Assembly of the herpes simplex virus capsid: preformed triplexes bind to the nascent capsid

The herpes simplex virus type 1 (HSV-1) capsid is a T=16 icosahedral shell that forms in the nuclei of infected cells. Capsid assembly also occurs in vitro in reaction mixtures created from insect cell extracts containing recombinant baculovirus-expressed HSV-1 capsid proteins. During capsid formation, the major capsid protein, VP5, and the scaffolding protein, pre-VP22a, condense to form structures that are extended into procapsids by addition of the triplex proteins, VP19C and VP23. We investigated whether triplex proteins bind to the major capsid-scaffold protein complexes as separate polypeptides or as preformed triplexes. Assembly products from reactions lacking one triplex protein were immunoprecipitated and examined for the presence of the other. The results showed that neither triplex protein bound unless both were present, suggesting that interaction between VP19C and VP23 is required before either protein can participate in the assembly process. Sucrose density gradient analysis was employed to determine the sedimentation coefficients of VP19C, VP23, and VP19C-VP23 complexes. The results showed that the two proteins formed a complex with a sedimentation coefficient of 7.2S, a value that is consistent with formation of a VP19C-VP23(2) heterotrimer. Furthermore, VP23 was observed to have a sedimentation coefficient of 4.9S, suggesting that this protein exists as a dimer in solution. Deletion analysis of VP19C revealed two domains that may be required for attachment of the triplex to major capsid-scaffold protein complexes; none of the deletions disrupted interaction of VP19C with VP23. We propose that preformed triplexes (VP19C-VP23(2) heterotrimers) interact with major capsid-scaffold protein complexes during assembly of the HSV-1 capsid.     

Deletion of the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5 from the herpes simplex virus type 1 genome substantially impairs productive viral infection in cell culture and pathogenesis in the rat central nervous system

A distinctive feature of the genetic make-up of herpes simplex virus type 1 (HSV-1), a human neurotropic virus, is that approximately half of the 81 known viral genes are not absolutely required for productive infection in Vero cells, and most can be individually deleted without substantially impairing viral replication in cell culture. If large blocks of contiguous viral genes could be replaced with foreign DNA sequences, it would be possible to engineer highly attenuated recombinant HSV-1 gene transfer vectors capable of carrying large cellular genes or multiple genes having related functions. We report the isolation and characterization of an HSV-1 mutant, designated d311, containing a 12 kb deletion of viral DNA located between the L-S Junction a sequence and the U(S)6 gene, spanning the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5. Replication of d311 was totally inhibited in rat B103 and mouse Neuro-2A neuroblastoma cell lines, and was reduced by over three orders of magnitude in human SK-N-SH neuroblastoma cells compared to wild-type (wt) HSV-1 KOS. This suggested that the deleted genes, while nonessential for replication in Vero cells, play an important role in HSV replication in neuronal cells, particularly those of rodent origin. Unlike wt KOS which replicated locally and spread to other regions of brain following stereotactic inoculation into rat hippocampus, d311 was unable to replicate and spread within the brain, and did not cause any apparent local neuronal cell damage. These results demonstrate that d311 is highly attenuated for the rat central nervous system. d311 and other mutants of HSV containing major deletions of the nonessential genes within U(S) have the potential to serve as useful tools for gene transfer applications to brain.     

Molecular interactions between the HSV-1 capsid proteins as measured by the yeast two-hybrid system

HSV-1 B capsids are composed of seven major proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26. VP indicates that the capsid protein is also a component of the infectious virion. Capsid proteins 21, 22a, and VP24 are specified by a single open reading frame (UL26) that encodes 635 amino acids. An objective of the work in our laboratory is to identify and map interactions among and between capsid proteins. In the present studies we employed the yeast GAL4 two-hybrid system developed by Fields and his colleagues (Nature 240, 245-246 (1989)) for this purpose. DNA corresponding to the capsid open reading frames was derived as a PCR product and fused to sequences of the GAL4 activation and DNA binding domains. Using this system each of the capsid proteins has been tested for interactions with all of the other capsid proteins. Three interactions have been identified: a relatively strong self-interaction between 22a molecules (residues 307-635 of UL26), bimolecular interactions between 22a and VP5, and another between VP19C and VP23. The interactions were detected by the expression of beta-galactosidase enzyme activity, and yielded 289, 86, and 63 units of enzyme activity, respectively. For the 22a self-interaction, elimination of residues 611-635 resulted in an approximately twofold decrease in enzyme activity. The C-terminal 25 amino acids of 22a were also essential for the bimolecular interaction between 22a and VP5.     

Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid

The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (Aequorea victoria) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An analysis of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) analysis of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a generalized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was observed, confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.     

Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis

Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.     

The PK domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is required for immediate-early gene expression and virus growth

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10DeltaPK). ICP10DeltaPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10DeltaPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10DeltaPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.     

A single amino acid substitution in the capsid protein VP1 of coxsackievirus B3 (CVB3) alters plaque phenotype in Vero cells but not cardiovirulence in a mouse model

We previously described a large plaque attenuant (p14V-1) derived from a cardiovirulent Coxsackievirus B3 (CVB3) and showed that there were no major determinants of either attenuation or plaque phenotype in the 5' nontranslated region (5'NTR). Part of the region encoding the last 124 amino acids of VP3 and the first 106 amino acids of VP1 of the attenuant was then sequenced and compared to the wild-type. Three nucleotide changes were found in the VP1 coding region: a silent single base change at nucleotide position 2467 (C to U) and a double-base change at position 2690-1 (AA to GT), which leads to a change from lysine to serine at amino acid position 80. This mutation maps to the begining of B-C loop of the three-dimensional structure of VP1 of CVB3, where a distinct surface projection is formed. Two infectious chimeric cDNA clones were constructed, based on a cardiovirulent cDNA construct. In one construct, the 5'NTR and the VP3-VP1 region were from p14V-1 and in the other, only the VP3-VP1 region was from this attenuant. Both chimeric viruses produced large plaques on Vero cell monolayers, similar to p14V-1 but larger than the prototypic cardiovirulent virus. In vivo experiments showed that both chimeric viruses induced myocarditis in a murine model, similar to wild-type virus. We conclude that mutation serine-80 in capsid protein VP1 of p14V-1 is a determinant of the large plaque phenotype but is not responsible for attenuation.     

The product of the herpes simplex virus type 1 UL25 gene is required for encapsidation but not for cleavage of replicated viral DNA

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278-283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246-259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.     

Increase of heat-shock protein and induction of gamma/delta T cells in peritoneal exudate of mice after injection of live Fusobacterium nucleatum

The herpes simplex virus-1 (HSV-1) capsid shell has 162 capsomers arranged on a T = 16 icosahedral lattice. The major capsid protein, VP5 MW = 149,075) is the structural component of the capsomers. VP5 is an unusually large viral capsid protein and has been shown to consist of multiple domains. To study the conformation of VP5 as it is folded into capsid promoters, we identified the sequence recognized by a VP5-specific monoclonal antibody and localized the epitope on the capsid surface by cryoelectron microscopy and image reconstruction. The epitope of mAb 6F10 was mapped to residues 862-880 by immunoblotting experiments performed with (1) proteolytic fragments of VP5, (2) GST-fusion proteins containing VP5 domains, and (3) synthetic VP5 peptides. As visualized in a three-dimensional density map of 6F10-precipitated capsids, the antibody was found to bind at sites on the outer surface of the capsid just inside the openings of the trans-capsomeric channels. We conclude that these sites are occupied by peptide 862-880 in the mature HSV-1 capsid.     

