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Biomedical subjects

J C Glorioso

Publications and source records attributed to J C Glorioso.

At least 55 records · Page 3Linked to original sources
PubMed1999

Antihyperalgesic effects of infection with a preproenkephalin-encoding herpes virus.

To test the utility of gene therapeutic approaches for the treatment of pain, a recombinant herpes simplex virus, type 1, has been engineered to contain the cDNA for an opioid peptide precursor, human preproenkephalin, under control of the human cytomegalovirus promoter. This virus and a similar recombinant containing the Escherichia coli lacZ gene were applied to the abraded skin of the dorsal hindpaw of mice. After infection, the presence of beta-galactosidase in neuronal cell bodies of the relevant spinal ganglia (lacZ-containing virus) and of human proenkephalin (preproenkephalin-encoding virus) in the central terminals of these neurons indicated appropriate gene delivery and expression. Baseline foot withdrawal responses to noxious radiant heat mediated by Adelta and C fibers were similar in animals infected with proenkephalin-encoding and beta-galactosidase-encoding viruses. Sensitization of the foot withdrawal response after application of capsaicin (C fibers) or dimethyl sulfoxide (Adelta fibers) observed in control animals was reduced or eliminated in animals infected with the proenkephalin-encoding virus for at least 7 weeks postinfection. Hence, preproenkephalin cDNA delivery selectively blocked hyperalgesia without disrupting baseline sensory neurotransmission. This blockade of sensitization was reversed by administration of the opioid antagonist naloxone, apparently acting in the spinal cord. The results demonstrate that the function of sensory neurons can be selectively altered by viral delivery of a transgene. Because hyperalgesic mechanisms may be important in establishing and maintaining neuropathic and other chronic pain states, this approach may be useful for treatment of chronic pain and hyperalgesia in humans.

Animals↗
PubMed1999

Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws.

Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery to diseased joints offers the prospect of achieving high, local concentrations of a therapeutic gene product in a sustained manner, while minimizing exposure of nontarget organs. We report that a single administration of a modified adenovirus encoding the Epstein-Barr-derived homologue of IL-10 can suppress the development of disease for extended periods of time when injected locally within the periarticular tissue surrounding the ankle joints of mice with collagen type II-induced arthritis. Furthermore, we show that injection of an adenoviral vector carrying the IL-10 gene into a single paw can suppress development of arthritis in other, noninjected paws of the same individual. The systemic protection resulting from local gene therapy occurred in the absence of detectable levels of viral IL-10 in the serum. Circulating Ab levels to heterologous collagen were unaffected; however, treatment with viral IL-10 significantly suppressed the development of Abs to autologous mouse type II collagen. Thus, the treatment of a single joint by local delivery of the vIL-10 gene may protect multiple joints of the same individual while avoiding deleterious side effects often associated with systemic therapy.

Adenoviridae↗
PubMed1999

Replication-defective herpes simplex virus vectors for neurotrophic factor gene transfer in vitro and in vivo.

We report here the construction and the use of two replication-defective herpes simplex virus vectors, SH FGF-2 and TH FGF-2, which efficiently transfer and express the cDNA for fibroblast-growth-factor-2 (FGF-2) in vitro and in vivo. One mutant was deleted in the immediate-early gene encoding ICP4; the other was deleted in ICP4, ICP22 and ICP27. FGF-2--or the control gene lacZ--were inserted in tk, under control of the human cytomegalovirus immediate-early promoter. Infection of Vero cells with SH FGF-2 induced a dramatic increase in FGF-2 protein levels in the first 2 days after infection, with a rapid return to baseline levels within day 4. In contrast, infection of Vero cells with TH FGF-2 displayed FGF-2 levels progressively increasing up to days 4-5, and slowly returning to baseline. Protein extracts of cells infected with TH FGF-2 induced neuronal differentiation of PC12 cells, indicating that the newly synthesized FGF-2 was biologically active. Robust transient transgene expression was also observed in the rat hippocampus after stereotaxical inoculation of TH FGF-2, but not of TH lacZ or of SH vectors. Enhanced gene expression both in vitro and in vivo by the triple immediate-early gene deletion mutant might be attributed to reduced vector cytotoxicity. The present data suggest that TH FGF-2 is suitable for studies of FGF-2 involvement in neurological disorders.

