The transcriptional activation domain of VP16 is required for efficient infection and establishment of latency by HSV-1 in the murine peripheral and central nervous systems.

The herpes simplex virus (HSV) transactivator VP16 is a structural component of the virion that activates immediate-early viral gene expression. The HSV-1 mutant in1814, which contains a 12-bp insertion that compromises the transcriptional function of VP16, replicated to a low level if at all in the trigeminal ganglia of mice (I. Steiner, J. G. Spivack, S. L. Deshmane, C. I. Ace, C. M. Preston, and N. W. Fraser (1990). J. Virol. 64, 1630-1638; Valyi-Nagy et al., unpublished data). However, in1814 did establish a latent infection in the ganglia after corneal inoculation from which it could be reactivated. In this study, several HSV-1 strains were constructed with deletions in the VP16 transcriptional activation domain. These viruses were viable in cell culture, although some were significantly reduced in their ability to initiate infection. A deletion mutant completely lacking the activation domain of VP16 (RP5) was unable to replicate to any detectable level or to efficiently establish latent infections in the peripheral and central nervous systems of immunocompetent mice. However, similar to in1814, RP5 formed a slowly progressing persistent infection in immunocompromised nude mice. Thus RP5 is severely neuroattenuated in the murine model of HSV infection. However, the activation domain of VP16 is not essential for replication in the nervous system, since we observed a slow progressive infection persisting in the absence of an immune response.

A neuroattenuated ICP34.5-deficient herpes simplex virus type 1 replicates in ependymal cells of the murine central nervous system.

Herpes simplex virus type 1 (HSV-1) variant 1716 is deleted in the gene encoding ICP34.5 and is neuroattenuated after intracranial inoculation of mice. Although the mechanism of attenuation is unclear, this property has been exploited to eliminate experimental brain tumours. Previously, it was shown that infectious 1716 was recoverable for up to 3 days after intracranial inoculation suggesting that there may be limited replication in the central nervous system (CNS). Here it is demonstrated that 1716 replicates in specific cell types (predominantly CNS ependymal cells) of BALB/c mice, using immunohistochemical, immunofluorescence, in situ hybridization and virus titration studies. While 1716-infected mice exhibited no overt signs of encephalitis, histological analysis showed a persistent loss of the ependymal lining. Thus, although ICP34.5-deficient viruses are neuroattenuated, they do retain the ability to replicate in and destroy the ependyma of the murine CNS. A detailed understanding of the mechanism(s) of neuroattenuation and limited replication could lead to the rational design of safe HSV vectors for cancer and gene therapy in the CNS.

Herpes simplex virus 1716, an ICP 34.5 null mutant, is unable to replicate in CV-1 cells due to a translational block that can be overcome by coinfection with SV40.

Herpes simplex virus (HSV) mutants lacking the gene encoding infected cell protein (ICP) 34.5 exhibit an attenuated phenotype in models of pathogenesis and have been used for experimental cancer therapy. Recently it was shown that the HSV ICP 34.5 protein functions to prevent the host cell-induced double-stranded RNA-activated protein kinase (PKR)-dependent translational block that normally occurs during virus infection. We now report that an HSV ICP 34.5 mutant called HSV-1716 is unable to replicate in the simian kidney cell-derived line CV-1, due to a translational block. Moreover, we find that this block can be overcome by simian virus 40 (SV40). This has been shown directly by infecting CV-1 cells with SV40 and HSV-1716 simultaneously, and indirectly via HSV-1716 infection of COS-1 cells (CV-1 cells transformed by an origin-defective mutant of SV40 that codes for wild-type T antigen). The translational block is restored when infections are done in the presence of the phosphatase inhibitor okadaic acid. These results support, but do not directly prove, contentions that HSV ICP 34.5 interacts with the PKR pathway to restore translation in non-permissive cells, and that SV40 large T antigen has a similar functional role, but acts downstream of the site of ICP 34.5 interaction (eIF2alpha) in the pathway. Study of this CV-1/COS-1 system should allow further clarification of the virus-host interactions that underlie the restricted replication of HSV-1 ICP 34.5 gene null mutants.

