WO1996038546A1

WO1996038546A1 - Herpes simplex type 1 protease mutants and vectors

Info

Publication number: WO1996038546A1
Application number: PCT/US1996/007795
Authority: WO
Inventors: R. Bruce Register; Jules A. Shafer
Original assignee: Merck & Co., Inc.
Priority date: 1995-06-01
Filing date: 1996-05-28
Publication date: 1996-12-05
Also published as: EP0828823A1; EP0828823A4; CA2222877A1

Abstract

Herpes Simplex Type 1 viruses having mutated protease genes are described, also are methods for introducing a point mutation in the protease gene, vectors used in this process, and host cells transformed with these vectors.

Description

TITLE OF THE INVENTION

HERPES SIMPLEX TYPE 1 PROTEASE MUTANTS AND VECTORS

DESCRIPTION OF THE INVENTION This invention relates to Herpes Simplex Virus type 1

(HSV-1) viruses which contain a mutation in the protease gene, and to vectors and host cells used in producing them.

BACKGROUND OF THE INVENTION The Herpes Simplex Type-1 (HSV-1) virus is a relatively large virus (152,260 bp). While much is known about the viral life cycle and its general activity, it has been difficult to study the relationship between biochemical and biophysiological properties of its gene products and the virus life cycle since its large size makes it difficult to create predetermined point mutations.

HSV-1 protease is a serine protease that has both a structural and enzymatic role in the assembly of the HSV-1 capsid. The protease and infected cell protein 35 (ICP-35) form a complex of approximately 1100 molecules in a ratio of 1 : 10 within the nucleus of the infected cell. Around this complex the capsid proteins assemble into B capsids. After assembly the protease cleaves itself twice and ICP-35 once, releasing the ICP-35 and the carboxyl terminal fragment of the protease from the capsid interior. The 247 amino acid protease remains within the capsid. Concurrently (or subsequently) the genomic HSV-1 DNA is packaged within the capsid.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to HSV-1 viruses which have a mutated protease gene. Preferred mutant viruses of this invention contain altered protease genes which include changes in amino acid sequences of the resulting proteases, and which confer phenotypes which are different from the wild-type virus. A further aspect of this invention are the vectors and sets of vectors used to create the mutant viruses of this invention and host cells which are transformed with these vectors.

The mutant viruses of this invention may also be made by methods which are described in co-pending U.S. Application Serial No. , (Attorney Docket No. 19458) filed herewith, which is hereby incorporated by reference.

The HSV-1 viruses of this invention are preferably made by transforming a host cell with a set of vectors comprising: a first vector comprising a HSV-1 mutated protease gene and overlapping DNA homologous with overlapping DNA of at least one additional vector; and additional vectors, each additional vector comprising a fragment of the substantially complete HSV-1 genome and also comprising overlapping DNA which is homologous with a sequential genomic fragment contained in at least one other additional vector, so that upon co- transfection of a host cell, replication of viral DNA, and recombination of the viral DNA, a virus having a mutated protease gene and which is replicable in a wild type or host range cell line is formed.

Preferably, the viruses of this invention may be made by a process comprising the steps of: (a) obtaining a set of starting vectors, each starting vector comprising a fragment of a substantially complete HSV-1 genome and also comprising DNA which is overlapping DNA with a sequential genomic fragment contained in other starting vectors, so that upon co-transfection of a host cell, replication of viral DNA, and recombination of the viral DNA, a virus is formed which is replicable in a wild type or host range cell line; (b) replacing a starting vector comprising a protease gene which is to be mutated with a first replacement vector, the first replacement vector comprising a mutated protease gene and overlapping DNA, and at least one additional replacement vector comprising genomic DNA which was present in the replaced starting vector, but is not present in the first replacement vector along with overlapping DNA; and (c) co-transfecting a host cell with the replacement vectors and the remaining starting vectors under conditions allowing replication of viral DNA and recombination of viral DNA to form a virus which is replicable in a wild type or host range cell line.

A further aspect of this invention is a set of vectors used to make the mutant viruses of this invention. The set of vectors comprises: a first vector which is a plasmid, comprising a HSV-1 mutated protease gene and overlapping DNA homologous with overlapping DNA of at least one additional vector; and additional vectors, each additional vector comprising a fragment of the substantially complete HSV-1 genome and also comprising overlapping DNA which is homologous with a sequential genomic fragment contained in at least one other additional vector, so that upon co-transfection of a host cell, replication of viral DNA, and recombination of the viral DNA, a virus having a mutated protease and which is replicable in a wild type or host range cell line is formed. The vectors of this invention are preferably made by a process comprising the steps of:

(a) obtaining a set of starting vectors, each starting vector comprising a fragment of the substantially complete HSV-1 genome and also comprising DNA which is overlapping DNA with a sequential genomic fragment contained in other starting vectors, so that upon co-transfection of a host cell, replication of viral DNA, and recombination of the viral DNA, a virus which is replicable in a wild type or host range cell line is formed;

(b) replacing a starting vector comprising a protease gene which is to be mutated with a first replacement vector, the first replacement vector comprising a mutated protease gene and overlapping DNA, and at least one additional replacement vector comprising genomic DNA which was present in the replaced starting vector, but is not present in the first replacement vector along with overlapping DNA; and

(c) co-transfecting a host cell with the replacement vectors and the remaining starting vectors under conditions allowing replication of viral DNA and recombination of viral DNA to form a mutant virus which is replicable in a wild type or host range cell line.

The first replacement vector may be made by a process comprising: (a) creating a vector comprising a protease gene site which is to be mutated and overlapping DNA; (b) defining a first restriction endonuclease site in a position 5' to the protease gene site which is to be mutated; (c) defining a second restriction endonuclease site in a position 3' to the protease gene site which is to be mutated to define a wild-type gene segment contained between the first and second restriction endonuclease sites; (d) creating a mutant protease gene segment substantially identical to the wild-type gene segment, except for comprising a desired mutation; and (e) replacing the wild-type gene segment with the mutant protease gene segment to obtain the first replacement vector. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is the DNA and amino acid sequences

(SEQ.ID.NO.: 1&2) of the HSV-1 (F) protease (Pra) BsmI fragment.

(The 82bρ upstream fragment is not shown).

Figure 2 is a diagram of HSV-1 protease (Pra) cleavage sites. Pra is a 635 amino acid serine protease which undergoes autolytic cleavage at Ala247 and Ala610. Products of this cleavage are shown. Figure 3 is a diagram of the plasmid/cosmid-based mutagenesis process of this invention.

As used in the specification and claims, the following definitions apply:

Null Mutant: an HSV-1 mutant which lacks the ability to grow or form plaques on Vero cells. Overlapping Vectors: two or more vectors, each containing a segment of a DNA which has sufficient common base pairs with the DNA contained in a second vector so that homologous recombination can occur when copies of the DNA are present in a common host.

Replacement Vector: a vector, generally a plasmid which contains a portion of a HSV-1 genomic fragment which was originally present in a starting vector. Generally, a starting vector will be replaced by two replacement vectors: the first one comprising the mutant gene and the second one comprising the remaining genomic DNA which was contained in the starting vector, but not present in the first vector. Additionally, replacement vectors also contain sufficient overlapping DNA so that homologous recombination can occur.

