthesis of this 3.0 kb mRNA was unaffected by the presence of cytosine arabinoside, but was made in greatly reduced amounts in cells infected with HSV-1 in the presence of cycloheximide: it was, therefore, classified as an early mRNA. By nuclease protection experiments, it was found that the 3.0 kb mRNA is unspliced and, further, that it is 3' co-terminal with a smaller 1.6 kb early mRNA which is transcribed from a DNA sequence 3' to the gD coding sequence. We describe the use of the Xenopus laevis oocyte system to produce HSV-1 gD in vitro. Oocytes injected with mRNA isolated from HSV-1-infected Vero cells synthesized gD, which was identified by immunoprecipitation. Injection of a plasmid clone containing the HSV-1 BamHI J fragment (0.89 to 0.93 map units) into the nuclei of Xenopus oocytes also resulted in synthesis of gD.

We have constructed two pseudorabies virus (PRV) mutants, each with a unique EcoRI restriction site in the nonessential gIII envelope glycoprotein gene. Since no natural PRV isolate has been reported to contain EcoRI sites, the isolation and single-step growth curve analysis of these mutants established that PRV can carry such a site with little ill effect in tissue culture. Virus carrying these defined mutations produced novel gIII proteins that enabled us to begin functional assignment of protein localization information within the gIII gene. Specifically, one viral mutant contained an in-frame synthetic EcoRI linker sequence that was flanked on one side by the first one-third of the gIII gene and on the other side by the last one-third of the gene. The resulting protein lacked the middle one-third of the parental species, including five of eight putative N-linked glycosylation signals, but was still glycosylated and found in enveloped virions; it was not secreted into the medium. A second viral mutant contained an in-frame synthetic EcoRI linker sequence that additionally specified a nonsense codon at position 158, producing a gIII protein that was glycosylated and secreted into the medium; the fragment was not found in enveloped virions. By endoglycosidase and pulse-chase analyses, we established a precursor-product relationship between the various forms of gIII expressed in the parental and mutant strains, and perhaps determined certain features of the gIII protein that are required for its efficient export within the cell.

We have studied the role of the red and gam genes in lambda replication, after infection of wild type and two recombination deficient hosts. Our results show that the rate of phage DNA replication is abnormally low in the absence of red function, in rec+ as well as rec− (A− and A−B−) bacteria. It appears that the virus general recombination proteins play some role in lambda replication that cannot be assumed by the general recombination proteins of its bacterial host. The red− defect in replication results in a decrease in the total amount of intracellular phage DNA. This DNA, nevertheless, seems normal in structure and is matured and packaged with good efficiency. In rec+ and recA− hosts infected with gam− mutants, the rate of lambda replication is also low, but in this case, abnormal DNA structures are produced at late times. The gam mutation seems to alter the program of replication such that circular molecules are produced not only at early times, but continuously, throughout the lytic cycle. This, and other facts, suggest that the gam protein is required for the transition from “early” to “late” replication. This requirement for gam function is not observed in recA−B− hosts, in which gam mutants replicate at a normal rate and produce DNA indistinguishable from that made by wild type phage. Thus, the gam requirement seems to involve an interaction of this phage protein with the product of the host's recB gene. Other evidence for such interaction comes from our finding that, in vivo, the gam protein does inhibit presumed action of the host's BC nuclease. In the gam− mutant infections, which are blocked in late replication, absence of a general recombination system seems to create a severe defect in maturation of intracellular phage DNA. This defect, unlike the one affecting λ replication rate, can be alleviated by either the red or rec functions and is correlated with the inability of the mutant phages to make DNA concatemers. Since other late functions (i.e. late messenger RNA production) appear to be normal, we conclude that concatemer formation, via replication or recombination, is an essential step in phage development.

Two nonsense mutants of Salmonella form a burst size in mixed infection of non-permissive bacteria which lies above that for non-complementing pairs but below that expected for complementation. The two mutants map closely together.

