CA2222877A1 - Herpes simplex type 1 protease mutants and vectors - Google Patents

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

W 096/38546 PCTlUS96J~779 TITLE OF THF INVENTION

I)ESCRIPTION OF THE INVENTION
This inventiion relates to Herpes Simplex Virus type 1 (HSV-1) viruses which ~,ontain a mutation in the protease gene, and to vectors and host cells used in producing them.

E~CKGROUND OF T~IE INVENTION
The Herpes Simplex Type-l (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 protlease is a serine protease that has both a structural and enzymatic role in the assembly of the HSV-1 capsid. The protease and infected ceil 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-l viruses which have a mutated protease gene. ~Preferred mutant viruses of this invention contain altered protease ~enes 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 W O 96/38546 PCTrUS96/07795 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 5 No. , (Attorney Docket No. 19458) filed herewith, which is hereby incorporated by reference.
The HSV-l viruses of this invention are preferably made by transforming a host cell with a set of vectors comprising: a first vector comprising a HSV-l mllt~ted protease gene and overlapping DNA
10 homologous with overlapping DNA of at least one additional vector; and additional vectors, each additional vector comprising a fragment of the subst~nti~lly complete HSV-l 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-15 transfection of a host cell, replication of viral DNA, and recombinationof the viral DNA, a virus having a mllt~ted protease gene and which is replicable in a wild type or host range cell line is forrned.
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 subst~nti~qlly complete HSV-l 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 W ~96~38546 P~TnUS9610779 ~ 3 ~
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 rem~ining 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 a$pect of this invention is a set of vectors used to 10 make the m~lt~nt viruses of this invention. The set of vectors comprises:
a first vector which is a plasmid, comprising a HSV-l 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 subst~n~i~lly complete HSV-1 genome and also comprising overlapping DNA which is homologous with a sequential genomic fragment conta~Lined 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 subst~nti~lly complete HSV-l 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) replaLcing a starting vector comprising a protease gene which is to be mutated with a first replacement vector, the first replacement vector comprising a W 096/38546 PCTrUS96/07795 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 rem~ining 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 mllt~ted and overlapping DNA;
(b) defining a first restriction endonuclease site in a position S' 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.

W 096/38S46 PCTrUS96/07795 BRIEF DESCRIPTIONI OF THE FIGURES
Figure 1 is the DNA and amino acid sequences (SEQ.ID.NO.:1&2) of the HSV-l (F) protease (Pra) BsmI fragment.
(The 82bp upstream fragment is not shown).
S Figure 2 is a diagram of HSV-l 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.
Overlappirg Vectors: two or more vectors, each cont~ining 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 ~f the DNA are present in a common host.
Replacement Vector: a vector, generally a plasmid which contains a portion of a ~ISV-l 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 rem~ining 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 subst~nti~lly complete genome of HSV-l along with overlapping DNA.
Subst~nti~lly 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-l virus is formed. This invention specifically el,lvisions: (1) an HSV-l virus cont~ining a complete genome cont~ining desired mutations and (2) an HSV-l virus W 096/38S46 PCTrUS96/07795 which does not have a complete genome, but the genes which are missing are not essential for virus replication; (3) an HSV-l virus missing genes which are essential for virus replication, but the missing gene product(s) are complemented by those produced in a host range 5 cell line; and (4) an HSV-l 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 10 too short nor too long, so ~at 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 subst~nti~lly identical to a segment in another vector.
15 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-l protease. Viruses 20 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, 25 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 30 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.

W 096/38546 PCTnUS96/0779 One or more of these vectors are replaced by one or more replacement vectors, each replacement vector cont~inin~ a smaller HSV-l DNA insert than in ~e starting vector, but together the replacement vectors contain the "equivalent amount" of unique, non-5 overlapping HSV-l genomic DNA as was present in the starting vector.
("Equivalent amount" as used in this content means subst:~nti~lly 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 mllt~ted is contained within one starting vector, then only this single 10 vector needs to be replaced. If, however, the protease gene which is to be mllt~ted is contained on two starting vectors (i.e., each starting vector cont~ining 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 lltili7ed, 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 rem~ining 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.