Starting Vector: one of a series of vectors, generally cosmids, which together comprise the substantially complete genome of HSV-1 along with overlapping DNA. Substantially Complete Genome: sufficient DNA is present so that upon transfection of a host cell, replication of the viral DNA and homologous recombination, a replicable HSV-1 virus is formed. This invention specifically envisions: (1) an HSV-1 virus containing a complete genome containing desired mutations and (2) an HSV-1 virus which does not have a complete genome, but the genes which are missing are not essential for virus replication; (3) an HSV-1 virus missing genes which are essential for virus replication, but the missing gene product(s) are complemented by those produced in a host range cell line; and (4) an HSV-1 virus according to 1), 2), or 3) and/or comprises additional DNA, regardless of source, which does not interfere with virus replication; or if replication is interfered with, which can be complemented by a host range cell line.

Replicable Virus: an HSV-1 virus whose genome is neither too short nor too long, so that functional capsid assembly and packaging occurs.

Overlapping DNA: a segment of DNA at least about 300 base pairs in length, more preferably about 2,000 to 5,000 base pairs in length, which is substantially identical to a segment in another vector. The vector generally contains two differing overlapping DNAs, one on the 5' end of the vector and one on the 3' end of the vector, and each overlapping DNA overlaps that of a different vector.

Host Range Cell line: a host cell line which has been transformed to express a viral gene, such as HSV-1 protease. Viruses which do not produce a functional version of this gene are able to utilize the protein produced by the transformed cell line.

One aspect of this invention is a convenient system which allows researchers to study the protease gene in the context of the virus, and to create any desired mutation(s) within the protease gene. The starting point for the method according to this invention is a set of vectors, such as cosmids. The total number of vectors in the set is not critical, but together the set of vectors contains a substantially complete HSV-1 genome. In general, the total number of vectors in the set should not be so large that it becomes cumbersome to co-transfect the host cell. Preferably the number of vectors in a set should be less than ten, and preferably, less than about eight, and most preferably about six. One or more of these vectors are replaced by one or more replacement vectors, each replacement vector containing a smaller HSV-1 DNA insert than in the starting vector, but together the replacement vectors contain the "equivalent amount" of unique, non- overlapping HSV-1 genomic DNA as was present in the starting vector. ("Equivalent amount" as used in this content means substantially the same amount, plus or minus any DNA which was intentionally added or deleted as mutations). If the complete protease gene which is to be mutated is contained within one starting vector, then only this single vector needs to be replaced. If, however, the protease gene which is to be mutated is contained on two starting vectors (i.e., each starting vector containing only a fragment of the protease gene), then the two starting vectors should be replaced. Replacement vectors make up one aspect of this invention. The first replacement vector may be a cosmid or a plasmid; plasmids are generally preferred. The vector may be any vector which is able to replicate in the host cell system. Any host cell may be utilized, but for general convenience, E. coli is preferred. The first replacement vector comprises a copy of the protease gene which is to be mutated along with a sufficient amount of overlapping DNA so that homologous recombination can occur. While homologous recombination can occur with a few base pairs (i.e., less than 20), it is preferred that at least about 300 base pairs of overlapping DNA be present, and even more preferred that at least about 2,000 to about 5,000 be present. It is preferred that overlapping DNA be overlapping with DNA of at least one vector, and it is preferred that it overlaps DNA of two vectors. Additional replacement vectors of this invention contain the remaining genes and/or gene fragments which were originally in the starting vector, along with overlapping DNA. Next, two restrictions sites should be defined in the replacement vector containing the protease gene to be mutated. These restriction sites, which may be naturally occurring or may be inserted as desired using known techniques, define a protease gene fragment which is to replaced by a newly synthesized mutated protease gene fragment. The first restriction site may be anywhere upstream of the position where the mutation or mutations are to be introduced. In a preferred embodiment, it is upstream of the initiation ATG site of the protease gene. The second restriction site may be anywhere within the protease gene, or even downstream of the gene, as long as it is downstream of the site where desired mutation or mutations are to be made. It is also desirable to choose a position for the second restriction site which is close enough to the first restriction site so that with currently available technology, the mutated gene fragment may be easily synthesized and sequenced as needed. Thus, the second restriction site is generally less than about 2,000 bp downstream of the first restriction site, and preferably less than about 1 ,100 bp downstream of the first restriction site.

The restriction sites may be the recognition sites for virtually any restriction endonuclease. It is preferred, however, that each site be unique. In order to ensure that the mutated gene fragment is cloned into the restriction sites having the correct orientation (i.e., can be "force-cloned"), it is particularly preferred that the enzyme recognizes different base pair sequences, and that the first restriction site and the second restriction site be differing base pair sequences, although recognized by the same enzyme. Numerous enzymes are known to have this characteristic, including Bsml.

The second replacement vector according to this invention comprises any viral DNA which was originally encoded in the first starting vector, but is not present in the first replacement vector, along with sufficient overlapping sequences so that homologous recombination can occur.

The remaining vectors in the series of vectors according to this invention may be any vectors, such that when the complete set of vectors is co-transfected into host cells, they are able to recombine to form a mutated virus which is replicable in a wild type or host range cell line.

In a preferred embodiment of this invention, a set of starting vectors to be used are the five cosmids: cos28, cosό, cos 14, cos48, and cos56, which were obtained from Dr. Andrew J. Davison. These cosmids and/or their equivalents can be made according to the description given in Cunningham and Davison Virology 797:116-124 (1993), which is hereby incorporated by reference. One of the cosmids of the Cunningham and Davison system, cosmid cos28, contains DNA encoding the protease and its substrate (the assembly protein ICP-35) on the overlapping genes (UL26 and UL26.5). This cosmid is replaced by two novel overlapping replacement vectors, both of which are further aspects of this invention. This is diagrammed in Figure 3B.

The first replacement vector should carry a copy of the HSV-1 protease gene which has at least two restriction sites that have been defined, according to the considerations mentioned above. One preferrred restriction enzyme is Bsml, a degenerate restriction endonuclease with a recognition sequence of GAATGΛCN^Λ (SEQ.ID.NO.:3).

In a preferred embodiment of this invention, the first replacement vector is plasmid pR700 (or a plasmid carrying the same inserts as pR700). Plasmid pR700 was made from the commercially available plasmid pGEM-4Z (Promega Corp, Madison, WT), and contains the UL26 protease gene in a 13.3 kb insert of HSV-1 (base pairs 44440-57747). Plasmid pR700 also contains two naturally occurring Bsml sites, a first one 82 base pairs 5'- of the HSV-1 protease start site and one at amino acid 348 of the protease. The "N" at the 5' Bsml site is "T" whereas at the 3' Bsml site, the "N" is "G", so that the mutant PCR fragments may be force-cloned into the vector. PCR mutagenesis of this 1.1 kb Bsml fragment was used to introduce various desired mutations into the HSV-1 protease gene fragment.

A second replacement vector according to this invention is plasmid pR710 (or a plasmid carrying the same inserts as pR710) which is derived from commercially available plasmid pNEB93 (New England Biolabs, Beverly, MA). Plasmid pR710 contains a 24.7 kb insert of HSV-1 (base pairs 24699-49435) that does not include the HSV-1 protease. Thus, a further aspect of this invention is a set of vectors comprising at least one vector selected from the group consisting of cos48, cos6, cosl4, cos56, and pR710 and at least one additional plasmid. Preferably the additional plasmid carries substantially the same insert as a plasmid selected from the group consisting of: pR700, pR711, pR712, pR713, pR714, pR715, pR716, pR717, pR718, pR725, pR728, pR729, and pR730. Preferred plasmids are selected from the group consisting of: pR700, pR711, pR712, pR713, pR714, pR715, pR716, pR717, pR718, pR725, pR728, pR729 and pR730. In this preferred embodiment, the two replacement plasmids and four remaining starting vectors, which together make up a further aspect of this invention, are introduced into HSV-1 host cells. The HSV-1 host cell chosen is generally not a critical aspect of this invention. Generally, any cell in which HSV-1 can replicate is an appropriate host cell. Particularly preferred host cells are Vero cells. DNA which is replicated during the virus life cycle homologously recombines in the host cells to create the mutant HSV-1 viruses of this invention. This is illustrated in Figure 3C.