Treatment of experimental subcutaneous human melanoma with a replication-restricted herpes simplex virus mutant.

Modified, non-neurovirulent herpes simplex viruses (HSV) have shown promise for the treatment of brain tumors, including intracranial melanoma. In this report, we show that HSV-1716, an HSV-1 mutant lacking both copies of the gene coding-infected cell protein 34.5 (ICP 34.5), can effectively treat experimental subcutaneous human melanoma in mice. In vitro, HSV-1716 replicated in all 26 human melanoma cell lines tested, efficiently lysing the cells. Therapeutic infection of subcutaneous human melanoma nodules with HSV-1716 led to viral replication that was restricted to tumor cells by immunohistochemistry. Moreover, HSV-1716 treatment significantly inhibited progression of preformed subcutaneous human melanoma nodules in SCID mice and caused complete regression of some tumors. This work expands the potential scope of HSV-1-based cancer therapy.

Herpes simplex 1716--an ICP 34.5 mutant--is severely replication restricted in human skin xenografts in vivo.

HSV-1716 is a replication-restricted, neuroattenuated ICP 34.5 gene mutant of herpes simplex virus type 1 (HSV-1). Because of the attenuated phenotype of ICP 34.5 mutants in rodent models of HSV disease, they have been promoted as potential vaccine strains and gene therapy vectors and have been used by us and others as therapeutic agents for the treatment of experimental malignant tumors. However, all data on the phenotype of HSV-1716 and other ICP 34.5 mutants are from animal model systems, while humans are the natural hosts of HSV-1. To achieve an initial characterization of the phenotype of 1716 in human tissue, we have studied its replication in mature human skin xenografts on SCID mice. We find that replication of 1716 is severely restricted in such human skin grafts relative to both parental wild-type HSV-1 strain 17+ and the HSV-1716 revertant virus 1716R, in which the 759-bp ICP 34.5 gene deletions have been repaired. Moreover, the replication of both 1716 and 17+ is significantly better in the human skin grafts than it is in mouse skin. The implications of these findings are discussed.

Therapy of experimental human brain tumors using a neuroattenuated herpes simplex virus mutant.

BACKGROUND: Engineered herpes simplex virus (HSV) strains previously have been shown to offer a potential therapeutic alternative to conventional treatment modalities for brain tumors. Because HSV Type 1 strain 1716 has a deletion in the gamma 34.5 neurovirulence gene that renders it avirulent in the mouse central nervous system, we have assessed its potential to induce selective lysis of tumor cells versus neurons in vitro and in vivo. EXPERIMENTAL DESIGN: To do this, we studied parental HSV-1 strain 17+ and strain 1716 using human embryonal carcinoma cells (NT2 cells). These cells resemble neuronal progenitor cells and can be induced to differentiate into neurons (NT2N) with retinoic acid. Intracerebral grafts of NT2 cells into the brains of nude mice resulted in lethal brain tumors, and grafts of NT2N cells resulted in the integration of NT2N cells. RESULTS: In vitro studies showed that strain 1716 replicates in and spreads on monolayers of NT2 cells but not in NT2N cells. In vivo, strain 1716 replicated preferentially in NT2 tumors as evidenced by immunohistochemical staining for viral antigens, by in situ hybridization for HSV-specific transcripts, and by titration of virus from brains with tumor after intracranial injection of the virus into these mice. The temporal regression of NT2 tumors in mice treated with strain 1716 was demonstrated in vivo by magnetic resonance imaging. Electron microscopy and studies of DNA fragmentation suggested that regression of NT2 brain tumors in strain 1716-treated mice was mainly caused by a nonapoptotic, lytic mode of cell death. Finally, strain 1716-treated NT2 tumor-bearing mice survived more than twice as long as mock-treated tumor-bearing mice, and these differences in survival (25 vs. 9 weeks) were statistically significant (p < 0.03). CONCLUSIONS: We conclude from these studies that strain 1716 induces regression of human neural tumors established in the brains of nude mice, resulting in their prolonged survival.