W 096138546 PCTrUS96/07795 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 5 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 10 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 15 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 20 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 BsmI.
The second replacement vector according to this invention comprises any viral DNA which was originally encoded in the first 25 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 30 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: cos2~, cos6, cosl4, W 096/38546 PCTnUS961~7795 cos48, and cosS6, which were obtained from Dr. Andrew J. Davison.
These cosmids and/or their equivalents can be made according to the description given in Cllnnin~h~m and Davison Virology 197:116-124 (1993), which is hereby incorporated by reference.
One of the cosmids of the Cllnningh~m 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 BsmI, a degenerate restriction endonuclease with a recognition sequence of GAATG/\CN'' (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, VVI), and contains the UL26 protease gene in a 13.3 kb insert of HSV-l (base pairs 44440-57747). Plasmid pR700 also contains two naturally occurring BsmI sites, a first one 82 base pairs 5'- of the HSV-l protease start site and one at amino acid 348 of the protease. The "N" at the 5' BsmI site is "T" whereas at the 3' BsmI 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 BsmI fragment was used to introduce various desired mutations into the HSV-l 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-l (base pairs 24699-49435) that does not include the HSV-l protease.

W 096/38546 PCTrUS96/07795 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 subst~nti~lly the 5 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 rem~ining starting vectors, which together make up a further aspect of this invention, are introduced into HSV-l host cells.
The HSV-l host cell chosen is generally not a critical aspect of this invention. Generally, any cell in which HSV-l can replicate is an 15 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 mllt~nt HSV-l viruses of this invention. This is illustrated in Figure 3C.
The above-described mutagenesis method allows one to 20 make the desired HSV-l protease mutations in the virus in a short period of time, i.e., within about 2 weeks. It has the further advantage that pure mllt~nt 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 25 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.), 30 Academic Press, Inc. San Diego, CA, pages 177-183, which is hereby incorporated by reference. However, since HSV-l 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 Fngl~nd S Biolabs) is a preferred polymerase for the PCR reactions used herein because of its proofre~clin~ ability and thermal stability at 99~C.
Virtually any mnt~tion 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 10 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 mllt~tion procedure of this invention, the following mutant viruses were made.
15 Throughout the specification and claims, the virus nomenclature is the same as that used for the replacement plasmid cont~inin~ the mutation, except that the virus uses the prefix "V" and the replacement plasmid uses "pR".

W 096/38546 PCTrUS96/07795 Representative protease mllt~nt~

VIRUS MUTATION ADDED SITE*
V711 His61 to Val61 AatII
V730 His61 to Ala61 Pspl406I
V715 His61 to Tyr61 none V717 Leul25 to Vall25 BsaAI
V718 Prol26 to Glyl26 BstXI
V713 Serl29 to Alal29 NheI
V714 Serl29 to Alal29 none V712 His148 to Alal48 PstI
V716 His148 to Tyrl48 none V725 His 148 to Argl48 MulI
V728 His148 to Glu148 Eco47III
V732** Alal 29 to Serl29 HindI~
V729 His 148 to Lys 148 StyI
5 * 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 Serl29.
10 Therefore, changes of amino acids at the active serine site and near the active serine site were of particular interest.

Mutations At Serl29:
A mutation was made in HSV- 1 protease gene to change the 15 protease amino acid Serl29 to Alal29. This virus is designated V713, and is a further aspect of this invention. Recombinant virus 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 ~0 kD protease (Pra) along with several -WO 96~38~46 PCT/US9fi/07795 other peptides r~ngin~ in molecular wieght from 29 kD to 75 kD. A 24 kD band seen in wild-type infections was absent.

Mutation At Leu 125 S V717 contains a mutation of Leul 25 to Val 125. This virus 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 virus.

Mutations At Pro 126 V718 contains a mutation of Prol26 to Glyl26. This virus 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 virus to replicate may reflect a requirement for proper structural assembly of the capsid. VVhile 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 pack~ging.
Mutations At His 14~s 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 30 activity. This result was unexpected because in the prior art, where the protease gene having the same mutation,but not contained within the virus showed no protease activity in in vitro assays. (Liu et al., 1992 Proc. Natl. Acad, Sci. USA 89:2076-20~s0 and Deckman et al., 1992 J.
Virology 66:7362-7367, both of which are incorporated by reference.) W 096/38546 PCTrUS96/07795 While not wishing to be bound by theory, this surprising result may be due to a difference in the three dimensional structure of the protein wi~in the virus environment, or the presence of a hi~erto unknown accessory protein which lends activity to the protease.
Viruses V716 (His148 to Tyrl48) V725 (His148 to Argl48) V729 (His148 to Lysl48) 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 (V711), Tyr61 (V715), and Ala61 (V730) all created null mutant viruses and in Western analysis had the same extra bands as the V713 mutant.
Taken with the observations of the His148 mutations, the results suggest ~at His61 is required for protease activity whereas His148 is not.