The above-described mutagenesis method allows one to make the desired HSV-1 protease mutations in the virus in a short period of time, i.e., within about 2 weeks. It has the further advantage that pure mutant virus cultures are generated; there are no wild type background viruses in the transfections of Vero cells.

In creating the mutant protease gene fragments of this invention, virtually any known method of synthesizing and mutating DNA may be used. PCR mutagenesis is a preferred method. In performing the PCR mutagenesis of the target DNA, standard PCR techniques may be used in general, such as those described in H. Russell, 1990, "Recombinant PCR" in PCR Protocols (Innis, et al, Eds.), Academic Press, Inc. San Diego, CA, pages 177-183, which is hereby incoφorated by reference. However, since HSV-1 DNA is quite GC rich and if the region which is to be mutated is also high in GC content (as is the case with the protease gene) it is preferred that a higher than usual melting temperature be employed during the PCR cycle, preferably at least about 99°C to maximize product formation. A second consideration with PCR mutagenesis in general is maintaining fidelity. While any suitable polymerase enzyme may be employed, VentR DNA polymerase (commercially available from New England Biolabs) is a preferred polymerase for the PCR reactions used herein because of its proofreading ability and thermal stability at 99°C.

Virtually any mutation which is desired may be introduced into the protease gene using the PCR mutagenesis method. For instance, in order to obtain viruses which have altered phenotypes, it is desirable to change an amino acid sequence. Further type of mutations which are preferred are those which introduce new restriction endonuclease recognition sites.

In order to demonstrate the versatility of the mutation procedure of this invention, the following mutant viruses were made. Throughout the specification and claims, the virus nomenclature is the same as that used for the replacement plasmid containing the mutation, except that the virus uses the prefix "V" and the replacement plasmid uses "pR".

TABLE 1

Representative protease mutants

VIRUS MUTATION ADDED SITE*

V711 His61 to Val61 Aatll

V730 His61 to Ala61 Pspl406I

V715 His61 to Tyrol none

V717 Leul25 to Vall25 BsaAI

V718 Prol26 to Glyl26 BstXI

V713 Serl29 to Alal29 Nhel

V714 Serl29 to Alal29 none

V712 Hisl48 to Alal48 PstI

V716 Hisl48 to Tyrl48 none

V725 Hisl48 to Argl48 Mull

V728 Hisl48 to Glul48 Eco47m

V732** Alal29 to Serl29 Hindffl

V729 Hisl48 to Lysl48 Styl

* restriction endonuclease site **back-mutation of V713

The active site serine of HSV-1 protease has been previously identified by chemical mutations methods to be Ser 129. Therefore, changes of amino acids at the active serine site and near the active serine site were of particular interest.

Mutations At Ser 129:

A mutation was made in HSV-1 protease gene to change the protease amino acid Ser 129 to Ala 129. This vims is designated V713, and is a further aspect of this invention. Recombinant vims could only be rescued on a host range cell line (PHS-23) which expresses protease. When V713 was used to super-infect Vero cells, the Western analysis showed an accumulation of the 80 kD protease (Pra) along with several other peptides ranging in molecular wieght from 29 kD to 75 kD. A 24 kD band seen in wild-type infections was absent.

Mutation At Leu 125 V717 contains a mutation of Leu 125 to Val 125. This vims did not grow on Vero cells at 31°, 34°, 37°, or 39°C, and showed by western blot analysis no protease activity at 20 hours after infection. A light 27 kd No protease band was observed in the western analysis. This band may reflect protease formed via recombination or carried over from the host range cell line PHS-23 during propagation of the vims.

Mutations At Pro 126

V718 contains a mutation of Prol26 to Glyl26. This vims did not grow on Vero cells at 31°, 34°, 37°, or 39°C, but after 20 hours, substantial processing of the 80 kd protease (Pra) occurred. However, even extended incubation for 7 days failed to produce plaques. The inability of the vims to replicate may reflect a requirement for proper structural assembly of the capsid. While not wishing to be bound by theory, this may result from the protease activity not being properly synchronized with the replication cycle, i.e., the protease may be cutting itself in the cytoplasm, or that the protease activity observed in this mutant is insufficient to digest all of the assembly protein within the capsid. If so, then the intact ICP-35 protein that is retained within the capsid may block DNA packaging.

Mutations At His 148

Mutations which changed the histidine at position 148 were mixed. Changing this amino acid to Ala (V712) resulted in a small plaque phenotype and Western analysis showed a reduction in protease activity. This result was unexpected because in the prior art, where the protease gene having the same mutation,but not contained within the vims showed no protease activity in in vitro assays. (Liu et al, 1992 Proc. Natl. Acad, Sci. USA 59:2076-2080 and Deckman et al, 1992 J. Virology 66:7362-7367 , both of which are incoφorated by reference.) While not wishing to be bound by theory, this suφrising result may be due to a difference in the three dimensional structure of the protein within the vims environment, or the presence of a hitherto unknown accessory protein which lends activity to the protease. Vimses V716 (Hisl48 to Tyrl48) V725 (Hisl48 to

Arg 148) V729 (His 148 to Lys 148) were not viable on Vero cells, but each exhibited a different level of protease activity. V729 showed no protease activity by Western blot analysis; V716 had greater than 50% protease activity, and V725 exhibited wild-type activity against Pra, but did not process ICP-35.

Mutations At His 61

Three mutations at His61 to Val61 (V71 1), Tyr61 (V715), and Ala61 (V730) all created null mutant vimses and in Western analysis had the same extra bands as the V713 mutant.

Taken with the observations of the His 148 mutations, the results suggest that His61 is required for protease activity whereas His 148 is not.

The following non-limiting Examples are presented to better illustrate the invention.

EXAMPLES

GENERAL METHODS

Viral Strains

Two strains of vimses were used, HSV-1 strain 17 [designated HSV-1(17)] and HSV-1 strain F [designated HSV-l(F)]. Mutations to the protease have been made in HSV-1 (F) (see Liu, F. et al, 1991 , J. Virol. 65:5149-5156, hereby incoφorated by reference) and temperature sensitive mutants have been isolated in HSV-1(17). (See Preston, V. et al, 1983, J. Virol. 45:1056-1064, hereby incoφorated by reference). Sequence analysis of the Bsml fragment revealed that the two strains differ by two amino acids (Leu300/Ser300 and Ser301/Pro301) and six silent mutations (in Prol5, Arg46, Gly84, Gln90, Gly 199 and His341). To make an equivalent comparison of in vitro and in vivo studies, a protease chimera (pR731) was made. Plasmid pRHS2, containing the HSV-l(F) protease was digested with Bsml and the l.lkb fragment was cloned into pR700 containing HSV- 1(17) protease. Both vimses were equivalent in vims titer and plaque moφhology on Vero cells.