Animals↗
PubMed1999

Herpes simplex virus type 1 vector-mediated expression of nerve growth factor protects dorsal root ganglion neurons from peroxide toxicity.

Nerve growth factor beta subunit (beta-NGF) transgene delivery and expression by herpes simplex virus type 1 (HSV-1) vectors was examined in a cell culture model of neuroprotection from hydrogen peroxide toxicity. Replication-competent (tk- K mutant background) and replication-defective (ICP4(-);tk- S mutant background) vectors were engineered to contain the murine beta-NGF cDNA under transcriptional control of either the human cytomegalovirus immediate-early gene promoter (HCMV IEp) (e.g., KHN and SHN) or the latency-active promoter 2 (LAP2) (e.g., KLN and SLN) within the viral thymidine kinase (tk) locus. Infection of rat B103 and mouse N2A neuronal cell lines, 9L rat glioma cells, and Vero cells with the KHN or SHN vectors resulted in the production of beta-NGF-specific transcripts and beta-NGF protein reaching a maximum at 3 days postinfection (p.i.). NGF protein was released into the culture media in amounts ranging from 10.83 to 352.86 ng/ml, with the highest levels being achieved in B103 cells, and was capable of inducing neurite sprouting of PC-12 cells. The same vectors produced high levels of NGF in primary dorsal root ganglion (DRG) cultures at 3 days. In contrast to HCMV IEp-mediated expression, the LAP2-NGF vectors showed robust expression in primary DRG neurons at 14 days. The neuroprotective effect of vector produced NGF was assessed by its ability to inhibit hydrogen peroxide-induced neuron toxicity in primary DRG cultures. Consistent with the kinetics of vector-mediated NGF expression, HCMV-NGF vectors were effective in abrogating the toxic effects of peroxide at 3 but not 14 days p.i. whereas LAP2-NGF vector transduction inhibited apoptosis in DRG neurons at 14 days p.i. but was ineffective at 3 days p.i. Similar kinetics of NGF expression were observed with the KHN and KLN vectors in latently infected mouse trigeminal ganglia, where high levels of beta-NGF protein expression were detected at 4 wks p.i. only from the LAP2; HCMV-NGF-driven expression peaked at 3 days but could not be detected during HSV latency at 4 weeks. Together, these results indicate that (i) NGF vector-infected cells produce and secrete mature, biologically active beta-NGF; (ii) vector-synthesized NGF was capable of blocking peroxide-induced apoptosis in primary DRG cultures; and (iii) the HCMV-IEp functioned to produce high levels of NGF for several days; but (iv) only the native LAP2 was capable of long-term expression of a therapeutic gene product in latently infected neurons in vivo.

Animals↗
PubMed1999

Genetic studies exposing the splicing events involved in herpes simplex virus type 1 latency-associated transcript production during lytic and latent infection.

Herpes simplex virus type 1 (HSV-1) establishes latency in sensory neurons, a state in which the viral lytic genes are silenced and only the latency locus is transcriptionally active, producing the 2. 0- and 1.5-kb latency-associated transcripts (LATs). Previous experimental evidence indicates that the LATs are stable introns, and it has been reported that LAT formation is abolished by debilitating substitution mutations in the predicted splice sites during lytic infection but not latency (J. L. Arthur et al., J. Gen. Virol. 79:107-116, 1998). We have independently studied a set of deletion mutations to explore the roles of the proposed splice sites during lytic and latent infection. HSV-1 mutant viruses missing the invariant intron-terminal 5'-G(T/C) or 3'-AG dinucleotides were analyzed for LAT formation during lytic infection in vitro, when only the 2-kb LAT is produced, and during latency in mouse trigeminal ganglia, where both LATs are expressed. Northern blot analysis of total RNAs from different productively infected cell lines showed that the lytic (2-kb) LAT was not expressed by the various splice site deletion mutants. In vivo studies using a mouse eye model of latency similarly showed that the latent (2- and 1. 5-kb) LATs were not expressed by the mutants. PCR analysis with primers flanking the LAT sequence revealed the expected splice junction for LAT excision in RNA from sensory neurons latently infected with wild-type but not mutant virus. Using a virus mutant deleted in the splicing signals flanking the 556-bp region of LAT whose absence distinguishes the 1.5- and 2-kb LATs, we observed selective elimination of 1.5-kb LAT expression in latency, supporting previous suggestions that the internal region is removed by splicing. Taken together, these results demonstrate that the 2-kb LAT is formed during both lytic and latent infection by splicing at the predicted splice sites and that an additional splicing event is involved in the latency-restricted production of the 1.5-kb LAT. We have also mapped the 3' end of the lytic 2-kb LAT and discuss our results in the context of previous models addressing the unusual stability of the LATs.