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

EXAMPLES
GENERAL METHODS
Viral Strains Two strains of viruses were used, HSV-l strain 17 [designated HSV- 1 (17)] and HSV- 1 strain F [designated HSV- 1 (F)] .
30 Mutations to the protease have been made in HSV-l(F) (see Liu, F.
etal., l991,J. Virol. 65:5149-5156, hereby incorporated 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 incorporated by reference). Sequence analysis of the BsmI fragment 35 revealed that the two strains differ by two amino acids (Leu300/Ser300 W 096/38546 PCTnUS96/0779 and Ser301/Pro301) and six silent mutations (in Prol5, Arg46, Gly84, Gln90, Glyl99 and His341). To make an equivalent comparison of in vitro and in vivo studies, a protease chimera (pR73 1) was made.
Plasmid pRHS2, cont~ining the HSV- l (F) protease was digested with 5 BsmI and the l.lkb fragment was cloned into pR700 cont~ining HSV-1(17) protease. Both viruses were equivalent in virus titer and plaque morphology 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 viruses. The first round of PCR mutagenesis was carried out in two separate reactions. In one reaction, a positive strand 15 oligonucleotide homologous to the DNA 5' to the first BsmI 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 BsmI site was paired with the positive strand oligonucleotide specified. The two specified oligonucleotides are 20 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 initi~ted. In the second round of the procedure, the DNA fragments 25 generated by the first round PCR reactions were gel purified and mixed together with oligonucleotides flanking the BsmI 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 30 Perkin Elmer Cetus. The cycle was melt for 1 minute at 99~C; anneal at 40~C for two minlltes; extend at 71~C for 3 minutes; for 30 cycles. The product of the second round PCR reaction and extended BsmI fragment, was digested with BsmI, gel purified and ligated into the BsmI sites of pR700.
-CA 02222877 1997-ll-28 W 096/38546 PCTrUS96/07795 Oli~onucleotides 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 5' and 3' oligonucleotides flanking the two BsmI cloning sites:
5'-GTACTCAAAAGGTCATAC-3' (SEQ.ID.NO.:4) (This oligo is 5' to the first BsmI site and was used for the generation of all mutations in the protease from amino acid 1 to 348).

10 5'-GGGAAACCAAACGCGGAATG-3' (SEQ.ID.NO.:5) (This oligo is 3' to the second BsmI site and was used in generation of mutations in the protease from amino acids 1 to 34~.) Oligonucleotides for the temperature sensitive protease mllt~nt pR701:
15 5'-GATACGGTGCGGGCAGTACTGCCTCCGGAT-3 ' (SEQ.ID.NO.:6) 5'-ATCCGGAGGCAGTACTGCCCGCACCGTATC-3' (SEQ.~.NO.:7) These oligos add a SacI site to the Ala48 to Val48 mutation.

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

25 Linker oligonucleotides (BspHI-PacI-HindIII) for pR710:
S'-CATGATTAATTA-3' (SEQ.ID.NO.:10) 5'-AGCTTAATTAAT-3' (SEQ.ID.NO.: 11) Oligonucleotides used for the His61 to Val61 mutation for pR711:
30 5'-CCCACTCCCGATTAACGTGGACGTCCGCGCTGGCTGCGAGG-TG-3' (SEQ.ID.NO.:12) 5 '-CCTCGCAGCCAGCGCGGACGTCCACGTTAATCGGGAGT-GGG-3' (SEQ.ID.NO.: 13) This also adds an AatII restriction site.

W 096/38~46 PCT~US96~77 Oligonucleotides used for the His148 to Alal48 mutation for pR712:
S ' -CCCCGATCGCACGCTGTTCGCTGCAGTCGCGCTGTGCGCGA-TCGGGCGG-3 ' (SEQ.ID.NO.: 14) S ' -GATCGCGCACAGCGCGACrGCAGCGAACAGCGTGCGATC-5 GGGG (SEQ.ID.NO.:15) This also adds a PstI restriction site.