PCR mutagenesis Four oligonucleotides and a DNA template were amplified in two rounds of PCR to create a variety of mutated DNA fragments which were subsequently cloned into plasmid pR700 and used to create the mutant vimses. The first round of PCR mutagenesis was carried out in two separate reactions. In one reaction, a positive strand oligonucleotide homologous to the DNA 5' to the first Bsml site, was paired with the negative strand oligonucleotide specified below. In the other reaction, a negative strand oligonucleotide homologous to the DNA 3' to the second Bsml site was paired with the positive strand oligonucleotide specified. The two specified oligonucleotides are complementary to each other, mutate the same amino acid residue, and most, but not all, concurrently introduce a new endonuclease restriction site. The specified DNA template (from pR700, pRHS2, or V713, below) was added to both reaction mixtures and PCR amplification initiated. In the second round of the procedure, the DNA fragments generated by the first round PCR reactions were gel purified and mixed together with oligonucleotides flanking the Bsml sites (SEQ.ID.NOS:4 and 5, below), and subjected to PCR amplification.

PCR mutagenesis was performed with VentR DNA polymerase (New England Biolabs) in a DNA thermal cycler from Perkin Elmer Cetus. The cycle was melt for 1 minute at 99°C; anneal at 40°C for two minutes; extend at 71°C for 3 minutes; for 30 cycles. The product of the second round PCR reaction and extended Bsml fragment, was digested with Bsml, gel purified and ligated into the Bsml sites of pR700. Oligonucleotides used for mutagenesis: Unless otherwise indicated, all oligos were from Midland Certified Reagent Co., Midland, TX. (In each pair, the plus stand oligo is listed first):

5' and 3' oligonucleotides flanking the two Bsml cloning sites:

5'-GTACTCAAAAGGTCATAC-3' (SEQ.ID.NO.:4) (This oligo is 5' to the first Bsml site and was used for the generation of all mutations in the protease from amino acid 1 to 348).

5'-GGGAAACCAAACGCGGAATG-3' (SEQ.ID.NO.:5) (This oligo is 3' to the second Bsml site and was used in generation of mutations in the protease from amino acids 1 to 348.)

Oligonucleotides for the temperature sensitive protease mutant pR701: 5'-GATACGGTGCGGGCAGTACTGCCTCCGGAT-3' (SEQ.ID.NO.:6) 5'-ATCCGGAGGCAGTACTGCCCGCACCGTATC-3' (SEQ.ID.NO.:7)

These oligos add a Sad site to the Ala48 to Val48 mutation.

Oligonucleotides for the temperature sensitive protease mutant pR701 : 5'_TTTTTGGCGCTCTTCGACAGCGGGGAC-3' (SEQ.ID.NO.:8) 5'-GTCCCCGCTGTCGAAGAGCGCCAAAAA-3' (SEQ.ID.NO.:9) These oligos add a Sapl site at the Thr30 to Phe30 mutation.

Linker oligonucleotides (BspHI-PacI-Hindlll) for pR710: 5'-CATGATTAATTA-3' (SEQ.ID.NO.:10) 5'-AGCTTAATTAAT-3' (SEQ.ID.NO.:l 1)

Oligonucleotides used for the His61 to Val61 mutation for pR711: 5'-CCCACTCCCGATTAACGTGGACGTCCGCGCTGGCTGCGAGG- TG-3' (SEQ.ID.NO.: 12)

5'-CCTCGCAGCCAGCGCGGACGTCCACGTTAATCGGGAGT- GGG-3' (SEQ.ID.NO.:13) This also adds an Aatπ restriction site. Oligonucleotides used for the His 148 to Ala 148 mutation for pR712: 5'-CCCCGATCGCACGCTGTTCGCTGCAGTCGCGCTGTGCGCGA- TCGGGCGG-3' (SEQ.ID.NO.:14)

5'-GATCGCGCACAGCGCGACTGCAGCGAACAGCGTGCGATC- GGGG (SEQ.ID.NO.:15)

This also adds a PstI restriction site.

Oligonucleotides used for the Ser 129 to Ala 129 mutation for pR713: 5'-CACCAACTACCTGCCCTCGGTCGCGCTAGCCACAAAACGCC- TGGGGGG-3' (SEQJD.NO.:16)

5^*-CAGGCGTTTTGTGGCTAGCGCGACCGAGGGCAGGTAG-

TTG-3'(SEQ.ID.NO.:17)

This also adds a Nhel restriction site.

Oligonucleotides used for the Serl29 to Alal29 mutation for pR714:

5'-CCAACTACCTGCCCTCGGTCGCCCTGGCCACAAAACGCCTG-

GGG-3' (SEQ.ID.NO.:18)

5'-GCCAGGGCGACCGAGGG-3^* (SEQ,ID.NO.:19) Oligonucleotides used for the His61 to Tyrol mutation for pR715: 5'-CCCACTCCCGATTAACGTGGACTACCGCGCTGGCTGCGAGG-

TG-3' (SEQ.ID.NO.:20)

5'-CGCGGTAGTCCACGTTA-3' (SEQ.ID.NO.:21)

Oligonucleotides used for the His 148 to Tyrl48 mutation for pR716: 5'-CCCCGATCGCACGCTGTTCGCGTACGTCGCGCTGTGCGCGA- TCGG-3' (SEQ.ID.NO.:22) 5'-GCGACGTACGCGAACAGC-3' (SEQ.ID.NO.:23)

Oligonucleotides used for the Leu 125 to Val 125 mutation for pR717: 5'-CACCAACTACGTGCCCTCGGTCTCCCTG-3' (SEQ.ID.NO.:24) 5'-CCGAGGGCACGTAGTTGGTGATCAGG-3' (SEQ.ID.NO.:25) This also adds a BsaAI restriction site. Oligonucleotides used for the Pro 126 to Gly 126 mutation for pR718: 5'-CAACTACCTGGGCTCGGTCTCCCTGGCC-3' (SEQ.ID.NO.:26) 5'-GAGACCGAGCCCAGGTAGTTGGTGATCAG-3' (SEQ.ID.NO.:27) This also adds a BstXI restriction site

Oligonucleotides used for the His 148 to Arg 148 mutation for pR725: 5'-CGCTGTTCGCACGCGTCGCGCTGTGCGCGATCG-3' (SEQ.ID.NO.:28) 5'-CAGCGCGACGCGTGCGAACAGCGTGCGATCGGG-3' (SEQ.ID.NO.:29) This also adds a Mull restriction site.

Oligonucleotides used for the His 148 to Glul48 mutation for pR728: 5'-CTGTTCGCGGAAGTAGCGCTGTGCGCGATCGG-3'

(SEQ.ID.NO.:30)

5'-CGCACAGCGCTACTTCCGCGAACAGCGTGCGATCGGG-3'

(SEQ.ID.NO.:31)

This also adds a Eco47Iϋ restriction site.

Oligonucleotides used for the His 148 to Lys 148 mutation for pR729:

5'-CGCTGTTCGCCAAGGTCGCGCTGTGCGCGATCG-3'

(SEQ.ID.NO.:32)

5'-CACAGCGCGACCTTGGCGAACAGCGTGCGATCGGG-3' (SEQ.ID.NO.:33)

This also adds a Styl restriction site.

Oligonucleotides used for the His61 to Ala61 mutation for pR730: 5'-CCGATTAACGTTGACGCCCGCGCTGGCTGCGAGGTGGG-3' (SEQ,ID.NO.:34)

5'-CAGCCAGCGCGGGCGTCAACGTTAATCGGGAGTGGG-3'

(SEQ.ID.NO.:35)

This also adds a Psp 14061 restriction site. Oligonucleotides used for the Alal29 to Serl29 back mutation for pR732 :

5'-CCTGCCCTCGGTAAGCTTGGCCACAAAACGCCTGG-3' (SEQ.ID.NO.:36) 5'-GGCGTTTTGTGGCCAAGCTTACCGAGGGCAGGTAG-3' (SEQ.ID.NO.:37) This also adds a Hindlll restriction site.

