Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have promise for genetic intervention in the brain, but several problems caused by the helper virus have compromised their utility. To develop a helper virus-free packaging system for these vectors, the DNA cleavage/packaging signals were deleted from a set of cosmids that represents the HSV-1 genome. Following cotransfection into cells, this modified cosmid set supported replication and packaging of vector DNA. However, in the absence of the DNA cleavage/packaging signals, the HSV-1 genome was not packaged, and consequently vector stocks were free of detectable helper virus. In the absence of helper virus, the vectors efficiently infected rat neural cells in culture or in the brain with minimal cytopathic effects. beta-galactosidase-positive cells were observed for at least 1 month in vivo, and vector DNA persisted for this period. This system may facilitate studies on neuronal physiology and potential therapeutic applications.     

Genetic editing of herpes simplex virus 1 and Epstein-Barr herpesvirus genomes by human APOBEC3 cytidine deaminases in culture and in vivo

Human APOBEC3 cytidine deaminases target and edit single-stranded DNA, which can be of viral, mitochondrial, or nuclear origin. Retrovirus genomes, such as human immunodeficiency virus (HIV) genomes deficient in the vif gene and the hepatitis B virus genome, are particularly vulnerable. The genomes of some DNA viruses, such as human papillomaviruses, can be edited in vivo and in transfection experiments. Accordingly, herpesviruses should be no exception. This is indeed the case for herpes simplex virus 1 (HSV-1) in tissue culture, where APOBEC3C (A3C) overexpression can reduce virus titers and the particle/PFU ratio ～10-fold. Nonetheless, A3A, A3G, and AICDA can edit what is presumably a small fraction of HSV genomes in an experimental setting without seriously impacting the viral titer. Hyperediting was found in HSV genomes recovered from 4/8 uncultured buccal lesions. The phenomenon is not restricted to HSV, since hyperedited Epstein-Barr virus (EBV) genomes were readily recovered from 4/5 established cell lines, indicating that episomes are vulnerable to editing. These findings suggest that the widely expressed A3C cytidine deaminase can function as a restriction factor for some human herpesviruses. That the A3C gene is not induced by type I interferons begs the question whether some herpesviruses encode A3C antagonists.     

A role for herpesvirus entry mediator as the receptor for herpes simplex virus 1 entry into primary human trabecular meshwork cells

The human eye is an important target for infection with herpes simplex virus 1 (HSV-1). Damage to cells forming the trabeculum of the eye by HSV-1 infection could contribute to the development of glaucoma, a major blinding disease. Primary cultures of human trabecular meshwork cells were used as an in vitro model to demonstrate the ability of HSV-1 to enter into and establish a productive infection of the trabeculum. Blocking of entry by anti-herpesvirus entry mediator (HVEM) antibody implicated HVEM as the major receptor for HSV-1 infection.     

Extensive homology between the herpes simplex virus type 2 glycoprotein F gene and the herpes simplex virus type 1 glycoprotein C gene.

The region of the herpes simplex virus type 2 (HSV-2) genome which maps colinearly with the HSV-1 glycoprotein C (gC) gene has been cloned, and the DNA sequence of a 2.29-kilobase region has been determined. Contained within this sequence is a major open reading frame of 479 amino acids. The carboxyterminal three-fourths of the derived HSV-2 protein sequence showed a high degree of sequence homology to the HSV-1 gC amino acid sequence reported by Frink et al. (J. Virol. 45:634-647, 1983). The amino-terminal region of the HSV-2 sequence, however, showed very little sequence homology to HSV-1 gC. In addition, the HSV-1 gC sequence contained 27 amino acids in the amino-terminal region which were missing from the HSV-2 protein. Computer-assisted analysis of the hydrophilic and hydrophobic properties of the derived HSV-2 sequence demonstrated that the protein contained structures characteristic of membrane-bound glycoproteins, including an amino-terminal signal sequence and carboxy-terminal hydrophobic transmembrane domain and charged cytoplasmic anchor. The HSV-2 protein sequence also contained seven putative N-linked glycosylation sites. These data, in conjunction with mapping studies of Para et al. (J. Virol. 45:1223-1227, 1983) and Zezulak and Spear (J. Virol. 49:741-747, 1984), suggest that the protein sequence derived from the HSV-2 genome corresponds to gF, the HSV-2 homolog of HSV-1 gC.     

