Homayon Ghiasi

Region of Herpes Simplex Virus Type 1 Latency-Associated Transcript Sufficient for Wild-Type Spontaneous Reactivation Promotes Cell Survival in Tissue Culture

ABSTRACT The latency-associated transcript (LAT) is the only abundant herpes simplex virus type 1 (HSV-1) transcript expressed during latency. In the rabbit eye model, LAT null mutants do not reactivate efficiently from latency. We recently demonstrated that the LAT null mutantdLAT2903 induces increased levels of apoptosis in trigeminal ganglia of infected rabbits compared to LAT+strains (G.-C. Perng, C. Jones, J. Ciacci-Zarella, M. Stone, G. Henderson, A. Yokht, S. M. Slanina, F. M. Hoffman, H. Ghiasi, A. B. Nesburn, and C. S. Wechsler, Science 287:1500–1503, 2000).The same study also demonstrated that a plasmid expressing LAT nucleotides 301 to 2659 enhanced cell survival of transfected cells after induction of apoptosis. Consequently, we hypothesized that LAT enhances spontaneous reactivation in part, because it promotes survival of infected neurons. Here we report on the ability of plasmids expressing different portions of the 5′ end of LAT to promote cell survival after induction of apoptosis. A plasmid expressing the first 1.5 kb of LAT (LAT nucleotides 1 to 1499) promoted cell survival in neuro-2A (mouse neuronal) and CV-1 (monkey fibroblast) cells. A plasmid expressing just the first 811 nucleotides of LAT promoted cell survival less efficiently. Plasmids expressing the first 661 nucleotides or less of LAT did not promote cell survival. We previously showed that a mutant expressing just the first 1.5 kb of LAT has wild-type spontaneous reactivation in rabbits, and a mutant expressing just the first 811 nucleotides of LAT has a reactivation frequency higher than that of dLAT2903 but lower than that of wild-type virus. In addition, mutants reported here for the first time, expressing just the first 661 or 76 nucleotides of LAT, had spontaneous reactivation indistinguishable from that of the LAT null mutantdLAT2903. In summary, these studies provide evidence that there is a functional relationship between the ability of LAT to promote cell survival and its ability to enhance spontaneous reactivation.

Identification of a major regulatory sequence in the latency associated transcript (LAT) promoter of herpes simplex virus type 1 (HSV-1)

The latency associated transcript (LAT) gene is the only viral genomic region that is abundantly transcribed during herpes simplex virus type 1 (HSV-1) neuronal latency. As such, it may play an important role in HSV-1 latency and/or reactivation. The regulation of the LAT gene is complex and appears to include a combination of positive and negative functional elements in and near the LAT promoter. In this study, transient CAT assays were used to map the minimal promoter necessary for constitutive activity in neuronal and nonneuronal cells to between nucleotide positions -161 and -2 (relative to the start of LAT transcription). The region from -283 to -161 was able to slightly increase promoter activity of the minimal promoter and appeared to have a larger effect in neuronal derived cells. Gel-shift experiments using nuclear extracts from neuronal and nonneuronal derived cells detected a major factor that bound specifically to the -161 to -2 probe. We designated this factor LAT promoter binding factor (LPBF). Two additional minor factors also bound specifically to the minimal promoter. DNase I footprint analysis and gel-shift competition experiments demonstrated that LPBF bound to a region that includes the palindromic sequence CCACGTGG located at nucleotides -72 to -65. Deletion of this palindrome resulted in a loss of binding of LPBF from the minimal promoter region and an 8- to 30-fold reduction in promoter activity in both neuronal and nonneuronal cells. Thus, LPBF appears to play a major role in LAT promoter regulation.

Vaccination of mice with herpes simplex virus type 1 glycoprotein D DNA produces low levels of protection against lethal HSV-1 challenge

The herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene was inserted into vectors pSVL or pRc/CMV under control of the SV40 late promoter or the human cytomegalovirus major immediate-early promoter, respectively. Intramuscular injection of mice with these gD-containing plasmids appeared to induce low levels of serum anti-gD antibody, as judged by the appearance of low levels of anti-HSV-1-neutralizing antibody and anti-gD ELISA responses in the serum of gD-DNA-vaccinated mice. As previously reported in other virus systems, vaccination with vector DNA also induced ELISA and neutralizing antibody titers. However, these titers were lower than those induced by the gD-containing plasmids. The ELISA and neutralization titers induced by the vectors appeared to be non-specific rather than directed at specific HSV-1 proteins, since serum from mice vaccinated with plasmid-gD immunoprecipitated significant amounts of gD from extracts of HSV-1-infected cells, while serum from mice vaccinated with vectors was unable to immunoprecipitate gD or any other obvious HSV-1 proteins. Neither pSVL-gD nor pRc/CMV-gD induced detectable lymphocyte proliferative or CTL responses. Vaccination with pSVL-gD provided a significant (P = 0.04, Fishers exact test), but low level of protection against lethal challenge with HSV-1. Vaccination with pRc/CMV-gD also appeared to provide a low level of protection against challenge, that was statistically significance at the 10% level (P = 0.054, Fishers exact test). Reports from numerous laboratories (including ours) have shown that vaccination with recombinantly expressed gD can provide very high levels of protection against HSV-1 lethal challenge. Thus, the results reported here suggest that vaccination with HSV-1 gD-DNA is not yet a useful alternative to a gD subunit vaccine.