Constmcts: Plasmids derived from HSV-1 (F) : pRHSl: This plasmid contains HSV-l(F) DNA base pairs 44590-54473, starting within the UL22 gene and ending within UL28. This was made by digesting HSV-1 (F) DNA with Xbal and Seal. The 9884 base pair fragment was gel purified and subcloned into pGEM-7Zf(-) (Promega) at the Xbal and Smal sites. pRHS2: This plasmid contains HS V- 1 (F) DNA base pairs 49126-53272, starting within UL25 and ending within UL27. To prepare this plasmid, pRHSl was digested with NotI and Nhel, and the 4148 base pair fragment was subcloned into the pGEM-7Zf(-) vector at the Bspl20I and Xbal sites. This clone was used for the creation of the host range cell line PHS23, and plasmids pR711, pR712, pR713, pR714, pR715, pR716, pR725, pR728, pR729 and pR730. pR731: pRHS-2 was digested with Bsml, and the 1.1 kb fragment was then subcloned into the Bsml sites of pR700. This created a F strain protease in the 17 strain vims. pR732: V713 vims DNA was digested with NotI and the 6.5 kb fragment containing the HSV-1 protease was gel purified. This fragment was used as a template for PCR to back-mutate the Ser 129 to Ala 129 back to Ser. The back mutation also created a new Hindlll site. pR732 exhibited a wild-type phenotype. The back mutation was performed to demonstrate that the mutant phenotypes observed for the various mutants of this invention were due to the mutagenesis process, and were not artifacts of the transfection procedure. Plasmids derived from HSV-1 (17): pR700: This plasmid contains HS V- 1 ( 17) DNA base pairs 44440- 57747, starting within UL22 and ending within UL28 to prepare HSV-1 cos-28 was digested with StuI and Ndel, the 13,308 base pair fragment was gel purified and ligated into pGEM-4Z (Promega) at the Ndel and Smal sites. This plasmid was use for both generation and sub-cloning of mutants pR701 , pR717 and pR718 into the Bsml sites. ρR710: This plasmid contains HS V- 1(17) DNA base pairs 24699- 49435, starting between UL10/UL11 and ending within UL25. Cos 28 was digested with Pad and BspHI and the resulting 24,736 bp fragment was subcloned with the two linker oligos (SEQ.ID.NOS. 10 & 11) containing BspHI-PacI-Hindlll into the PacI/HindUI sites of New England Biolabs vector pNEB93. pR701: HSV-1 temperature sensitive mutant was created from pR700 by PCR mutagenesis. It has a Thr30 to Phe30 mutation which contains a Sapl site and an Ala48 to Val48 mutation a containing a new Seal site.

Sequencing

Sequencing reactions were done using a Sequenase® Quick Denature Plasmid Sequencing kit (United States Biochemical) according to the manufacturer's instructions. S-35 dATP was obtained from Amersham.

Host Range Cell Line PHS-23 (Expressing Protease). pRHS2 was co-transfected with pSVNeo (Southern et al,

1982. J. Mol. Appl. Gen. 1 :327-341) into Vero cells and cultured in 800 μg/ml of G418 sulphate (GIBCO). Drug resistant cell lines were screened for the ability to complement the temperature sensitive protease vims, V701, at 39°C.

Digests

Prior to transfection, cosmid DNA and pR710 were digested with Pad. Plasmids pR700, pR701, pR71 1 , pR712, pR713, pR714, pR715, pR716, pR717, pR718, pR725, pR728, pR729, pR730, and pR731 were digested with Hindm and Ndel, while pR732 was digested with Xbal. The digested DNA was precipitated in 2M final NH4OAC pH 7.5, and 2 volumes of isopropanol, centrifuged 10 minutes then washed in 70% ethanol and dried. The DNA was re-suspended in 10 mM Tris, 1 mM EDTA pH 7.8. Restriction endonucleases were purchased from New England Biolabs and Promega (Madison, WI).

Western Blots

12% SDS-PAGE gels were transferred to Immobilon-P (Milhpore, Bedford, MA) and blocked in phosphate buffered saline, 2% bovine fetal calf serum (FCS) (Hyclone Laboratories, Logan, UT), 2% nonfat dry milk, and 0.1 % Tween-20. A peptide made to correspond to the N-terminus of the protease, DAPGDRMEEPLPDRAC-NH2 (SEQ.ID.NO.:38), was conjugated to keyhole limpet hemocyanin, and was used to generate a polyclonal rabbit antibody (Multiple Peptide Systems, San Diego, CA). The second antibody was Goat Anti-Rabbit IgG (H+L) alkaline phosphatase conjugate (Bio-Rad, Hercules, CA). Western blots were developed with an alkaline phosphatase conjugate substrate kit from Bio Rad or with a ECL kit from Amersham.

Southern Blots

Viral DNA was digested with the restriction endonuclease corresponding to those sites which were added at the site of mutation. Agarose gels were transferred to Zeta Probe (Millipore) in 0.4M NaOH, and hybridized at room temperature with P-32 kinased oligonucleotides (below) in 5 X SSC, 20 mM Na2HPθ4 pH 7.2, 7% SDS, 1 X Denhardts and 100 μg/ml herring sperm DNA, for two hours, then washed with 5X SSC at 50°C for four changes at 15 minutes. Oligonucleotides used to probe Southerns: SH-2 5'-CAGCGCTGGGATTTTCG-3' (SEQ.ID.NO.-.39) SH-10 5'-GTTAACAACATGATGCTG-3' (SEQ,ID.NO.:40) Transfections

Vero or PHS-23 cells were plated at 3 x 10^ cells per well in six well clusters the day before transfection. The following day the cells were washed in Delbeco's Modified Eagles Medium (DMEM) (from GIBCO, Gaithersburg, MD) without FCS and then 1 ml of transfection cocktail was added. Transfection cocktail was made as follows. To 100 μl of DMEM media, 0.5 μg of digested DNA was added, followed by 14 μl of LiptofectAMINETM. (GIBCO) This transfection mixture was incubated for 30 minutes at room temperature, then 900 μl of DMEM was added. The cells (90% confluent) were washed twice with DMEM without FCS and then the one ml of transfection mixture was added. The transfection was incubated for 18 hours at 37°C, 5% C02. Transfected cells were then washed and fresh media, DMEM, 4% FCS, 100 units/ml penicillin and 100 μg/ml streptomycin, was added. At day six or seven the recombinant vims were harvested and the vims was plaque purified.

Plaque Purification.

After transfection with LipofectAMINETM Reagent (GIBCO/BRL) the cells were scraped off the plates and were either frozen and thawed three times, or sonicated. Serial dilutions 1 :10, 1 :100, 1 :1000, 1 :10,000, 1 : 100,000, and 1 :1 ,000,000 were done in DMEM. Cells in six well clusters were incubated with 0.5 ml of each dilution and were rocked every 15 minutes for 2 hours. The cells were then over-layed with 0.5% agarose, DMEM without phenol red, and 10% FCS and incubated at 37 °C in 5% CO2 for three to five days. Plaques were picked with a cotton-plugged sterile Pasteur pipette by piercing the agarose and lifting a plug containing the recombinant vims. The plug was placed in a sterile eppendorf tube containing 0.5 ml of DMEM and 20% FCS. The plaque was sonicated and then repuified as described. Recombinant vims expansion

After plaque purification the vims was expanded on Vero cells, or if the the mutant was a null mutant, it was expanded on the host range cell line PHS-23.