Self-association of herpes simplex virus type 1 ICP35 is via coiled-coil interactions and promotes stable interaction with the major capsid protein

The ordered copolymerization of viral proteins to form the herpes simplex virus (HSV) capsid occurs within the nucleus of the infected cell and is a complex process involving the products of at least six viral genes. In common with capsid assembly in double-stranded DNA bacteriophages, HSV capsid assembly proceeds via the assembly of an outer capsid shell around an interior scaffold. This capsid intermediate matures through loss of the scaffold and packaging of the viral genomic DNA. The interior of the HSV capsid intermediate contains the viral protease and assembly protein which compose the scaffold. Proteolytic processing of these proteins is essential for and accompanies capsid maturation. The assembly protein (ICP35) is the primary component of the scaffold, and previous studies have demonstrated it to be capable of intermolecular association with itself and with the major capsid protein, VP5. We have defined structural elements within ICP35 which are responsible for intermolecular self-association and for interaction with VP5. Yeast (Saccharomyces cerevisiae) two-hybrid assays and far-Western studies with purified recombinant ICP35 mapped a core self-association domain between Ser165 and His219. Site-directed mutations in this domain implicate a putative coiled coil in ICP35 self-association. This coiled-coil motif is highly conserved within the assembly proteins of other alpha herpesviruses. In the two-hybrid assay the core self-association domain was sufficient to mediate stable self-association only in the presence of additional structural elements in either N- or C-terminal flanking regions. These regions also contain conserved sequences which exhibit a high propensity for alpha helicity and may contribute to self-association by forming additional short coiled coils. Our data supports a model in which ICP35 molecules have an extended conformation and associate in parallel orientation through homomeric coiled-coil interactions. In additional two-hybrid experiments we evaluated ICP35 mutants for association with VP5. We discovered that in addition to the C-terminal 25 amino acids of ICP35, previously shown to be required for VP5 binding, an additional upstream region was required. This region is between Ser165 and His234 and contains the core self-association domain. Site-directed mutations and construction of chimeric molecules in which the self-association domain of ICP35 was replaced by the GCN4 leucine zipper indicated that this region contributes to VP5 binding through mediating self-association of ICP35 and not through direct binding interactions. Our results suggest that self-association of ICP35 strongly promotes stable association with VP5 in vivo and are consistent with capsid formation proceeding via formation of stable subassemblies of ICP35 and VP5 which subsequently assemble into capsid intermediates in the nucleus.     

The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D

The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR/PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1. 302 to HIgR/PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR/PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR/PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.     

Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell

Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation determinant on capsid protein VP1, and we showed that at least one other capsid protein, VP2, could also express a mouse adaptation determinant. Both determinants are located in the inside of the three-dimensional structure of the viral capsid. They may be involved in the early steps of mouse nerve cell infection subsequent to receptor attachment.     

The herpes simplex virus type 1 U(L)17 gene encodes virion tegument proteins that are required for cleavage and packaging of viral DNA

Previous studies have suggested that the U(L)17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZ expression cassette in place of 1,490 bp of the 2,109-bp U(L)17 open reading frame [HSV-1(deltaU(L)17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5' end of the U(L)17 open reading frame [HSV-1(U(L)17-stop)] were plaque purified on engineered cell lines containing the U(L)17 gene. A virus derived from HSV-1(U(L)17-stop) but containing a restored U(L)17 gene was also constructed and was designated HSV-1(U(L)17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(deltaU(L)17) nor HSV-1(U(L)17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(deltaU(L)17) compared to wild-type virus show no detectable differences. These data indicate that the U(L)17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the U(L)17 gene product, an anti-U(L)17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent Mr 77,000 and weakly with a protein of apparent Mr 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted U(L)17 protein. We therefore conclude that the U(L)17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.     

The inverted repeat regions of the simian varicella virus and varicella-zoster virus genomes have a similar genetic organization

Simian varicella virus (SVV) causes a varicella-like disease in nonhuman primates. The DNA sequence and genetic organization of the inverted repeat region (RS) of the SVV genome was determined. The SVV RS is 7559 bp in size with 56% guanine+cytosine (G+C) content and includes 3 open reading frames (ORFs). The SVV RS1 ORF encodes a 1279 amino acid (aa) protein with 58 and 39% identity to the varicella-zoster virus (VZV) gene 62 and herpes simplex virus type 1 (HSV-1) ICP4 homologs, respectively. The predicted 261 aa SVV RS2 polypeptide possesses 52% identity with the VZV gene 63 homolog and 23% identity with the HSV-1 ICP22. The SVV RS3 encodes a 187 aa polypeptide with 56% and 28% identity to the VZV gene 64 and the HSV-1 US10 homologs, respectively, and includes an atypical zinc finger motif. A G+C-rich 16 base-pair (bp) sequence which is repeated 7 times and a putative SVV origin of replication were identified between the RS1 and RS2 ORFs. Comparison with the VZV RS indicates the SVV and VZV RS regions are similar in size and genetic organization.     