SummaryMitochondria are dynamic organelles that are essential for cellular metabolism but can be functionally disrupted during pathogen infection. In neurons, mitochondria are transported on microtubules via the molecular motors kinesin-1 and dynein and recruited to energy-requiring regions such as synapses. Previous studies showed that proteins from pseudorabies virus (PRV), an alphaherpesvirus, localize to mitochondria and affect mitochondrial function. We show that PRV and herpes simplex virus type 1 (HSV-1) infection of rodent superior cervical ganglion (SCG) neurons disrupts mitochondrial motility and morphology. During PRV infection, glycoprotein B (gB)-dependent fusion events result in electrical coupling of neurons and increased action potential firing rates. Consequently, intracellular [Ca2+] increases and alters mitochondrial dynamics through a mechanism involving the Ca2+-sensitive cellular protein Miro and reduced recruitment of kinesin-1 to mitochondria. This disruption in mitochondrial dynamics is required for efficient growth and spread of PRV, indicating that altered mitochondrial transport enhances alphaherpesvirus pathogenesis and infection.

Summary After replicating in epithelial cells, alphaherpesviruses such as pseudorabies virus (PRV) invade axons of peripheral nervous system neurons and undergo retrograde transport toward the distant cell bodies. Although several viral proteins engage molecular motors to facilitate transport, the initial steps and neuronal responses to infection are poorly understood. Using compartmented neuron cultures to physically separate axon infection from cell bodies, we found that PRV infection induces local protein synthesis in axons, including proteins involved in cytoskeletal remodeling, intracellular trafficking, signaling, and metabolism. This rapid translation of axonal mRNAs is required for efficient PRV retrograde transport and infection of cell bodies. Furthermore, induction of axonal damage, which also induces local protein synthesis, prior to infection reduces virion trafficking, suggesting that host damage signals and virus particles compete for retrograde transport. Thus, similar to axonal damage, virus infection induces local protein translation in axons, and viruses likely exploit this response for invasion.

THOMAS et al.1 have constructed a mutant strain of bacteriophage λ (λgt.λC) which is especially well suited to studies involving the cloning of recombinant DNA molecules. Specifically, this bacteriophage, deleted of 15% of its genome, has only two EcoRI restriction endonuclease sites which, when cleaved, reduce the phage chromosome to three DNA fragments. The larger of these, the right- and left-hand arms of the chromosome, encode all functions necessary for lytic infection. The smaller, central fragment consisting of approximately 5.000 base pairs encodes the λ attachment site, the Int and Xis functions and a necessary part of the λ-generalised recombination system, but no genes necessary for phage propagation. Although left- and right-hand restriction fragments have all necessary λ genes, they seem to lack sufficient length of DNA for packaging into a viable phage particle1. This provides a powerful positive selection for recombinant DNA molecules consisting of isolated and rejoined right and left restriction fragments which have also incorporated a fragment of foreign DNA restoring the λ chromosome to viable size.

Regions of herpes simplex virus type 1 (HSV-1) DNA with variation in the size of restriction endonuclease fragments were identified by comparison of the BamHI, KpnI or SalI restriction endonuclease digestion patterns among 15 HSV-1 isolates after hybridization with specific 32P-labeled cloned HSV-1 DNA fragments. Of the types of restriction fragment polymorphism identified, one was a strain with a distinctly different restriction fragment than the prototype (loss or gain of restriction sites). Another type, the specific fragment varied only in size among strains. Thirteen distinct variations were identified. Ten were mapped to the unique sequence of the L component; two to the inverted repeat of the L component and one to the inverted repeat of the S component. The presence of a common ancestor from which some isolates of HSV-1 might derive was deduced from an analysis of the distribution of the thirteen variations among the 15 HSV-1 isolates.