Treatment of experimental intracranial murine melanoma with a neuroattenuated herpes simplex virus 1 mutant.

Brain metastases occur commonly in the setting of a variety of human cancers. At present, such cases are invariably fatal and highlight a need for research on new therapies. We have developed a mouse brain tumor model utilizing the Harding-Passey melanoma cell line injected intracranially into C57Bl/6 mice. Tumors develop in 100% of the mice and can be detected by magnetic resonance imaging as early as 5 days post cell injection. Death from tumor progression occurs between 12 and 16 days post cell injection. Stereotactic injection of the neuroattenuated HSV-1 strain 1716 into brain tumors 5 or 10 days postinjection of the melanoma cells results in a statistically significant increase in the time to development of neurological symptoms and in complete tumor regression and the long-term survival of some treated animals. Moreover, viral titration studies and immunohistochemistry suggest that replication of this virus is restricted to tumor cells and does not occur in the surrounding brain tissue. These results suggest that HSV-1 mutant 1716 shows particular promise for use as a therapeutic agent for the treatment of brain tumors.

A thymidine kinase-negative HSV-1 strain establishes a persistent infection in SCID mice that features uncontrolled peripheral replication but only marginal nervous system involvement.

A detailed knowledge of the pathogenesis of infections caused by thymidine-kinase (TK)-deficient herpes simplex virus type 1 (HSV-1) strains is important because such mutants can arise during treatment of HSV infections with acyclovir--especially in immunocompromised patients--and also because TK-negative mutants may become useful for the therapy of intracranial tumors. In this work, we studied the pathogenesis of a genetically engineered TK-negative HSV-1 strain dlsptk, in SCID mice (mice with severe combined immunodeficiency) after corneal infection. We found that dlsptk established a persistent infection that kills SCID mice within 80.2 +/- 21.3 days. The cause of death seemed to be related to uncontrolled viral replication in the superficial and deep facial tissues of the animals. Viremia probably did not occur, as judged by the inability to detect infectious virus and viral gene expression in various internal organs. However, the virus did reach the nervous system, most probably by axonal transport from the primary site of the infection. Virus-specific DNA reached low but detectable levels in the trigeminal ganglia and the brainstems by 7 days p.i. and remained at low levels for up to 50 days p.i. as determined by spot blot analysis. By in situ hybridization and immunostaining we determined that, in some of the neurons of the trigeminal ganglia infected by the virus, viral latency was established. However, our results suggested that in other infected neurons viral replication occurred and virus spread to surrounding nonneuronal cells and to the central nervous system. This work provides a new model in which the pathogenesis of infections caused by TK-deficient HSV strains in immunocompromised hosts can be effectively studied and which may also help to identify the potential side effects of the therapy of intracranial tumors with TK-negative HSV strains.

Expression of functional cell surface C1-inactivator by U937 cells.

We have previously shown that the human monocyte-like cell line U937 synthesizes C1-INA and expresses cell surface C1-INA. In this report we provide evidence that this surface-expressed C1-INA is functionally active. Intact U937 cells demonstrated functional C1-INA activity in a hemolytic assay. This activity was blocked when the cells were incubated with monospecific antibody to C1-INA, and was not detectable in cell-free supernatants of U937 cells. SDS-PAGE analysis of radiolabeled U937 cell surface proteins purified by anti-C1-INA affinity chromatography revealed two distinct bands. One protein had a Mr of 105 kDa identical to plasma C1-INA, and the second had a Mr of 200 kDa. We were unable to determine the identity of the 200 kDa protein by Western blotting with anti-C1-INA. However, the possibility exists that this 200 kDa molecule may represent a C1-INA receptor, a dimeric form of C1-INA, or an unrelated cell surface protein with affinity for C1-INA. Furthermore, we show that treatment of U937 cells with phorbol ester resulted in an increase in the percentage of cells expressing surface C1-INA. These results suggest that U937 cells express functional cell surface C1-INA, which could function in vivo to protect these human tumor cells from lysis by host complement.

Synthesis of C1 inhibitor (C1-INA) by a human monocyte-like cell line, U937.