Oligonucleotides used for the Serl29 to Alal29 mutation for pR713:
5 '-CACCAACTACCTGCCCTCGGTCGCGCTAGCCACAAAACGCC-10 TGGGGGG-3' (SEQ.ID.NO.: 16) 5'-CAGGCG l l~GTGGCTAGCGCGACCGAGGGCAGGTAG-TTG-3'(SEQ.ID.NO.: 17) This also adds a NheI restriction site.

15 Oligonucleotides used for the Serl29 to Alal29 mllt~hon for pR714:
5 '-CCAACTACCTGCCCTCGGTCGCCCTGGCCACAAAACGCCTG-GGG-3' (SEQ.ID.NO.: 18) 5'-GCCAGGGCGACCGAGGG-3' (SEQ.ID.NO.:l9) Oligonucleotides used for the His61 to Tyr61 mutation for pR715:
20 5'-CCCACTCCCGATTAACGTGGACTACCGCGCTGGCTGCGAGG-TG-3' (SEQ.ID.NO.:20) 5 ' -CGCGGTAGTCCACGTTA-3 ' (SEQ.~D.NO. :21) Oligonucleotides used for the His148 to Tyrl48 mutation for pR716:
25 5'-CCCCGATCGCACGCTGTTCGCGTACGTCGCGCTGTGCGCGA-TCGG-3' (SEQ.ID.NO.:22) 5'-GCGACGTACGCGAACAGC-3' (SEQ.ID.NO.:23) Oligonucleotides used for the Leul25 to Vall25 mutation for pR717:
30 5 '-CACCAACTACGTGCCCTCGGTCTCCCTG-3 ' (SEQ.ID.NO. :24) 5'-CCGAGGGCACGTAGTTGGTGATCAGG-3' (SEQ.ID.NO.:25) This also adds a BsaAI restriction site.

W O 96/38546 PCTrUS96/07795 Oligonucleotides used for the Prol26 to Glyl26 mutation for pR718:
5'-CAACTACCTGGGCTCGGTCTCCCTGGCC-3' (SEQ.ID.NO.:26) 5 '-GAGACCGAGCCCAGGTAGTTGGTGATCAG-3 ' (SEQ.ID.NO.:27) 5 This also adds a BstXI restriction site Oligonucleotides used for the His148 to Argl48 mutation for pR725:
5 ' -CGCTGTTCGCACGCGTCGCGCTGTGCGCGATCG-3 ' (SEQ.ID.NO.:28) 10 5 ' -CAGCGCGACGCGTGCGAACAGCGTGCGATCGGG-3 ' (SEQ.ID.NO.:29) This also adds a MulI restriction site.

Oligonucleotides used for the His148 to Glu148 mutation for pR728:
15 5 ' -CTGTTCGCGGAAGTAGCGCTGTGCGCGATCGG-3 ' (SEQ.ID.NO.:30) S ' -CGCACAGCGCTA( ~TTCCGCGAACAGCGTGCGATCGGG-3 ' (SEQ.ID.NO.:31) This also adds a Eco47III restriction site.
Oligonucleotides used for the His148 to Lysl48 mutation for pR729:
5 ' -CGCTGTTCGCCAAGGTCGCGCTGTGCGCGATCG-3 ' (SEQ.ID.NO.:32) 5 ' -CACAGCGCGACCTTGGCGAACAGCGTGCGATCGGG-3 ' 25 (SEQ.ID.NO.:33) This also adds a StyI restriction site.

Oligonucleotides used for the His61 to Ala61 mutation for pR730:
5 ' -CCGATTAACGTTGACGCCCGCGCTGGCTGCGAGGTGGG-3 ' 30 (SEQ.ID.NO.:34) 5 ' -CAGCCAGCGCGGGCGTCAACGTTAATCGGGAGTGGG-3 ' (SEQ.ID.NO.:35) This also adds a Pspl406I restriction site.

W 096138546 PCTnU~96/0779 Oligonucleotides used for the Alal29 to Serl29 back mutation for pR732:
J 5'-CCTGCCCTCGGTAAGCTTGGCCACAAAACGCCTGG-3' (SEQ.ID.NO.:36) 5'-GGCGlYmGTGGCCAAGCTTACCGAGGGCAGGTAG-3' (SEQ.ID.NO.:37) This also adds a HindIII restriction site.