We have constructed a pseudorabies virus mutant that contains virtually a complete deletion of the predicted signal sequence coding region for a nonessential envelope glycoprotein, gIII. No signal sequence mutants have been reported previously for a herpesvirus glycoprotein. Through endoglycosidase treatments and pulse-chase analysis, we have determined that the mutant gIII protein is not posttranslationally modified like the wild-type polypeptide, but rather is present as a single, stable species within the infected cell. The mutant polypeptide cannot be detected in the virus envelope, nor is it aberrantly localized to the tissue culture medium. Immunofluorescence studies have indicated that the mutant protein also is not localized to the surfaces of infected cells. In addition, Northern (RNA) and slot blot analyses, as well as in vitro translation experiments using infected-cell cytoplasmic RNA, have indicated that the mutant gIII allele is expressed at lower levels than the wild-type gene is. This is despite the fact that no alterations have been made upstream of the gIII coding sequence. From these results, it appears that the first 22 amino acids of the wild-type gIII protein define a necessary signal peptide that is responsible for at least the correct initiation of translocation and subsequent glycosylation of the gIII envelope glycoprotein within infected cells.

The 15.4 kb Eco RI-H fragment of Herpes simplex virus type 1 (HSV-1) strain Patton, which contains the entire short unique (U s ) region, has been cloned in bacteriophage λ. The fragment contains a terminal redundancy of about 900 bp that represents the S region terminal-repeat sequences. The restriction enzyme Sma I cleaves the Eco RI-H fragment at more than 30 sites. We have constructed an Sma I map of this fragment using thirteen isolates of λgt WES hybrid bacteriophage that carry various deletions of the Eco RI-H fragment.

We describe the nucleotide sequence of a herpes simplex virus type 1 DNA fragment containing the intron of the immediate-early mRNA-5 (IE mRNA-5) gene. The location of the intron within this fragment was determined by a Berk & Sharp nuclease S1 protection analysis, and by cloning and sequencing cDNA containing sequences overlapping t he IE mRNA-5 splice point. We found that the 149 base pair (bp) intron contained four copies of an identical 23 bp GC rich tandem repeat followed by a further reiteration consisting of the first 15 bp only.

gIII, the major envelope glycoprotein of pseudorabies virus (PRV), shares approximately 20% amino acid similarity with glycoprotein gC of herpes simplex virus type 1 (HSV-1) and HSV-2. We describe here our first experiments on the potential conservation of function between these two genes and gene products. We constructed PRV recombinants in which the gIII gene and regulatory sequences have been replaced with the entire HSV-1 gC gene and its regulatory sequences. The gC promoter functions in the PRV genome, and authentic HSV-1 gC protein is produced, albeit at a low level, in infected cells. The gC protein is present at the cell surface but cannot be detected in the PRV envelope.

We have asked, "What is the mechanism of strand exchange during site-specific recombination of phage lambda?" Crosses carried out in vivo have shown that the recombination joint can be extended rather than flush and that the four-strand breaks and rejoinings needed to from a recombinant can occur asynchronously. Crosses carried out in vitro have shown that all the nucleotides at the site of crossover are conserved during recombination, as are most or all of the superhelical turns present in the substrate molecules. We have presented new data showing that topoisomerase activity of Int protein relaxes DNA by making transient single-strand, rather than double-strand, breaks in the phosphodiester back-bone. These findings are incorporated into a model for strand exchange that has as its central intermediate a four-strand structure.

When the entire Us region of HSV-1 (Patton) was cloned as an EcoRI fragment in bacteriophage λgtWES, the BamHI B6B5 fragment was observed to vary in size among independent isolates [Umene and Enquist, Gene 13 (1981) 251–268]. This fragment polymorphism also occurred in DNA of HSV-1 single plaque isolates. We report here that this heterogeneity is due to variation in copy number of a 15-bp tandem repeat of sequence 5'-CCACTCCCCACCCAC-3', which apparently lies in an intergenic region of the HSV-1 DNA.