Animals↗
PubMed1998

Enhanced tumor cell killing in the presence of ganciclovir by herpes simplex virus type 1 vector-directed coexpression of human tumor necrosis factor-alpha and herpes simplex virus thymidine kinase.

Past studies have documented the promise of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) suicide gene therapy as a potential antitumor treatment. HSV-TK converts the pro-drug ganciclovir (GCV) into a toxic nucleotide analogue, the incorporation of which into cellular DNA blocks cell proliferation. In this report, we have examined the hypothesis that the effectiveness of HSV-TK suicide gene therapy can be enhanced by coexpression of the antitumor cytokine human tumor necrosis factor-alpha (TNF-alpha) from the same replication-defective HSV-1 vector. In vitro testing demonstrated that TNF-alpha expression from this vector potentiated the killing of both TNF-alpha-sensitive L929 tumor cells and TNF-alpha-resistant U-87 MG cells in the presence of GCV. Furthermore, treatment of established intradermal L929 tumors in vivo with the TNF-alpha/TK vector and GCV resulted in prolonged animal survival compared with treatment with parental HSV-TK vector in the presence or absence of GCV. Treatment of intracerebral U-87 MG tumors showed a clear benefit of TK therapy, but a significant further increase in survival using the TNF-alpha vector could not be demonstrated. We found that potentiation of cell killing in vitro required intracellular TNF-alpha because purified protein added to the culture medium of cells infected with HSV-TK vector failed to have the same effect. Accordingly, potentiation in vivo should depend on efficient infection, but immunohistochemical analysis indicated that virus administration by U-87 MG intratumoral injection was inadequate, resulting in an estimated <1% infection of all tumor cells. Moreover, the majority of infected tumor cells were localized at the tumor margin. Together, these results suggest that TNF-enhanced tk gene therapy should provide a useful treatment for TNF-alpha-sensitive tumors and perhaps also for TNT-alpha-resistant tumors if vector delivery can be improved to increase the percentage of transduced tumor cells.

Animals↗
PubMed1998

Efficient gene delivery into epstein-barr virus (EBV)-transformed human B cells mediated by replication-defective herpes simplex virus-1 (HSV-1): A gene therapy model for EBV-related B cell malignancy.

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.

Antiviral Agents↗
PubMed1998

Herpes virus-mediated preproenkephalin gene transfer to the amygdala is antinociceptive.

To evaluate the role of the amygdala in pain modulation and opioid-mediated antinociception, a recombinant, replication-defective herpes virus carrying the human preproenkephalin cDNA was injected bilaterally into the rat amygdala. Four days after gene delivery nociceptive behavior was assessed by the formalin test. Rats infected with the virus expressing preproenkephalin showed a selective, naloxone-reversible abolition of phase 2 flinching behavior compared to rats infected with a control virus. The results implicate the amygdala in the control of pain and in opioid analgesia and demonstrate the use of recombinant herpes viruses as tools for studying gene function in specific neural pathways of the central nervous system.

Amygdala↗
PubMed1998

Modulation of mucosal and systemic immunity by enteric administration of nonreplicating herpes simplex virus expressing cytokines.