Expression of herpes simplex virus type 1 glycoproteins in interferon-treated human neuroblastoma cells.

Human alpha interferon (IFN) significantly inhibits the replication of herpes simplex virus type 1 in human neuroblastoma cells. This inhibitory effect can be blocked by pretreatment with antiserum to IFN. We observed no significant differences in the expression of major nucleocapsid proteins, including VP5, between IFN-treated and untreated neuroblastoma cells. Electron micrographs demonstrated that there were distinct viral nucleocapsids within IFN-treated neuroblastoma cells. The expression of glycoproteins B and E was significantly reduced in these IFN-treated cells. On the other hand, glycoprotein D, although reduced in quantity, was expressed after IFN treatment. An immunofluorescence assay of the IFN-treated and virus-infected cells detected glycoprotein D in the Golgi complexes and in the nuclear membranes. Our results indicate that human alpha IFN may be useful in the study of gene expression in IFN-treated cells of neuronal origin.     

Human natural killer cells limit replication of herpes simplex virus type 1 in vitro

Studies were undertaken to determine whether natural killer (NK) cells could inhibit the replication of herpes simplex virus type 1 (HSV-1) in culture. In the absence of effector cells, HSV-1 was found to replicate in fibroblasts with up to a 100-fold increase in virus titer from 4 to 16 hr after incubation at 37 degrees C. Human peripheral blood mononuclear cells were found to limit virus replication in a dose-dependent manner, with the greatest inhibition being observed at the highest concentration evaluated: i.e., an effector:target ratio of 800:1. The antiviral effect was not observed when nonactivated or virus-activated mononuclear cells were added to the virus preparations at the end (instead of the beginning) of the assay period, indicating that the observed effect was not due to a nonspecific toxicity of soluble factors released from freeze-thawed effectors. Neither was inhibition of HSV-1 replication due to the generation of interferon (IFN) during the NK assay, because the addition of anti-IFN did not abrogate the antiviral effect. Thus, the inhibition of viral replication was most likely due to a cytotoxic effector rather than to release of soluble factors. The effector cells responsible for limiting HSV-1 replication were shown to be NK cells by a number of criteria. Mononuclear cells from both HSV-1 seropositive and seronegative donors limited virus replication; their activity could be boosted by pretreatment of effector cells with IFN; the effector cells which limited virus replication were found in Percoll gradient fractions enriched for large granular lymphocytes; and the effector cells shared the cell surface phenotype of NK cells--they were enriched in populations depleted of T cells by panning with Leu-4 and were depleted of activity by treatment with the anti-NK antibody Leu-11b plus complement. We conclude that human NK cells are capable of recognizing and lysing HSV-1-infected target cells before infectious virus progeny are generated. These results suggest that NK cells, acting early in the course of an infection, might serve to limit HSV-1 replication and therefore reduce the virus load in the host before the development of the adaptive immune response and clearance of the infection.     

The mode of entry of herpes simplex virus type 1 into Vero cells

The mode of entry of herpes simplex virus type 1 (HSV-1) into Vero cells was investigated quantitatively with biological techniques. The entry of virus occurred rapidly when the virus-adsorbed cells were incubated at 37 C. The kinetics of virus entry was found to be similar to that of the process of uncoating, indicating that the uncoating of HSV-1 occurs simultaneously with the entry of virus into the cell. Experiments with ammonium chloride revealed that acidity in endosomes is not necessary for the entry or uncoating of HSV-1, in contrast with the cases of enveloped RNA viruses. In addition, endocytosis of the virus seems to be one of the processes of entry for HSV-1. However, the kinetics of endocytosis showed that the cell-bound virus is endocytosed gradually and suggested that the endocytosis of HSV-1 does not lead the virus to an uncoating process. These results are most consistent with a mechanism of entry for HSV-1 involving fusion of the viral envelope with the plasma membrane of the host cell.     