Expression of the int gene after phage λ infection normally requires the products of genes c II and c III. However, when the phage carries a deletion in the nonessential b 2 region adjacent to int , efficient synthesis of active Int protein does not require c II and c III function. This inhibition of Int synthesis by nucleotide sequences downstream from the int structural gene behaves in a cis -dominant fashion in mixed infections. It is specific for P L - and not p I-initiated transcripts. Based on these observations, and those of others, a model is proposed in which Int translation from the p L transcript is inhibited by the interaction of downstream b 2 nucleotide sequences and nucleotide sequences in the int region. The data imply a novel temporal mechanism regulating prophage λ induction: circularization of the prophage genome results in the transposition of inhibitory b 2 region sequences next to int and blocks further Int protein synthesis beyond the low level required for excision. As a consequence of this process, the control of int expression is transferred from the p L promoter to p I and the c II/ c III system. Such a genetic regulatory mechanism involving the rearrangement of genetic elements downstream from a structural gene may be of general use during development in other systems.

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) cause both persistent and latent infections, including recurrent cutaneous disease, lethal neonatal disease, central nervous system disease and other clinical syndromes. Modified live vaccines or conventionally prepared subunit vaccines have generally been unsuccessful in the treatment of HSV-1 and HSV-2 infections from the standpoints of safety and efficacy. It has been established that HSV-1 and HSV-2 infectivity may be neutralized in vitro with antisera directed specifically against each of the four major glycoproteins of the virus (gA/gB, gC, gD and gE) and antisera against glycoprotein gD, of either HSV-1 or HSV-2, are capable of neutralizing both HSV-1 and HSV-2 infectivity in vitro and in vivo. We have previously reported on the identification, DNA sequence and expression at low level in Escherichia coli of the gD gene of HSV-1 strain Patton. Here we describe construction of a hybrid gene encoding a chimaeric protein containing HSV-1 gD, bacteriophage lambda Cro and E. coli beta-galactosidase (gD-beta-gal) protein, which is expressed at high level in E. coli. Moreover, the chimaeric protein elicits antibodies in rabbits that not only immunoprecipitate gD from cells infected with HSV-1 and HSV-2 but also neutralize HSV-1 and HSV-2 infectivity in vitro.

Glycoprotein gIII of pseudorabies virus is a major antigen found in the envelopes of virus particles as well as in and on the surfaces of infected cells. It is not an essential gene product for virus growth in tissue culture. In this report, we provide evidence that, although it is not essential, the gIII protein is required for efficient virus growth and that gIII mutants are quickly outgrown by wild-type virus in mixed infections.

We have constructed a hybrid plasmid by insertion of the thymidine kinase (TK) gene of Herpes simplex virus (HSV) type I at the BamHI site on Escherichia coli plasmid pBR322. The restriction endonuclease cleavage site map for the viral DNA fragment was determined for ten nucleases, and the insert in the recombinant plasmid has the same restriction nuclease digestion pattern as bona fide viral DNA. This result indicates that the plasmid contains an accurate copy of the viral DNA. The viral TK gene carried on the plasmid can be introduced into mammalian cells where it is expressed. This source of DNA with a selectable marker should be of considerable practical use in gene-transfer experiments in mammalian cells.

We have combined techniques of genetic and physical mapping with rapid DNA sequence analysis to identify the nucleotide change in lambda int mutations. These mutations define two dominant phenotypic classes: (i) recombination that is partially independent of accessory factors, and (ii) inhibition of wild-type Int by missense or nonsense proteins, i.e., negative complementation.

We have isolated and characterized a novel int mutant of phage lambda. This mutant promotes efficient recombination between the phage and bacterial attachment sites, but, unlike wild type, does not promote efficient recombination of any other pair of attachment sites tested in most conditions. In particular, recombination between two phage or two prophage attachment sites is poor relative to the wild type frequency. We attribute this unusual phenotype to differences in the distribution of int protein binding sites among different attachment sites (Ross and Landy 1982, 1983). We suggest that int protein molecules bound to one of two recombining DNAs interact with empty sites or with bound proteins on the other, and that the mutant protein acts efficiently only if the distribution of protein binding sites within the two attachment sites is that of the attP-attB pair. Similar discrimination among attachment site pairs by wild type int protein may also modulate recombination frequencies.