Constructs:
Plasmids derived from HSV-l (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-l (F) DNA with XbaI and ScaI. The 9884 base pair fragment was gel purified and subcloned into pGEM-7Zf(-) (Promega) at the XbaI and SmaI sites.
pRHS2: This plasmid contains HSV-l(F) DNA base pairs 49126-53272, starting within UL25 and ending within UL27. To prepare this plasmid, pRHS 1 was digested with NotI and NheI, and the 414~ base pair fragment was subcloned into the pGEM-7Zf(-) vector at the Bspl20I
and XbaI 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 BsmI, and the 1.1 kb fragment was then subcloned into the BsmI sites of pR700. This created a F strain protease in the 17 strain virus.
pR732: V713 virus DNA was digested with NotI and the 6.5 kb fragment cont~ining the HSV-l protease was gel purified. This fragment was used as a template for PCR to back-mutate the Serl29 to Alal29 back to Ser. The back mutation also created a new HindIII 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.

W 096/38546 PCTrUS96/07795 Plasmids derived from HSV-l (17):
pR700: This plasmid contains HSV-1(17) DNA base pairs 44440-57747, starting within UL22 and ending within UL28 to prepare HSV-l cos-28 was digested with StuI and NdeI, the 13,308 base pair fragment was gel purified and ligated into pGEM-4Z (Promega) at the NdeI and SmaI sites. This plasmid was use for both generation and sub-cloning of m~lt~nts pR701, pR717 and pR718 into the BsmI sites.
pR710: This plasmid contains HSV-1(17) DNA base pairs 24699-49435, starting between UL10/ULl l and ending within UL25. Cos 28 was digested with PacI and BspHI and the resulting 24,736 bp fragment was subcloned with the two linker oligos (SEQ.ID.NOS. 10 & 11) cont~ining BspHI-PacI-HindIII into the PacI/HindIII sites of New England Biolabs vector pNEB93.
pR701: HSV-l temperature sensitive mutant was created from pR700 by PCR mutagenesis. It has a Thr30 to Phe30 mutation which contains a SapI site and an Ala48 to Val48 mutation a cont~ining a new ScaI 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 ,ug/ml of G418 sulphate (GIBCO). Drug resistant cell lines were screened for the ability to complement the temperature sensitive protease virus, V701, at 39~C.
Digests Prior to transfection, cosmid DNA and pR710 were digested with PacI. Plasmids pR700, pR701, pR711, pR712, pR713.
pR714, pR715, pR716, pR717, pR71~, pR725, pR728, pR729, pR730, W 096J38546 PCT~US961077 and pR73 1 were digested with Hinclm and NdeI, while pR732 was digested with XbaI. The digested DNA was precipitated in 2M final NH40Ac pH 7.5, and 2 volumes of isopropanol, centrifuged l0 minlltes then washed in 70% ethanol and dried. The DNA was re-suspended in 10 mM Tris, 1 mM EDTA pH 7.g. Restriction endonucleases were purchased from New Fngl~nd Biolabs and Promega (Madison, WI).

Western Blots 12% SDS-PAGE gels were transferred to Immobilon-P
(Millipore, 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) ~lk~line phosphatase conjugate (Bio-Rad, Hercules, CA).
Western blots were developed with an ~lk~line 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 Na2HPO4 pH 7.2, 7% SDS, 1 X Denhardts and 100 ,ug/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'-CAGCGCTGGGAl~ lCG-3' (SEQ.ID.NO.:39) SH-10 5'-GTTAACAACATGATGCTG-3' (SEQ.ID.NO.:40) W O 96/38546 PCTrUS96/07795 Transfections Vero or PHS-23 cells were plated at 3 x 105 cells per well in six well clusters the day before transfection. The following day the cells were washed in Delbeco's Modi~1ed Eagles Medium (DMEM) S (from GIBCO, Gaithersburg, MD) without FCS and then 1 ml of transfection cocktail was added. Transfection cocktail was made as follows. To 100 ,~Ll of DMEM media, 0.5 ,ug of digested DNA was added, followed by 14 ,ul of LiptofectAM~ETM. (GIBCO) This transfection mixture was incubated for 30 minlltes at room temperature, 10 then 900 ,ul 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% CO2. Transfected cells were then washed and fresh media, DMEM, 4% FCS, 100 units/ml penicillin and 100 ~lg/ml 15 streptomycin, was added. At day six or seven the recombinant virus were harvested and the virus was plaque purified.