Vims titers

Expanded vims stocks were titered on Vero and PHS-23 cells. Serial dilutions 1 : 10, 1 :100, 1 :1000, 1 :10,000, 1 :100,000, and

1 :1,000,000 were done in DMEM. Six well clusters were then infected with 0.5 ml of each dilution, rocked every 15 minutes, and adsorbed for

2 hours at 37°C. The cells were then fed with DMEM, 4% FCS and

0.16% human IgG (Armour, Kankakee, IL). Two to six days later the cells were fixed in 1 ml of methanol for 7 minutes and then air dryed.

Fixed cells were stained with Gemsa stain for 45 minutes, washed with water and dryed. The plaques were then counted under a microscope.

Vims DNA Mini Preps

Mini preps of vims DNA were made as follows: a T-225 flask of vero or PHS-23 cells was infected at a MOI of 5 and harvested at 18 hours post infection. Cells were pelleted and then washed in PBS three times. The cells were re-suspended in 400 μl lOmM Tris pH 8.0, 5mM NaCl, 5mM EDTA and incubated on ice for 10 minutes. NP-40 was added to a final concentration of 1 % and incubated for ten minutes on ice. The nuclei were pelleted at 10,000 x g for 15 minutes. The resulting supernatant solution was incubated with proteinase K

(Boehringer Mannheim, Indianopolis, IN) 100 μg/ml at 37°C overnight. DNA was extracted twice with phenol and once with chloroform and precipitated

EXAMPLE 2

In order to test the mutagenesis process of this invention, the temperature sensitive mutation described by Preston et al, 1983 J. Virol. 45: 1056-1064, which is hereby incoφorated by reference, was made. This required the introduction of two amino acid changes. Four separate transfections were done. Vero or host range cell line PHS-23 were plated at approximately 3.0 x 10^ cells per well in six well clusters. This resulted in a cell density of 80-90% the following day. The cells were washed in DMEM without FCS just prior to transfection. The transfection mix was then added to the cells and incubated for 18 hours at 37°C. The cells were washed IX and 3 ml of DMEM with 4% FCS was added. At day six or seven, plaques were observed and recombinant vims was harvested. Each transfection gave rise to 50 or more plaques five to six days post transfection. Recombinant vims was titered on both Vero and PHS-23 cell lines. A minimum of four plaques were picked per transfection, all of the isolates grew and plaqued similarly. Table 2 shows the titer of both wild type HSV-1 (17) from one of the temperature sensitive (ts) mutant isolates on Vero and PHS-23 cells at 31⁰C and 39°C.

Table 2. HSV-1 Temperature Sensitive Protease Mutants V701 and HSV-1 titer on Host Range And Vero Cells

Host Cell Line Vims Temp°C Titer (pfu)

Vero V701 31 6.2 x 105

Vero V701 39 1.2 x 102

Vero HSV-1(17) 31 1.0 x 107

Vero HSV-1(17) 39 2.1 x 107

PHS-23 V701 31 1.3 x lθ6

PHS-23 V701 39 1.0 x 106

PHS-23 HSV-K17) 31 2.5 x 107

PHS-23 HSV-1(17) 39 1.0 x 107 SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: MERCK & CO. , INC.

Register, Robert B. Shafer, Jules A.

(ii) TITLE OF INVENTION: HERPES SIMPLEX TYPE 1 PROTEASE MUTANTS AND VECTORS

(iii) NUMBER OF SEQUENCES: 40

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Ms. Joanne M. Giesser

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(D) SOFTWARE: PatentIn Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Giesser, Joanne M.

(B) REGISTRATION NUMBER: 32,838

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(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (908) 594-3046

(B) TELEFAX: (908) 594-4720

(2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1050 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO [ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1050

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATG GCA GCC GAT GCC CCG GGA GAC CGG ATG GAG GAG CCC CTG CCA GAC 48 Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp 1 5 10 15

AGG GCC GTG CCC ATT TAC GTG GCT GGG TTT TTG GCC CTG TAT GAC AGC 96 Arg Ala Val Pro lie Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser 20 25 30

GGG GAC TCG GGC GAG TTG GCA TTG GAT CCG GAT ACG GTG CGT GCG GCC 144 Gly Asp Ser Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala 35 40 45

CTG CCT CCG GAT AAC CCA CTC CCG ATT AAC GTG GAC CAC CGC GCT GGC 192 Leu Pro Pro Asp Asn Pro Leu Pro lie Asn Val Asp His Arg Ala Gly 50 55 60

TGC GAG GTG GGG CGG GTG CTG GCC GTG GTC GAC GAC CCC CGC GGG CCG 240 Cys Glu Val Gly Arg Val Leu Ala Val Val Asp Asp Pro Arg Gly Pro 65 70 75 80

TTT TTT GTG GGA CTG ATC GCC TGC GTG CAA CTG GAG CGC GTC CTC GAG 288 Phe Phe Val Gly Leu lie Ala Cys Val Gin Leu Glu Arg Val Leu Glu 85 90 95

ACG GCC GCC AGC GCT GCG ATT TTC GAG CGC CGC GGG CCG CCG CTC TCC 336 Thr Ala Ala Ser Ala Ala lie Phe Glu Arg Arg Gly Pro Pro Leu Ser 100 105 110

CGG GAG GAG CGC CTG TTG TAC CTG ATC ACC AAC TAC CTG CCC TCG GTC 384 Arg Glu Glu Arg Leu Leu Tyr Leu lie Thr Asn Tyr Leu Pro Ser Val 115 120 125

TCC CTG GCC ACA AAA CGC CTG GGG GGC GAG GCG CAC CCC GAT CGC ACG 432 Ser Leu Ala Thr Lys Arg Leu Gly Gly Glu Ala His Pro Asp Arg Thr 130 135 140

CTG TTC GCG CAC GTC GCG CTG TGC GCG ATC GGG CGG CGC CTC GGC ACT 480 Leu Phe Ala His Val Ala Leu Cys Ala lie Gly Arg Arg Leu Gly Thr 145 150 155 160

ATC GTC ACC TAC GAC ACC GGT CTC GAC GCC GCC ATC GCG CCC TTT CGC 528 lie Val Thr Tyr Asp Thr Gly Leu Asp Ala Ala lie Ala Pro Phe Arg 165 170 175

CAC CTG TCG CCG GCG TCT CGC GAG GGG GCG CGG CGA CTG GCC GCC GAG 576 His Leu Ser Pro Ala Ser Arg Glu Gly Ala Arg Arg Leu Ala Ala Glu 180 185 190 GCC GAG CTC GCG CTG TCC GGA CGC ACC TGG GCG CCC GGC GTG GAG GCG 624

Ala Glu Leu Ala Leu Ser Gly Arg Thr Trp Ala Pro Gly Val Glu Ala

195 200 205

CTG ACC CAC ACG CTG CTT TCC ACC GCC GTT AAC AAC ATG ATG CTG CGG 672

Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg 210 215 220

GAC CGC TGG AGC CTG GTG GCC GAG CGG CGG CGG CAG GCC GGG ATC GCC 720

Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gin Ala Gly lie Ala 225 230 235 240