We have constructed two strains of bacteriophage λ to meet the requirements of an EK2 host vector system as established by the NIH Advisory Committee on Recombinant DNA Research. Both strains rely on the λgt·λC system devised originally by Davis and his colleagues (1). Here we describe some of the properties of these vectors and their use in cloning a specific Eco Rl fragment of mouse DNA which encodes extensive portions of the mouse ribosomal genes.

A pseudorabies virus (Becker strain) glycoprotein gene was located in the UL region at map position 0.40. The gene was identified by using open reading frame Escherichia coli plasmid expression vectors and specific antibody reagents. A 1.55-kilobase unspliced transcript from the gene was detected in pseudorabies virus-infected tissue culture cells. The DNA sequence revealed a single open reading frame of 1,437 base pairs encoding 479 amino acids. The predicted primary translation product has a molecular weight of 50,860 and contains features of a typical herpesvirus glycoprotein. An E. coli expression plasmid was constructed that contained essentially all of the open reading frame for this gene. Antibodies raised in rabbits against the protein expressed in bacteria by this plasmid immunoprecipitated pseudorabies virus-specific glycoproteins of 92,000 and 74,000 daltons from infected cell extracts. It is likely that these two forms represent different glycosylation states of the protein.

Pseudorabies virus (PRV) vaccine strain Bartha has a diminished capacity to cause disease and harbors a variety of mutations affecting virulence. It has been reported that PRV Bartha produces virions with reduced amounts of the major envelope glycoprotein gIII. One hypothesis was that this phenotype was due to reduced expression of the gIII gene. In this report, we demonstrate that the reduced amount of gIII in virions was not mediated at the level of transcription, but rather reflected a defect in protein localization. We describe experiments with gene replacement technology to prove that the expression defect was closely linked to the gIII gene itself. Using pulse-chase experiments, we found a defect similar to that observed for certain signal sequence mutations of PRV Becker gIII. The Bartha gIII protein was translated, but was inefficiently introduced into the membrane protein export pathway. Consequently, only a fraction of the primary Bartha gIII translation product was glycosylated and matured. The remaining fraction stayed presumably in the cytoplasm, where it never became glycosylated or inserted into cell or virus membranes. The result was that Bartha-infected cells produced virions with reduced amounts of gIII in their envelopes. Comparison of the DNA sequence of the promoter and amino-terminal coding regions of Becker and Bartha gIII genes revealed a single base pair difference in Bartha, changing codon 14 of the signal sequence from a leucine (CTC) to a proline (CCC) codon. We suggest that the signal sequence mutation is responsible for the apparent reduced expression phenotype of this attenuated strain. This mutation represents, to our knowledge, the first reported natural signal sequence mutation in a herpesvirus glycoprotein.

We have identified integrated proviral DNA sequences of m1 and HT-1 isolates of Moloney sarcoma virus (MuSV) in EcoRI digests of transformed mink cell genomic DNA and have cloned these fragments in bacteriophage lambda. Both the lambda-HT1 phage recombinant, containing a 12.3-kilobase MuSV pair (kb) fragment, and the lambda-m1 phage recombinant, containing a 7.0-kb fragment, possess full copies of the sarcoma viruses along with 5' and 3' host flanking sequences. The MuSV proviral DNA sequences, 6.7 kb for HT-1 and 5.2 kb for m1, are colinear by heteroduplex microscopy with the 1.5-kb difference in size accounted for by two approximately equal to 0.8-kb deleted regions in m1. Both integrated viral genomes are terminally redundant and have integrated at the same site in the provirus but at different sites on the host chromosome. The host sequence flanking integrated HT-1 MuSV have been identified as a single EcoRI restriction fragment of 5.6 kb in normal mink cells.