In this report the ability of enteric immunization with recombinant replication deficient (ICP4-/-) HSV expressing IFN gamma to generate protection and modulate mucosal and systemic immunity was evaluated. ICP4-/-HSV, ICP4-/-HSV expressing IL4, live replicating, and uv HSV were used as controls. Following enteric administration of live HSV, a Th1 cytokine response was induced in the spleen, while both Th1 and notable Th2 cytokine production were detected at mucosal sites. Modulation of mucosal and systemic immune response was achieved when nonreplicating recombinant HSV viruses expressing cytokines were used. Compared to the control replication defective viruses, decreased frequency of Th2 cytokine producing cells in Peyer's patches was observed following enteric administration of nonreplicating HSV expressing IFN gamma. When IFN gamma expressing virus was given enterically, modulation was observed at the systemic level, measured by ELISPOT for cytokine producing cells, ELISA from the in vitro restimulated splenic cell cultures, and by the increase of the IgG2a/IgG1 ratio in the serum. This report provides evidence that replication defective viruses expressing cytokine genes in contrast to uv HSV, are immunogenic when administered enterically and can generate significant immunomodulatory effects at the mucosal and systemic levels.

Animals↗
PubMed1998

High-efficacy thymidine kinase gene transfer to ovarian cancer cell lines mediated by herpes simplex virus type 1 vector.

In this report, a replication-defective herpes simplex virus type 1 (HSV-1) vector has been employed to deliver the Escherichia coli LacZ and HSV thymidine kinase (HSVtk) genes to six human ovarian carcinoma cell lines and the efficacy of gene transfer compared to that of adenoviral vectors in vitro. The transduction efficiency of the LacZ-containing virus TOZ.1 was evaluated qualitatively and quantitatively following infection of the different ovarian cancer cell lines. The therapeutic ability of the HSV-T3 vector, which contains the HSVtk gene, was additionally investigated in comparison to the AdCMVHSVTK. Our results show that HSV-1-mediated gene transfer is quantitatively superior to adenoviral vector in five of the six ovarian cancer cell lines at a 100-fold lower dose in vitro. Our preliminary studies suggest that HSV-1 may be a promising alternative vector for ovarian cancer gene therapy.

Cell Division↗
PubMed1998

Implications of maturation for viral gene delivery to skeletal muscle.

Different viral vectors have been analyzed as gene delivery vehicles to skeletal muscle for potentially therapeutic purposes. In this review, we evaluate the application of retroviral, adenoviral, and herpes simplex viral vectors to deliver genes to skeletal muscle and focus on the dramatic loss of viral transduction detected throughout muscle maturation. Recent results suggested that there are several factors involved in the reduced viral transducibility of mature skeletal muscle: muscle cells become post-mitotic in an early stage, the extracellular matrix develops into a physical barrier, and a loss of myoblast mediation occurs since myoblasts progressively become quiescent. Approaches to improve viral gene delivery to mature skeletal muscle may include the use of particular enzymes to increase the permeability of the extracellular matrix, the pre-treatment of the muscle with a myonecrotic agent to induce myoblast mediation, or the application of the myoblast-mediated ex vivo gene transfer.

Adenoviridae↗
PubMed1998

Drug inducible transgene expression in brain using a herpes simplex virus vector.

The ability to regulate transgene expression is likely to be important in the use of gene transfer to treat diseases of the central nervous system (CNS). In order to achieve regulatable gene expression we created a replication-incompetent genomic herpes simplex vector containing a RU486-inducible transactivator and a lacZ reporter gene under transcriptional control of a minimal promoter. Reporter gene expression from the vector was regulated by administration of RU486 in vitro and in vivo. In cell culture half maximal expression was achieved with 10(-8) M RU486, and maximal expression was achieved by 24 h. Following stereotactic inoculation of the vector into rat hippocampus, expression was increased 150-fold by i.p. administration of RU486. This demonstrates that the RU486 system functions as a tight on/off switch for regulating expression of a transgene delivered to the brain via an HSV vector.

Animals↗
PubMed1998

Development of herpes simplex virus replication-defective multigene vectors for combination gene therapy applications.