Vaccination with a cocktail of seven recombinantly expressed HSV-1 glycoproteins protects against ocular HSV-1 challenge more efficiently than vaccination with any individual glycoprotein.

Our previous studies have shown that of seven HSV-1 glycoproteins (gB, gC, gD, gE, gG, gH and gI) individually expressed in baculovirus, vaccination with gD provides the best protection against HSV-1 challenge. To establish whether vaccination with a mixture of these seven expressed glycoproteins would provide better protection against HSV-1 challenge than vaccination with gD alone, we determined the level of protection afforded by vaccination with a cocktail of the seven expressed glycoproteins. The amount of each of the seven expressed glycoproteins in the mixture was equivalent to one-seventh the amount of gD used in the gD alone vaccination. Thus, the total amount of glycoprotein was the same for the cocktail and gD alone vaccine. For neutralizing antibody titer, delayed-type hypersensitivity (DTH), and survival following lethal challenge, no difference was observed between mice vaccinated with all seven glycoproteins and those vaccinated with gD. However, for other criteria, vaccination with all seven glycoproteins appeared to provide better protection than vaccination with gD. Following ocular challenge, virus was not detected at any time in the tears of mice vaccinated with all seven glycoproteins. In contrast, virus was detected in the tears of gD vaccinated mice for up to 3 days post challenge. Mock vaccinated mice had virus in their tears for as long as 10 days. Mice vaccinated with all seven glycoproteins had no eye disease, while gD vaccinated mice had a significant amount of blepharitis. Finally, compared to gD vaccinated mice, the mice vaccinated with all seven glycoproteins were more efficiently protected against the establishment of HSV-1 latency following ocular infection. Our results therefore suggest that while for some protective criteria there was no significant difference between vaccination with gD or seven glycoproteins, vaccination with seven glycoproteins was more efficient in protecting challenged mice against some forms of eye disease, the duration of infection and the establishment of latency.

The effect of latency-associated transcript on the herpes simplex virus type 1 latency-reactivation phenotype is mouse strain-dependent.

Herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) null mutants reactivate poorly in the rabbit ocular model. The situation in mice is less clear. Reports concluding that LAT null mutants reactivate poorly in the mouse explant-induced reactivation (EIR) model are contradicted by a similar number of reports of normal EIR of LAT(-) mutants in mice. To determine if the EIR phenotype might be mouse strain-dependent we infected BALB/c and Swiss Webster mice with LAT(-) or LAT(+) virus and assessed EIR in individual trigeminal ganglia. Compared to LAT(+) virus, LAT(-) virus reactivated poorly in Swiss Webster mice (P<0.05). In contrast, the EIR phenotype of these viruses was similar in BALB/c mice (P>0.1). Thus, LAT appeared to have a much greater impact on the EIR phenotype in Swiss Webster mice than in BALB/c mice. The mouse strain therefore appeared consequential in the HSV-1 EIR phenotype in mice.

Expression of herpes simplex virus type 1 glycoprotein B in insect cells: Initial analysis of its biochemical and immunological properties

A recombinant baculovirus (vAc-gB1) was constructed which expresses the glycoprotein B (gB) gene of herpes simplex virus type 1 (HSV-1). When Sf9 cells were infected with these recombinant viruses, a protein that was close in size to authentic HSV-1 gB was detected by gB polyclonal antibody. The recombinant gB was found on the membrane of Sf9 cells and was susceptible to tunicamycin, glycosidase F (PNGase F) and partially susceptible to Endo-H. Antibodies raised in mice to this recombinant recognized viral gB and neutralized the infectivity of HSV-1 in vitro. Mice inoculated with the recombinant gB were protected from lethal challenge with HSV-1.