A novel human artificial chromosome gene expression system using herpes simplex virus type 1 vectors

Human artificial chromosome (HAC) vectors are an important gene transfer system for expression and complementation studies. We describe a significant advance in HAC technology using infectious herpes simplex virus type 1 (HSV-1) amplicon vectors for delivery. This highly efficient method has allowed gene-expressing HACs to be established in glioma-, kidney- and lung-derived cells. We also developed an HSV-1 hypoxanthine phosphoribosyltransferase (HPRT) HAC vector, which generated functional HPRT-expressing HACs that complemented the genetic deficiency in human cells. The transduction efficiency of the HSV-1 HAC amplicons is several orders of magnitude higher than lipofection-mediated delivery. Studies on HAC stability between cell types showed important differences that have implications for HAC development and gene expression in human cells. This is the first report of establishing gene-expressing HACs in human cells by using an efficient, high-capacity viral vector and by identifying factors that are involved in cell-type-specific HAC instability. The work is a significant advance for HAC technology and the development of HAC gene expression systems in human cells.     

Persistence of herpes simplex virus type 1 in rat neurotumor cells.

Herpes simplex virus type 1 (HSV-1) infection of a rat central nervous system tumor cell line led to almost complete destruction of the cells. Cells that survived the infection could be isolated and shown to produce infectious HSV particles for variable lengths of time in culture ranging from 20 to 57 passages. Even though infectious virus production eventually ceased, the cell lines continued to produce herpes-specified proteins as measured by immunological techniques. These cells also showed herpesvirus-like structures in the electron microscope. The persistently infected cells that produced HSV antigens and bore HSV sequences were resistant to superinfection by HSV-1. The resistance was not due to failure of adsorption of the virus or to the production of interferon by the cells. The nature of the block in HSV replication in these neurotumor cells, which contain and partially express the HSV genome, is unknown, but may offer an interesting parallel to the known latency of HSV in neural tissues.     

Imaging herpes simplex virus type 1 amplicon vector-mediated gene expression in human glioma spheroids

Vectors derived from herpes simplex virus type 1 (HSV-1) have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector-mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fluorescent protein (HSV-GFP). After infection or microscopy-guided vector injection of glioma spheroids at various spheroid sizes, injection pressures and injection times, the extent of HSV-1 vector-mediated gene expression was investigated via laser scanning microscopy. Infection of spheroids with HSV-GFP demonstrated a maximal depth of vector-mediated GFP expression at 70 to 80 μm. A > 80% transduction efficiency was reached only in small spheroids with a diameter of < 150 μm. Guided vector injection into the spheroids showed transduction efficiencies ranging between < 10 and > 90%. The results demonstrated that vector-mediated gene expression in glioma spheroids was strongly dependent on the mode of vector application-injection pressure and injection time being the most important parameters. The assessment of these vector application parameters in tissue models will contribute to the development of safe and efficient gene therapy protocols for clinical application.     

Latent herpes simplex virus type 1 gene expression in ganglia innervating the human gastrointestinal tract.

Using in situ hybridization, we demonstrated that latent herpes simplex virus type 1 (HSV-1) gene expression is prevalent in human adult nodose ganglia. This suggests that infection of gastrointestinal sensory nerves, probably through swallowed virus-laden oral secretions, occurs commonly and that HSV-1 reactivating from this site may play a role in recurrent gastrointestinal disorders.     

Entry pathways of herpes simplex virus type 1 into human keratinocytes are dynamin- and cholesterol-dependent

Herpes simplex virus type 1 (HSV-1) can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s). Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol.     

High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.