Some gene therapy applications will require simultaneous expression of multiple gene products to achieve a therapeutic effect. In this study we describe the generation and characterization of replication incompetent herpes simplex virus type 1 (HSV-1) vectors (HX86Z or HX86G) carrying distinct and independently regulated expression cassettes for five transgenes (hIL-2, hGM-CSF, hB7.1, HSV-tk and lacZ or hIFN gamma). The transgenes, representing 12 kb of DNA sequence, were recombined into separate loci of a single mutant virus vector deleted for 11.6 kb of vector sequences representing portions of nine viral genes, ICP4, ICP22, ICP27, ICP47, UL24, UL41, UL44, US10 and US11. Deletion of the immediate--early genes ICP4, ICP22 and ICP27 substantially reduced vector cytotoxicity, prevented early and late viral gene expression and left intact MHC class I antigen expression. Simultaneous expression of multiple transgenes was obtained for up to 7 days in primary human melanoma cells with peak expression at 2-3 days after infection. The transgenes were chosen for their potential to function synergistically in tumor destruction and vaccine gene therapy applications, but the method and vector employed could be applied to other multigene therapy strategies. This study demonstrates the potential for engineering large transgene capacity DNA viruses such as HSV-1 for expression of multiple transgenes.

Defective Viruses↗
PubMed1998

Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons.

Herpes simplex virus type 1 (HSV-1) has many attractive features that suggest its utility for gene transfer to neurons. However, viral cytotoxicity and transient transgene expression limit practical applications even in the absence of viral replication. Mutant viruses deleted for the immediate early (IE) gene, ICP4, an essential transcriptional transactivator, are toxic to many cell types in culture in which only the remaining IE genes are expressed. In order to test directly the toxicity of other IE gene products in neurons and develop a mutant background capable of longterm transgene expression, we generated mutants deleted for multiple IE genes in various combinations and tested their relative cytotoxicity in 9L rat gliosarcoma cells, Vero monkey kidney cells, and primary rat cortical and dorsal root neurons in culture. Viral mutants deleted simultaneously for the IE genes encoding ICP4, ICP22 and ICP27 showed substantially reduced cytotoxicity compared with viruses deleted for ICP4 alone or ICP4 in combination with either ICP22, ICP27 or ICP47. Infection of neurons in culture with these triple IE deletion mutants substantially enhanced cell survival and permitted transgene expression for over 21 days. Such mutants may prove useful for efficient gene transfer and extended transgene expression in neurons in vitro and in vivo.

Animals↗
PubMed1998

Recombinant herpes simplex virus type 1 engineered for targeted binding to erythropoietin receptor-bearing cells.

The utility of recombinant herpes simplex virus type 1 (HSV-1) vectors may be expanded by manipulation of the virus envelope to achieve cell-specific gene delivery. To this end, an HSV-1 mutant virus deleted for glycoprotein C (gC) and the heparan sulfate binding domain of gB (KgBpK-gC-) was engineered to encode different chimeric proteins composed of N-terminally truncated forms of gC and the full-length erythropoietin hormone (EPO). Biochemical analyses demonstrated that one gC-EPO chimeric molecule (gCEPO2) was posttranslationally processed, incorporated into recombinant HSV-1 virus (KgBpK-gCEPO2), and neutralized with antibodies directed against gC or EPO in a complement-dependent manner. Moreover, KgBpK-gCEPO2 recombinant virus was specifically retained on a soluble EPO receptor column, was neutralized by soluble EPO receptor, and stimulated proliferation of FD-EPO cells, an EPO growth-dependent cell line. FD-EPO cells were nevertheless refractory to productive infection by both wild-type HSV-1 and recombinant KgBpK-gCEPO2 virus. Transmission electron microscopy of FD-EPO cells infected with KgBpK-gCEPO2 showed virus endocytosis leading to aborted infection. Despite the lack of productive infection, these data provide the first evidence of targeted HSV-1 binding to a non-HSV-1 cell surface receptor.

Animals↗
PubMed1998

Herpes simplex virus type 1 glycoprotein B requires a cysteine residue at position 633 for folding, processing, and incorporation into mature infectious virus particles.