Cell surface expression of herpes simplex virus type 1 glycoprotein H in recombinant baculovirus-infected cells

DNA encoding the complete sequence for the herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) was inserted into a baculovirus transfer vector under control of the baculovirus polyhedrin gene promoter. After co-transfection with wild-type baculovirus DNA, recombinants expressing gH were isolated by plaque purification. The baculovirus-expressed HSV-1 gH represented a significant portion of total cellular protein and was several hundred fold more abundant than gH in HSV-1-infected Vero cells. The expressed gH appeared to be glycosylated, since it was similar in size to wild-type HSV-1 gH, was susceptible to both tunicamycin and endoglycosidase-H treatment, and was labeled by [3H]mannose. In contrast to previous reports of gH expressed in mammalian cells, the baculovirus recombinant-expressed gH was abundant on the cell surface as judged by indirect immunofluorescence. To our knowledge, this is the first report of expressed HSV-1 gH being transported to the cell surface in the absence of other HSV-1 gene products and the first report of expressed gH with an apparent molecular weight similar to authentic HSV-1 gH.

Baculovirus-expressed glycoprotein E (gE) of herpes simplex virus type-1 (HSV-1) protects mice against lethal intraperitoneal and lethal ocular HSV-1 challenge

We have constructed a recombinant baculovirus expressing high levels of the herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) in Sf9 cells. The expressed gE migrated on gels as a double band with apparent molecular weights of 68 and 70 kDa. The recombinant gE was glycosylated based on its susceptibility to tunicamycin treatment and was transported to the membrane of Sf9 cells based on indirect immunofluorescence. Mice vaccinated with gE developed high serum titers of HSV-1-neutralizing antibodies based on plaque reduction assays. gE vaccination also induced a strong delayed type hypersensitivity (DTH) response to HSV-1. In addition, mice vaccinated with the recombinant gE were protected from both intraperitoneal and ocular lethal HSV-1 challenge. To our knowledge, this is the first report in which vaccination with gE was shown to induce high neutralizing antibody titers, a DTH response, or protection against lethal HSV-1 challenge.

Perforin pathway is essential for protection of mice against lethal ocular HSV-1 challenge but not corneal scarring

Perforin (cytolysin; pore-forming protein) is expressed in both CD8(+) cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, and is a major factor responsible for the cytolytic activities of these cells. Both CD8(+) T-cells and NK cells are important in eliminating cells infected with certain viruses. We examined the role of perforin in a mouse model of HSV-1 infection using perforin-deficient mice. Naïve perforin knockout (perforin(0/0)) mice were more susceptible to lethal HSV-1 ocular challenge (60% survival), than naïve parental C57BL/6 (100% survival). In contrast, both C57BL/6 and perforin(0/0) mice had similar levels of HSV-1 induced corneal scarring. Vaccination of perforin(0/0) mice induced a significantly higher HSV-1 neutralizing antibody titer than vaccination of C57BL/6 mice, and the mice were completely protected against lethal ocular challenge. These results suggest that in naïve mice ocularly challenged with HSV-1, the perforin pathway was involved in protection against death, but not in protection against corneal scarring.

Vaccination with different HSV-1 glycoproteins induces different patterns of ocular cytokine responses following HSV-1 challenge of vaccinated mice.

We previously reported that vaccination of BALB/c mice with different baculovirus expressed HSV-1 glycoproteins induced varying degrees of protection against HSV-1 ocular challenge, ranging from complete protection to no protection, to exacerbation of eye disease. To correlate specific local immune responses with protection and exacerbation of corneal scarring, we examined immune cell infiltrates in the cornea after ocular HSV-1 challenge of vaccinated mice. Mice were vaccinated with gD, which completely protects against corneal scarring, gG, which produces no protection against corneal scarring, or gK, which exacerbates corneal scarring. Cryostat sections of cornea were taken at different times after challenge and examined for infiltrating cells containing IL-2, IL-4, IFN-gamma, IL-6, or TNF-alpha. No corneal infiltrates were seen before challenge or 1 day after ocular challenge in any groups. By days 3-7, many cells containing IL-4 and IFN-gamma, but few cells containing IL-2, had infiltrated into the corneas of gG or mock vaccinated mice. At the same times, many cells containing IL-2, but few cells containing IL-4 or IFN-gamma, were seen in the corneas of gD vaccinated mice. In contrast, the corneas of mice vaccinated with gK contained large amounts of IL-2, IFN-gamma, and IL-4. Our results suggest that: (1) corneas from gD vaccinated mice had no corneal disease and developed a response highly biased toward IL-2 responses; (2) corneas from gG or mock vaccinated eyes had significant corneal disease and developed a mostly IL-4 and IFN-gamma cytokine response; and (3) corneas from gK vaccinated mice had exacerbated corneal disease and developed strong IL-2, IL-4 and IFN-gamma cytokine responses.