A derivative of bacteriophage lambda has been modified and tested together with an appropriate host system to meet the criteria of EK2 biologic containment for cloning DNA from higher organisms. In this report certain of the safety features are summarized and some of the tests carried out to confirm the containment properties of the vector are described. The cloning efficiency of this system, together with available gene purification and hybrid screening technology, indicate that it can be used to clone DNA fragments carrying specific, unique mammalian genes.

DNA isolated from defective and nondefective virions of herpes simplex type 1 (HSV-1) (strain Patton) was digested with restriction endonucleases, and the resulting DNA fragments were inserted in the EK2 coliphage vector lambdagtWES . lambdaB. The recombinant DNA was encapsidated in vitro under P4 maximum containment conditions. These lambda-HSV1 hybrids were purified and amplified, and the DNA was isolated in the P4 facility. DNA, free of viable phage and bacteria, was removed from P4 conditions and analyzed. Represented among the hybrids studied to date are DNA fragments from about 50 percent of the normal HSV-1 genome. The hybrids derived from defective HSV-1 DNA fragments demonstrate the existence of many similar but not identical classes of defective genomes.

Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion. This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences. Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences. One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site. This integration site sequence from normal mink cells was also cloned into phage lambda. An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus.

We have used elements of the E. coli lactose ( lac ) Operon to produce a collection of herpes simplex virus types 1 and 2 glycoprotein D (gD-1 and gD-2) antigens. Our approach employed recombinant DNA techniques to construct plasmids with various segments of the gD-1 and gD-2 coding sequences fused to the lac Z gene. Such hybrid genes were expressed in a regulated manner in E. coli by joining them to the lac promoter-operator region. Efficient translation of these hybrid genes was facilitated by incorporating a coding sequence specifying a short peptide leader ( λcro ) in the plasmid expression vectors resulting in synthesis of chimeric Cro-gD-β-galactosidase proteins. In addition, insertion of synthetic translation terminators at the junction of gD and lac Z enabled us to produce specific truncated gD polypeptide sequences unfused to β-galactosidase. The gD antigens produced in E. coli were not glycosylated and were generally recovered as dense insoluble aggregates. Proteins containing portions of gD-1 or gD-2 were analyzed by immunoprecipitation using anti-HSV rabbit serum and a number of monoclonal antibodies recognizing different epitopes of gD-1. Initial animal studies were done with antigens that reacted with neutralizing antisera or monoclonal antibodies. When these bacterially produced proteins were injected into rabbits, antibodies were produced that specifically immunoprecipitated authentic gD polypeptides and neutralized the infectivity of both virus types. These studies suggest that gene fusion techniques can be used to produce immunogenic proteins in large quantity. These polypeptides are not only useful in analyses of gene structure and function, but also can provide novel diagnostic reagents and well-defined pure antigens for vaccine development.

DNA fragments encoding herpes simplex virus type 1 and type 2 glycoprotein D (gD-1 and gD-2, respectively) have been inserted into plasmid vectors and expressed under the transcriptional control of the Escherichia coli lac promoter-operator. The proteins expressed in this system comprised gD sequences fused to a small bacteriophage λ Cro leader (i.e. Cro-gD). Such Cro-gD fusion proteins were found to be intrinsically unstable in E. coli and accumulated to low levels only. We found that fusion of this Cro-gD coding sequence to the 5′ end of a sequence encoding β-galactosidase (β-gal), resulted in high levels of synthesis of Cro-gD-β-gal fusion proteins provided that certain carboxy-terminal gD coding sequences were deleted. These Cro-gD-β-gal proteins accumulated to levels comprising approximately 10% of the total cell protein and were found to form intracellular insoluble aggregates. Insertion of an in-phase amber nonsense codon between the gD and β-gal coding sequences, resulted in synthesis of both Cro-gD and Cro-gD-β-gal proteins in cells containing suppressors. Under these conditions, the Cro-gD amber fragment was stabilized by the Cro-gD-β-gal protein. Chimaeric gD-1 and gD-2 containing proteins were found to be immunologically active and induced antibodies in rabbits which immunoprecipitated authentic gD polypeptides and neutralized the infectivity of both virus types.