Human monocytes are known to synthesize many of the components of complement, including C1-INA. In this report we demonstrate that the human monocyte-like cell line U937 is also capable of synthesizing functional C1-INA. This was shown in several ways, including 1) incorporation of tritiated amino acids into antigenic C1-INA, immunoprecipitation, and detection by fluorography; 2) a sensitive ELISA, which allowed quantitation of antigenic C1-INA in cell lysates, and 3) a C2-dependent hemolytic assay in which the functional activity of U937 C1-INA was assayed. Data from the ELISA indicate that U937 cells contain between 2.1 to 12.8 ng of C1-INA per 1 X 10(6) cells. Furthermore, fluorescence-activated cell sorter analysis revealed that approximately 16% of U937 cells carry C1-INA as a surface bound antigen. Other proteins found to be synthesized by U937 cells include C1r, C8, and possibly alpha-2-macroglobulin. These results suggest that the U937 cell line could be a convenient and valuable model for the study of monocyte C1-INA synthesis and physiology.

Studies of complement autoactivatability in hereditary angioedema: direct relationship to functional C-1-INA and the effect of classical pathway activators.

It has been observed earlier that the hemolytic complement in diluted sera obtained from patients with hereditary angioedema (HAE) undergoes spontaneous decay when incubated at 37 degrees C. Employing individual serum from patients at different stages of this disease it was demonstrated that this spontaneous loss of hemolytic complement also occurs without dilution and is directly linked to the absence of functional C-1-INA. Incubation of HAE serum resulted in a loss of activity which appears to be dependent upon the concentration of functional C-1-INA. While C-1-INA levels less than 50 micrograms/ml lead to rapid depletion with time, reconstitution of deficient sera with highly purified C-1-INA or of undiluted NHS inhibited spontaneous activation. Furthermore, NHS was rendered susceptible to autoactivation when its C-1-INA was depleted by passage over an anti-C-1-INA Sepharose 4B affinity column in the presence of 10 mM EDTA, indicating that in the absence of functional C-1-INA, C1 undergoes an uninhibited spontaneous autoactivation which leads to the consumption of C4 and C2 but not C3. Consumption of C3 was observed, however, in HAE sera that contained a significant amount of immune complexes. Incubation of HAE sera with highly purified Hageman factor fragment (5 micrograms/ml), or aggregated IgG (2 mg/ml) was found to accelerate the rate of decay when compared to untreated samples while sera from patients under treatment with Danazol or Stanozolol failed to autoactive. These results suggest that, the absence of C-1-INA, may, by itself trigger the dissociation and autoactivation of C1 in the sera of such patients; however, the presence of other complement activators accelerates the reaction. This inherent property of HAE sera, i.e., spontaneous autoactivation at 37 degrees C, may be a useful screening test but direct determination of C-1-INA activity is required to establish the precise diagnosis.

Mechanisms of activation of the classical pathway of complement by Hageman factor fragment.

The mechanism by which a fragment of activated Hageman factor (HFf) activates the classical pathway of complement in serum or platelet-poor plasma has been further delineated. When serum or platelet-poor plasma was incubated with various concentrations of HFf, the total complement hemolytic activity was reduced in a dose-dependent manner. This activation appears to be due to the direct interaction of HFf with macromolecular C1, since incubation of purified C1 with HFf resulted in dissociation of the subunits with concomitant reduction of C1r antigenicity that is indicative of C1 activation. HFf-dependent activation was prevented by prior treatment of HFf with the active site-directed inhibitor, H-D-proline-phenylalanine-arginine chloromethyl ketone or with a specific inhibitor of activated HF derived from corn. Incubation of HFf with highly purified C1r also resulted in activation of C1r as assessed directly using a synthetic substrate or indirectly by activation of C1s and consumption of C2. However, incubation of HFf with highly purified C1s resulted in formation of activated C1s (C1s-) but this was less efficient than HFf activation of C1r. We therefore conclude that activation of C1 in macromolecular C1 is the result of HFf conversion of C1r to C1r; activation of C1s then occurs primarily by C-1r and to a lesser degree by the direct action of HFf.