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) resides in the virus envelope in an oligomeric form and plays an essential role in virus entry into susceptible host cells. The oligomerizing domain is a movable element consisting of amino acids 626 to 653 in the gB external domain. This domain contains a single cysteine residue at position 633 (Cys-633) that is predicted to form an intramolecular disulfide bridge with Cys-596. In this study, we examined gB oligomerization, processing, and incorporation into mature virus during infection by two mutant viruses in which either the gB Cys-633 [KgB(C633S)] or both Cys-633 and Cys-596 [KgB(C596S/C633S)] residues were mutated to serine. The result of immunofluorescence studies and analyses of released virus particles showed that the mutant gB molecules were not transported to the cell surface or incorporated into mature virus envelopes and thus infectious virus was not produced. Immunoprecipitation studies revealed that the mutant gB molecules were in an oligomeric configuration and that these mutants produced hetero-oligomers with a truncated form of gB consisting of residues 1 to 43 and 595 to 904, the latter containing the oligomerization domain. Pulse-chase experiments in combination with endoglycosidase H treatment determined that the mutant molecules were improperly processed, having been retained in the endoplasmic reticulum (ER). Coimmunoprecipitation experiments revealed that the cysteine mutations resulted in gB misfolding and retention by the molecular chaperones calnexin, calreticulin, and Grp78 in the ER. The altered conformation of the gB mutant glycoproteins was directly detected by a reduction in monoclonal antibody recognition of two previously defined distinct antigenic sites located within residues 381 to 441 and 595 to 737. The misfolded molecules were not transported to the cell surface as hetero-oligomers with wild-type gB, suggesting that the conformational change could not be corrected by intermolecular interactions with the wild-type molecule. Together, these experiments confirmed that a disulfide bridge involving Cys-633 and Cys-596 is not essential for oligomerization but rather is required for proper folding and maintenance of a gB domain essential to complete posttranslational modification, transport, and incorporation into mature virus particles.

Animals↗
PubMed1998

Heparan sulfate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread.

Herpes simplex virus type 1 (HSV-1) mutants defective for envelope glycoprotein C (gC) and gB are highly impaired in the ability to attach to cell surface heparan sulfate (HS) moieties of proteoglycans, the initial virus receptor. Here we report studies aimed at defining the HS binding element of HSV-1 (strain KOS) gB and determining whether this structure is functionally independent of gB's role in extracellular virus penetration or intercellular virus spread. A mutant form of gB deleted for a putative HS binding lysine-rich (pK) sequence (residues 68 to 76) was transiently expressed in Vero cells and shown to be processed normally, leading to exposure on the cell surface. Solubilized gBpK- also had substantially lower affinity for heparin-acrylic beads than did wild-type gB, confirming that the HS binding domain had been inactivated. The gBpK- gene was used to rescue a KOS gB null mutant virus to produce the replication-competent mutant KgBpK-. Compared with wild-type virus, KgBpK- showed reduced binding to mouse L cells (ca. 20%), while a gC null mutant virus in which the gC coding sequence was replaced by the lacZ gene (KCZ) was substantially more impaired (ca. 65%-reduced binding), indicating that the contribution of gC to HS binding was greater than that of gB. The effect of combining both mutations into a single virus (KgBpK-gC-) was additive (ca. 80%-reduced binding to HS) and displayed a binding activity similar to that observed for KOS virus attachment to sog9 cells, a glycosaminoglycan-deficient L-cell line. Cell-adsorbed individual and double HS mutant viruses exhibited a lower rate of virus entry following attachment, suggesting that HS binding plays a role in the process of virus penetration. Moreover, the KgBpK- mutant virus produced small plaques on Vero cells in the presence of neutralizing antibody where plaque formation depended on cell-to-cell virus spread. These studies permitted the following conclusions: (i) the pK sequence is not essential for gB processing or function in virus infection, (ii) the lysine-rich sequence of gB is responsible for HS binding, and (iii) binding to HS is cooperatively linked to the process of efficient virus entry and lateral spread but is not absolutely required for virus infectivity.

Animals↗