Cloned DNA fragments from herpes simplex virus (HSV) type 1 (strain Patton) were tested for activation of endogenous mouse retrovirus in BALB/3T3 cells. Activation within the L region of HSV-1 DNA was observed with the ∼3.4-kilobase pair (kbp) Bam HI fragment which contains the virus thymidine kinase (TK) gene, and the ∼5.3-kbp Eco RI L fragment. Activation by the TK-containing Bam HI fragment was abrogated by digestion with Eco RI. Activation within the S region of HSV-1 DNA was observed with the ∼15.2-kbp Eco RI H ragment and the ∼8.4-kbp Eco RI/ Hin dIII H/G fragment. Assaying for retrovirus activation serves as an additional parameter for mapping biological functions within the HSV genome.

Two naturally occurring variations of herpes simplex virus type 1 (Patton strain) with novel tandem DNA sequence duplications in the S component were isolated, and the DNA was characterized. These variants were identified among a number of plaque isolates by the appearance of new restriction enzyme fragments that hybridized with radiolabeled DNA from the BamHI Z fragment (map coordinates 0.936 to 0.949) located in the unique S region. One isolate, SP26-3, carried a 3.1-kilobase-pair duplication defined by recombination between a site in the BamHI Z fragment and a site near the origin of replication in the inverted repeat sequence of the S component carried by the EcoRI H fragment. The other isolate, SP22-4, carried a 3.5-kilobase-pair duplication defined by a recombination event between a tandem repeat array in the BamHI Z fragment and a site near the amino terminus of the Vmw175 gene in the S-region inverted repeat sequence contained in the EcoRI K fragment. Both duplicated segments contained the entire immediate early mRNA-5 coding region as well as the origin of replication located in the inverted repeat sequence of the S component. The DNA sequence of each duplication joint was determined.

The pseudorabies virus (PRV) gE gene encodes a multifunctional membrane protein found in infected cell membranes and in the virion envelope. Deletion of the gE gene results in marked attenuation of the virus in almost every animal species tested that is permissive for PRV. A common inference is that gE mutants are less virulent because they have reduced ability to spread from cell to cell; e.g., gE mutants infect fewer cells and, accordingly, animals live longer. In this report, we demonstrate that this inference does not hold in a rat experimental model for virus invasion of the brain. We find that animals infected with gE mutants live longer despite extensive retrograde, transneuronal spread of virus in the rat brain. In this model of brain infection, virus is injected into the stomach musculature and virions spread to the brain in long axons of brain stem neurons that give rise to the tenth cranial nerve (the vagus). The infection then spreads from neuron to neuron in well-defined, and physically separated, areas of the brain involved in autonomic regulation of the viscera. We examined the progression of infection of five PRV strains in this circuitry: the wild-type PRV-Becker strain, the attenuated PRV-Bartha vaccine strain, and three gE mutants isogenic with the PRV-Becker strain. By 60 to 67 h after infection, all PRV-Becker-infected animals were dead. Analysis of Becker-infected rats killed prior to virus-induced death demonstrated that the virus had established an infection only in the primary vagal neurons connected directly to the stomach and synaptically linked neurons in the immediate vicinity of the caudal brain stem. There was little spread to other neurons in the vagus circuitry. In contrast, rats infected with PRV-Bartha or PRV-Becker gE mutants survived to at least 96 h and exhibited few overt signs of disease. Despite this long survival and the lack of symptoms, brains of animals sacrificed at this time revealed extensive transsynaptic infection not only of the brain stem but also of areas of the forebrain synaptically linked to neurons in the brain stem. This finding provides evidence that the gE protein plays a role in promoting symptoms of infection and death in animals that is independent of neuron-to-neuron spread during brain infection. When this early virulence function is not active, animals live longer, resulting in more extensive spread of virus in the brain.