Plaque Purification.
After transfection with LipofectAMINETM Reagent 20 (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 25 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 cont~ining the recombinant virus.
The plug was placed in a sterile eppendorf tube cont:~ining 0.5 ml of 30 DMEM and 20% FCS. The plaque was sonicated and then repuified a.s described.

Wo 96138S46 PCT/US96107795 Recombinant virus expansion After plaque purification the virus was expanded on Vero cells, or if the the mllt~nt was a null mllt~nt, it was expanded on the host range cell line PHS-23.
s Virus titers Expanded virus 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 10 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% hllm~n IgG (Armour, ~nk~kee, LL). Two to six days later the cells were fixed in 1 ml of methanol for 7 minlltes and then air dryed.
Fixed cells were stained with Gemsa stain for 45 minlltes, washed with 15 water and dryed. The plaques were then counted under a microscope.

Virus DNA Mini Preps Mini preps of virus DNA were made as follows: a T-225 flask of vero or PHS-23 cells was infected at a MOI of S and harvested 20 at 18 hours post infection. Cells were pelleted and then washed in PBS
three times. The cells were re-suspended in 400 ,ul 10mM Tris pH 8.0, SmM NaCl, SmM 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 25 resulting supern~t~nt solution was incubated with proteinase K
(Boehringer ~nnheim, Indianopolis, IN) 100 ,ug/ml at 37~C overnight.
DNA was extracted twice with phenol and once with chloroform and precipitated 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 incorporated by reference, was 35 made. This required the introduction of two amino acid changes. Four W 096/38546 PCTrUS96/07795 separate transfections were done. Vero or host range cell line PHS-23 were plated at approximately 3.0 x 105 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.
S The transfection mix was then added to the cells and incubated for 1~
hours at 37~C. The cells were washed lX and 3 ml of DMEM with 4%
FCS was added. At day six or seven, plaques were observed and recombinant virus was harvested. Each transfection gave rise to 50 or more plaques five to six days post transfection. Recombinant virus was 10 titered on both Vero and PHS-23 cell lines. A minimllm 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-l (17) from one of the temperature sensitive (ts) mutant isolates on Vero and PHS-23 cells at 31~C and 39~C.
Table 2. HSV-l Temperature Sensitive Protease Mutants V701 and HSV-l titer on Host Range And Vero Cells HostCell Line Virus 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 106 PHS-23 V701 39 1.0 x 106 PHS-23 HSV-1(17) 31 2.5 x 107 PHS-23 HSV-1(17) 39 1.0 x 107 CA 02222877 l997-ll-28 W 096/38546 PCTnUS96/~779 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 (B) STREET: 126 East Lincoln Avenue, P.O. Box 2000-0907 (C) CITY: Rahway (D) STATE: New Jersey (E) COUNTRY: US
(F) ZIP: 07065-0907 (v) COMPUTER READABLE FORM:
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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 (C) REFERENCE/DOCRET NUMBER: 19457 PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (908) 594-3046 (B) TELEFAX: (908) 594-4720 (2) INFORMATION FOR SEQ ID NO:l:
(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