Regulated expression of a Sindbis virus replicon by herpesvirus promoters

We describe the use of herpesvirus promoters to regulate the expression of a Sindbis virus replicon (SINrep/LacZ). We isolated cell lines that contain the cDNA of SINrep/LacZ under the control of a promoter from a herpesvirus early gene which requires regulatory proteins encoded by immediate-early genes for expression. Wild-type Sindbis virus and replicons derived from this virus cause death of most vertebrate cells, but the cells discussed here grew normally and expressed the replicon and beta-galactosidase only after infection with a herpesvirus. Vero cell lines in which the expression of SINrep/LacZ was regulated by the herpes simplex virus type 1 (HSV-1) infected-cell protein 8 promoter were generated. One Vero cell line (V3-45N) contained, in addition to the SINrep/LacZ cDNA, a Sindbis virus-defective helper cDNA which provides the structural proteins for packaging the replicon. Infection of V3-45N cells with HSV-1 resulted in the production of packaged SINrep/LacZ replicons. HSV-1 induction of the Sindbis virus replicon and packaging and spread of the replicon led to enhanced expression of the reporter gene, suggesting that this type of cell could be used to develop sensitive assays to detect herpesviruses. We also isolated a mink lung cell line that was transformed with SINrep/LacZ cDNA under the control of the promoter from the human cytomegalovirus (HCMV) early gene UL45. HCMV carries out an abortive infection in mink lung cells, but it was able to induce the SINrep/LacZ replicon. These results, and those obtained with an HSV-1 mutant, demonstrate that this type of signal amplification system could be valuable for detecting herpesviruses for which a permissive cell culture system is not available.     

Peptide display on functional tailspike protein of bacteriophage P22

The tailspike protein (TSP) of Salmonella typhimurium P22 bacteriophage is a multifunctional homotrimer, 6 copies of which are non-covalently attached to the capsid to form the virion tail in the last reaction of phage assembly. An antigenic peptide of foot-and-mouth disease virus (FMDV), aa 134-156 of protein VP1, has been joined to the carboxy terminus of TSP, and produced as a fusion protein in Escherichia coli directed by the trp promoter. The resulting fusion protein is soluble, stable, non-toxic, and can be easily purified by standard procedures. Moreover, both the endorhamnosidase and capsid assembly activities of the TSP are conserved, permitting the fusion protein to reconstitute infectious viruses by in vitro association with tailless particles. In both free TSP and P22 chimeric virions, the foreign peptide is solvent-exposed and highly antigenic, indicating that P22 TSP could be an appropriate carrier protein for multimeric peptide display.     

A novel herpesvirus amplicon system for in vivo gene delivery

For gene therapy approaches to succeed, improved vector systems are needed that combine a large carrying capacity with high transduction efficiency in vivo. Towards this goal, we have developed a novel herpes simplex virus (HSV) amplicon vector, pHE, which contains an HSV-1 replication origin (ori S) and packaging sequence that permit vector replication and packaging into HSV-1 capsids. The vector also contains the Epstein-Barr virus (EBV) unique latent replication origin (ori P) sequence and a modified EBNA-1 gene to allow the vector to be maintained as an episome in transfected E5 helper cells. This system allows for efficient packaging of high-titer vector since the E5 cells are first selected for the presence of the pHE vector before helper virus infection. The infectious pHE vector has efficient transgene expression in a variety of human cell lines in vitro. Stereotactic injection of pHE vector supernatant into the rat brain resulted in high, localized reporter gene expression. Finally, the pHE vector could carry a stable 21 kb DNA payload into HSV virions. This pHE vector system should have a broad range of gene transfer applications.