In this work we used brefeldin A (BFA), a specific inhibitor of export to the Golgi apparatus, to study pseudorabies virus viral glycoprotein processing and virus egress. BFA had little effect on initial synthesis and cotranslational modification of viral glycoproteins in the endoplasmic reticulum (ER), but it disrupted subsequent glycoprotein maturation and export. Additionally, single-step growth experiments demonstrated that after the addition of BFA, accumulation of infectious virus stopped abruptly. BFA interruption of virus egress was reversible. Electron microscopic analysis of infected cells demonstrated BFA-induced disappearance of the Golgi apparatus accompanied by a dramatic accumulation of enveloped virions between the inner and outer nuclear membranes and also in the ER. Large numbers of envelope-free capsids were also present in the cytoplasm of all samples. In control samples, these capsids were preferentially associated with the forming face of Golgi bodies and acquired a membrane envelope derived from the trans-cisternae. Our results are consistent with a multistep pathway for envelopment of pseudorabies virus that involves initial acquisition of a membrane by budding of capsids through the inner leaf of the nuclear envelope followed by deenvelopment and release of these capsids from the ER into the cytoplasm in proximity to the trans-Golgi. The released capsids then acquire a bilaminar double envelope containing mature viral glycoproteins at the trans-Golgi. The resulting double-membraned virus is transported to the plasma membrane, where membrane fusion releases a mature, enveloped virus particle from the cell.

Pseudorabies virus (PRV) has been used extensively to map synaptic circuits in the CNS and PNS. A fundamental assumption of these studies is that the virus replicates within synaptically linked populations of neurons and does not spread through the extracellular space or by cell- to-cell fusion. In the present analysis we have used electron microscopy to characterize pathways of viral replication and egress that lead to transneuronal infection of neurons, and to document the non-neuronal response to neuronal infection. Three strains of PRV that differ in virulence were used to infect preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV). The data demonstrate that viral replication and transneuronal passage occur in a stepwise fashion that utilizes existing cellular processes, and that the non-neuronal response to infection serves to restrict nonspecific spread of virus by isolating severely infected neurons. Specifically, capsids containing viral DNA replicate in the cell nucleus, traverse the endoplasmic reticulum to gain access to the cytoplasm, and acquire a bilaminar membrane envelope from the trans cisternae of the Golgi. The outer leaf of this envelope fuses with the neuron membrane to release virus adjacent to axon terminals that synapse upon the infected cell. A second fusion event involving the viral envelope and the afferent terminal releases the naked capsid into the bouton. Systematic analysis of serial sections demonstrated that release of virus from infected neurons occurs preferentially at sites of afferent contact. Nonspecific diffusion of virus from even the most severely infected cells is restricted by astrocytes and other non-neuronal elements that are mobilized to the site of viral infectivity. The ability of glia and macrophages to restrict spread of virus from necrotic neurons is the product of (1) temporal differences in the mobilization of these cells to the site of infection, (2) differential susceptibility of these cells to PRV infection, and (3) abortive viral replication in cells that are permissive for infection. The findings provide further insight into the intracellular routes of viral assembly and egress and support the contention that transneuronal spread of virus in the brain results from specific passage of virions through synaptically linked neurons rather than through cell fusion or release of virus into the extracellular space.

THOMAS et al.1 have constructed a derivative of phage λ (λgt·λC) that is useful for cloning DNA fragments 1,000 to 15,000 base pairs long from other organisms. Their cloning procedure involves cutting the phage DNA into three fragments with EcoRI nuclease, removing the central (or “C”) fragment, and annealing the outside fragments with an RI digest of foreign DNA. We have recently introduced three amber mutations—Wam403, Eam1100, and Sam100—into λgt·λC with the object of making the phage less likely to encounter a susceptible host in nature2. The NIH Advisory Committee on Recombinant DNA Research, on the basis of appropriate tests, has certified λgtWES·λC as an EK2 vector3,4.