GGA CAC ACC TAC CTC CAG GCG AGC GAA AAA TTC AAA ATG TGG GGG GCG 768

Gly His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys Met Trp Gly Ala 245 250 255

GAG CCT GTT TCC GCG CCG GCG CGC GGG TAT AAG AAC GGG GCC CCG GAG 816

Glu Pro Val Ser Ala Pro Ala Arg Gly Tyr Lys Asn Gly Ala Pro Glu

260 265 270

TCC ACG GAC ATA CCG CCC GGC TCG ATC GCT GCC GCG CCG CAG GGT GAC 864

Ser Thr Asp lie Pro Pro Gly Ser lie Ala Ala Ala Pro Gin Gly Asp

275 280 285

CGG TGC CCA ATC GTC CGT CAG CGC GGG GTC GCC TCG CCC CCG GTA CTG 912

Arg Cys Pro lie Val Arg Gin Arg Gly Val Ala Ser Pro Pro Val Leu 290 295 300

CCC CCC ATG AAC CCC GTT CCG ACA TCG GGC ACC CCG GCC CCC GCG CCG 960

Pro Pro Met Asn Pro Val Pro Thr Ser Gly Thr Pro Ala Pro Ala Pro 305 310 315 320

CCC GGC GAC GGG AGC TAC CTG TGG ATC CCG GCC TCC CAT TAC AAC CAG 1008

Pro Gly Asp Gly Ser Tyr Leu Trp lie Pro Ala Ser His Tyr Asn Gin 325 330 335

CTC GTC GCC GGC CAC GCC GCG CCC CAA CCC CAG CCG CAT TCC 1050

Leu Val Ala Gly His Ala Ala Pro Gin Pro Gin Pro His Ser

340 345 350

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 350 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :

Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp 1 5 10 15 Arg Ala Val Pro lie Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser 20 25 30

Gly Asp Ser Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala 35 40 45

Leu Pro Pro Asp Asn Pro Leu Pro lie Asn Val Asp His Arg Ala Gly 50 55 60

Cys Glu Val Gly Arg Val Leu Ala Val Val Asp Asp Pro Arg Gly Pro 65 70 75 80

Phe Phe Val Gly Leu He Ala Cys Val Gin Leu Glu Arg Val Leu Glu 85 90 95

Thr Ala Ala Ser Ala Ala He Phe Glu Arg Arg Gly Pro Pro Leu Ser

100 105 110

Arg Glu Glu Arg Leu Leu Tyr Leu He Thr Asn Tyr Leu Pro Ser Val

115 120 125

Ser Leu Ala Thr Lys Arg Leu Gly Gly Glu Ala His Pro Asp Arg Thr 130 135 140

Leu Phe Ala His Val Ala Leu Cys Ala He Gly Arg Arg Leu Gly Thr 145 150 155 160

He Val Thr Tyr Asp Thr Gly Leu Asp Ala Ala He Ala Pro Phe Arg 165 170 175

His Leu Ser Pro Ala Ser Arg Glu Gly Ala Arg Arg Leu Ala Ala Glu 180 185 190

Ala Glu Leu Ala Leu Ser Gly Arg Thr Trp Ala Pro Gly Val Glu Ala 195 200 205

Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg 210 215 220

Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gin Ala Gly He Ala 225 230 235 240

Gly His Thr Tyr Leu Gin Ala Ser Glu Lys Phe Lys Met Trp Gly Ala 245 250 255

Glu Pro Val Ser Ala Pro Ala Arg Gly Tyr Lys Asn Gly Ala Pro Glu 260 265 270

Ser Thr Asp He Pro Pro Gly Ser He Ala Ala Ala Pro Gin Gly Asp 275 280 285

Arg Cys Pro He Val Arg Gin Arg Gly Val Ala Ser Pro Pro Val Leu 290 295 300

Pro Pro Met Asn Pro Val Pro Thr Ser Gly Thr Pro Ala Pro Ala Pro 305 310 315 320 Pro Gly Asp Gly Ser Tyr Leu Trp He Pro Ala Ser His Tyr Asn Gin 325 330 335

Leu Val Ala Gly His Ala Ala Pro Gin Pro Gin Pro His Ser 340 345 350

(2) INFORMATION FOR SEQ ID NO:3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 GAATGCN (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : GTACTCAAAA GGTCATAC (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO ( iv) ANTI -SENSE : NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGGAAACCAA ACGCGGAATG 20

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GATACGGTGC GGGCAGTACT GCCTCCGGAT 30

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : ATCCGGAGGC AGTACTGCCC GCACCGTATC 30

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TTTTTGGCGC TCTTCGACAG CGGGGAC 27

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GTCCCCGCTG TCGAAGAGCG CCAAAAA 27

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CATGATTAAT TA 12

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: AGCTTAATTA AT 12

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CCCACTCCCG ATTAACGTGG ACGTCCGCGC TGGCTGCGAG GTG 43

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CCTCGCAGCC AGCGCGGACG TCCACGTTAA TCGGGAGTGG G 41

(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 49 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CCCCGATCGC ACGCTGTTCG CTGCAGTCGC GCTGTGCGCG ATCGGGCGG 49

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GATCGCGCAC AGCGCGACTG CAGCGAACAG CGTGCGATCG GGG 43

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CACCAACTAC CTGCCCTCGG TCGCGCTAGC CACAAAACGC CTGGGGGG 48 (2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CAGGCGTTTT GTGGCTAGCG CGACCGAGGG CAGGTAGTTG 40

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CCAACTACCT GCCCTCGGTC GCCCTGGCCA CAAAACGCCT GGGG 44

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GCCAGGGCGA CCGAGGG 17

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CCCACTCCCG ATTAACGTGG ACTACCGCGC TGGCTGCGAG GTG 43

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CGCGGTAGTC CACGTTA 17

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CCCCGATCGC ACGCTGTTCG CGTACGTCGC GCTGTGCGCG ATCGG 45

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GCGACGTACG CGAACAGC 18

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CACCAACTAC GTGCCCTCGG TCTCCCTG (2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: CCGAGGGCAC GTAGTTGGTG ATCAGG 26

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CAACTACCTG GGCTCGGTCT CCCTGGCC 28

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GAGACCGAGC CCAGGTAGTT GGTGATCAG 29

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: CGCTGTTCGC ACGCGTCGCG CTGTGCGCGA TCG 33

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CAGCGCGACG CGTGCGAACA GCGTGCGATC GGG 33

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

CTGTTCGCGG AAGTAGCGCT GTGCGCGATC GG 32

(2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: CGCACAGCGC TACTTCCGCG AACAGCGTGC GATCGGG 37

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CGCTGTTCGC CAAGGTCGCG CTGTGCGCGA TCG 33

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: CACAGCGCGA CCTTGGCGAA CAGCGTGCGA TCGGG 35

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CCGATTAACG TTGACGCCCG CGCTGGCTGC GAGGTGGG 38

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: CAGCCAGCGC GGGCGTCAAC GTTAATCGGG AGTGGG 36

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: CCTGCCCTCG GTAAGCTTGG CCACAAAACG CCTGG 35

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GGCGTTTTGT GGCCAAGCTT ACCGAGGGCA GGTAG 35

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp Arg Ala Cys 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: CGTATCCGGA TCCAATC 17

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: GTTAACAACA TGATGCTG

Claims

WHAT IS CLAIMED IS:

1. Mutant Heφes Simplex Type 1 vims, (HSV-1) made by a process comprising: transforming a host cell with a set of vectors comprising: a first vector comprising a HSV-1 mutated protease gene and overlapping DNA homologous with overlapping DNA of at least one additional vector; and additional vectors, each additional vector comprising a fragment of the substantially complete HSV-1 genome and also comprising overlapping DNA which is homolo.gous with a sequential genomic fragment contained in at least one other additional vector, so that upon co-transfection of a host cell, replication of viral DNA, and recombination of the viral DNA, a replicable vims having a mutated protease and which is replicable in a wild type or host range cell line is formed.