CA 02222877 l997-ll-28 ( ix ) FEATURE:
( A ) NAME /KEY: CDS
(B) LOCATION: 1. .1050 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp Arg Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser Gly Asp Ser Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala Leu Pro Pro Asp Asn Pro Leu Pro Ile Asn Val Asp His Arg Ala Gly Cys Glu Val Gly Arg Val Leu Ala Val Val Asp Asp Pro Arg Gly Pro Phe Phe Val Gly Leu Ile Ala Cys Val Gln Leu Glu Arg Val Leu Glu Thr Ala A1A Ser Ala Ala Ile Phe Glu Arg Arg Gly Pro Pro Leu Ser Arg Glu Glu Arg Leu Leu Tyr Leu Ile Thr Asn Tyr Leu Pro Ser Val Ser Leu Ala Thr Lys Arg Leu Gly Gly Glu Ala His Pro Asp Arg Thr Leu Phe Ala His Val Ala Leu Cys Ala Ile Gly Arg Arg Leu Gly Thr ATC GTC ACC TAC GAC ACC GGT CTC GAC GCC GCC ATC GCG CCC TTT CGC ~ 528 Ile Val Thr Tyr Asp Thr Gly Leu Asp Ala Ala Ile Ala Pro Phe Arg CAC CTG TCG CCG GCG TCT CGC GAG GGG GCG ~GG CGA CTG GCC GCC GAG 576 His Leu Ser Pro Ala Ser Arg Glu Gly Ala Arg Arg Leu Ala Ala Glu CA 02222877 l997-ll-28 WO 96138546 PCTtUS96107795 Ala Glu Leu Ala Leu Ser Gly Arg Thr Trp Ala Pro Gly Val Glu Ala Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gln Ala Gly Ile Ala Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly Ala Glu Pro Val Ser Ala Pro Ala Arg Gly Tyr Lys Asn Gly Ala Pro Glu Ser Thr Asp Ile Pro Pro Gly Ser Ile Ala Ala Ala Pro Gln Gly Asp Arg Cys Pro Ile Val Arg Gln Arg Gly Val Ala Ser Pro Pro Val Leu Pro Pro Met Asn Pro Val Pro Thr Ser Gly Thr Pro Ala Pro Ala Pro Pro Gly Asp Gly Ser Tyr Leu Trp Ile Pro Ala Ser His Tyr Asn Gln Leu Val Ala Gly His Ala Ala Pro Gln Pro Gln Pro His Ser (2) INFORMATION FOR SEQ ID NO:2:
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Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp CA 02222877 l997-ll-28 W O 96/38546 PCTrUS96/07795 ~rg Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser ~ly Asp Ser Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala .
Leu Pro Pro Asp Asn Pro Leu Pro Ile Asn Val Asp His Arg Ala Gly Cys Glu Val Gly Arg Val Leu Ala Val Val Asp Asp Pro Arg Gly Pro ~he Phe Val Gly Leu Ile Ala Cys Val Gln Leu Glu Arg Val Leu Glu ~hr Ala Ala Ser Ala Ala Ile Phe Glu Arg Arg Gly Pro Pro Leu Ser Arg Glu Glu Arg Leu Leu Tyr Leu Ile Thr Asn Tyr Leu Pro Ser Val Ser Leu Ala Thr Lys Arg Leu Gly Gly Glu Ala His Pro Asp Arg Thr Leu Phe Ala His Val Ala Leu Cys Ala Ile Gly Arg Arg Leu Gly Thr ~le Val Thr Tyr Asp Thr Gly Leu Asp Ala Ala Ile Ala Pro Phe Arg ~is Leu Ser Pro Ala Ser Arg Glu Gly Ala Arg Arg Leu Ala Ala Glu Ala Glu Leu Ala Leu Ser Gly Arg Thr Trp Ala Pro Gly Val Glu Ala Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gln Ala Gly Ile Ala ~ly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly Ala ~lu Pro Val Ser Ala Pro Ala Arg Gly Tyr Lys Asn Gly Ala Pro Glu Ser Thr Asp Ile Pro Pro Gly Ser Ile Ala Ala Ala Pro Gln Gly Asp Arg Cys Pro Ile Val Arg Gln 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 W 096138546 PCT~US96/07795 Pro Gly Asp Gly Ser Tyr Leu Trp Ile Pro Ala Ser His Tyr Asn Gln Leu Val Ala Gly His Ala Ala Pro Gln Pro Gln Pro His Ser (2) INFORMATION FOR SEQ ID NO:3:
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(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:

(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

W 096/38546 PCTrUS96/07795 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

(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:

(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:

(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

CA 02222877 l997-ll-28 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
GGC~llll~l GGCCAAGCTT ACCGAGGGCA GGTAG 35 (2) INFORMATION FOR SEQ ID NO:38:
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(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 A1A Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp Arg Ala Cys (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNES5: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

(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 W 096/38546 PCTrUS96/07795 (iii) HYPOTHETICAL: NO
(iv) AWTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
GTTAACAACA TGATGCTG = 18

Claims (30)

WHAT IS CLAIMED IS:

1. Mutant Herpes Simplex Type 1 virus, (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 virus having a mutated protease and which is replicable in a wild type or host range cell line is formed.

2. A virus 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 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 mutant virus which is replicable in a wild type or host range cell line.

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

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

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

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

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

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

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

13. A virus 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 virus according to Claim 2 wherein the starting vectors are chosen from the group consisting of: cos28, cos6, cos14, cos48 and cos56.

15. A virus 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 virus 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 virus 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, Leu125, Pro126, Ser129, and His148.

22. A set of vectors comprising at least one vector selected from the group consisting of: cos28, cos6, cos14, 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 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.

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 virus 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, cos14, cos48, and cos56.