2. A vims according to Claim 1 wherein the process further comprises:

(a) obtaining a set of starting vectors, each starting vector comprising a fragment of the substantially complete HSV-1 genome and also comprising DNA which is overlapping DNA with a sequential genomic fragment contained in other starting vectors, so that upon co- transfection of a host cell, replication of viral

DNA, and recombination of the viral DNA, a vims is formed which is replicable in a wild type or host range cell line;

(b) replacing a starting vector comprising a protease gene which is to be mutated with a first replacement vector, the first replacement vector comprising a mutated protease gene and overlapping DNA, and at least one additional replacement vector comprising genomic DNA which was present in the replaced starting vector, but is not present in the first replacement vector along with overlapping DNA; and (c) co-transfecting a host cell with the replacement vectors and the remaining starting vectors under conditions allowing replication of viral DNA and recombination of viral DNA to form a mutant vims which is replicable in a wild type or host range cell line.

3. A vims according to Claim 2 wherein the first replacement vector is made by a process comprising:

(a) creating a vector comprising a protease gene site which is to be mutated and overlapping

DNA;

(b) defining a first restriction endonuclease site in a position 5' to the protease gene site which is to be mutated; (c) defining a second restriction endonuclease site in a position 3' to the protease gene site which is to be mutated to define a wild-type gene segment contained between the first and second restriction endonuclease sites; (d) creating a mutant protease gene segment substantially identical to the wild-type gene segment, except for comprising a desired mutation; and (e) replacing the wild-type gene segment with the mutant protease gene segment to obtain the first replacement vector.

4. A vims according to Claim 3 wherein the starting vectors are cosmids.

5. A vims according to Claim 4 wherein the replacement vectors are plasmids.

6. A vims according to Claim 5 wherein the second restriction endonuclease site is up to approximately 2,000 base pairs downstream of the first endonuclease restriction site.

7. A vims according to Claim 6 wherein the second restriction site is up to approximately 1 ,100 base pairs downstream of the first endonuclease restriction site.

8. A vims according to Claim 7 wherein the first restriction site and the second restriction site are both recognized by the same restriction endonuclease.

9. A vims according to Claim 8 wherein the first restriction site and the second restriction site have different nucleic acid sequences.

10. A vims according to Claim 7 wherein the mutant protease gene segment is made by PCR mutagenesis.

11. A vims according to Claim 10 wherein the mutant protease gene segment further comprises a new restriction endonuclease restriction site.

12. A vims according to Claim 10 wherein the mutant protease gene encodes a different amino acid than wild-type HSV-1 protease.

13. A vims according to Claim 12 wherein the mutant protease gene encodes a different amino acid at a site selected from the group of: His 61, Leu 125, Pro 126, Ser 129, and His 148.

14. A vims according to Claim 2 wherein the starting vectors are chosen from the group consisiting of: cos28, cos6, cos 14, cos48 and cos56.

15. A vims according to Claim 2 selected from the group consisting of: V711, V712, V713, V714, V715, V716, V717, V718, V725, V728, V729, and V730.

16. A HSV-1 vims selected from the group consisting of: V711 , V712, V713, V714, V715, V716, V717, V718, V725, V728,

V729, and V730.

17. A set of vectors comprising: a first vector which is a plasmid, comprising a HSV-1 mutated protease gene and overlapping DNA homologous with overlapping DNA of at least one additional vector; and additional vectors, each additional vector comprising a fragment of the substantially complete HSV-1 genome and also comprising overlapping DNA which is homologous with a sequential genomic fragment contained in at least one other additional vector, so that upon co-transfection of a host cell, replication of viral DNA, and recombination of the viral DNA, a vims having a mutated protease and which is replicable in a wild type or host range cell line is formed.

18. A set of vectors according to Claim 17 wherein at least one of the additional vectors is a plasmid.

19. A set of vectors according to Claim 18 wherein at least one of the additional vectors is a cosmid.

20. A set of vectors according to Claim 17 wherein the mutant protease gene encodes a different amino acid than wild-type HSV-1 protease.

21. A set of vectors according to Claim 20 wherein the mutant protease encodes a different amino acid at a site selected from the group of: His61, Leul25, Prol26, Serl29, and Hisl48.

22. A set of vectors comprising at least one vector selected from the group consisting of: cos28, cos6, cosl4, cos48, cos56, pR710 and at least one vector which carries substantially the same insert as a plasmid selected from the group consisting of: pR700, pR711, pR712, pR713, pR714, pR715, pR716, pR717, pR718, pR725, pR728, pR729, and pR730.

23. A vector selected from the group consisting of: pR700, pR710, pR711, pR712, pR713, pR714, pR715, pR716, pR717, pR718, pR725, pR728, pR729, and pR730.

24. A vector made by a process comprising the steps of:

(a) obtaining a set of starting vectors, each starting vector comprising a fragment of the substantially complete HSV-1 genome and also comprising DNA which is overlapping DNA with a sequential genomic fragment contained in other starting vectors, so that upon co- transfection of a host cell, replication of viral DNA, and recombination of the viral DNA, a vims which is replicable in a wild type or host range cell line is formed;

(b) replacing a starting vector comprising a protease gene which is to be mutated with a first replacement vector, the first replacement vector comprising a mutated protease gene and overlapping DNA, and at least one additional replacement vector comprising genomic DNA which was present in the replaced starting vector, but is not present in the first replacement vector along with overlapping DNA.

25. A vector according to Claim 24 wherein the first replacement vector may be made by a process comprising:

(a) creating a vector comprising a protease gene site which is to be mutated and overlapping DNA; (b) defining a first restriction endonuclease site in a position 5' to the protease gene site which is to be mutated;

(c) defining a second restriction endonuclease site in a position 3' to the protease gene site which is to be mutated to define a wild-type gene segment contained between the first and second restriction endonuclease sites;

(d) creating a mutant protease gene segment substantially identical to the wild-type gene segment, except for comprising a desired mutation; and

(e) replacing the wild-type gene segment with the mutant protease gene segment to obtain the first replacement vector.

26. A host cell transformed with a set of vectors comprising: a first vector which is a plasmid, comprising a HSV-1 mutated protease gene and overlapping DNA homologous with overlapping DNA of at least one additional vector; and additional vectors, each additional vector comprising a fragment of the substantially complete HSV-1 genome and also comprising overlapping DNA which is homologous with a sequential genomic fragment contained in at least one other additional vector, so that upon co-transfection of a host cell, replication of viral DNA, and recombination of the viral DNA, a vims having a mutated protease and which is replicable in a wild type or host range cell line is formed.

27. A host cell according to Claim 26 wherein at least one of the additional vectors is a plasmid.

28. A host cell according to Claim 27 wherein at least one of the additional vectors is a cosmid.

29. A host cell transformed with a vector selected from the group consisting of: pR700, pR710, pR711, pR712, pR713, pR714, pR715, pR716, pR717, pR718, pR725, pR728, pR729, and pR730.

30. A host cell according to Claim 29 which is additionally transformed with at least one vector selected from the group consisting of: cos28, cos6, cos 14, cos48, and cos56.