An open reading frame (ORF) situated between the U(L)20 and U(L)21 genes encodes a protein designated as U(L)20.5. The U(L)20.5 ORF lies 5' and in the same orientation as the U(L)20 ORF. The expression of the U(L)20.5 ORF was verified by RNase protection assays and by in-frame insertion of an amino acid sequence encoding an epitope of an available monoclonal antibody. The tagged U(L)20.5 protein colocalized in small dense nuclear structures with products of the alpha22/U(S)1.5, U(L)3, and U(L)4 genes. Expression of the U(L)20.5 gene was blocked in cells infected and maintained in the presence of phosphonoacetate, indicating that it belongs to the late, or gamma(2), kinetic class. U(L)20.5 is not essential for viral replication inasmuch as a recombinant virus made by insertion of the thymidine kinase gene into the U(L)20.5 ORF replicates in all cell lines tested [J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman (1991) J. Virol. 65, 6414-6424]. The genomic location of the recently discovered genes illustrates the compact nature of the viral genome.

Infection of Vero and HEp-2 but not of 143TK−cells with herpes simplex virus 1 results in fragmentation and dispersal of the Golgi apparatus. Concurrently, in all three infected cell lines the microtubular network is disrupted, suggesting that the disruption of microtubules is essential but not sufficient to induce the fragmentation of the Golgi apparatus. We now report the following: (i) In polykaryocytes formed in Vero cells infected with HSV-1syn−mutant viruses, intact Golgi stacks were readily detected by electron microscopy. These aggregated in the center of large polykaryocytes. (ii) The distribution of viral glycoprotein D, examined in both fixed and nonfixed cells, appeared to match the distribution of the Golgi stacks, suggesting that the aggregated Golgi stacks funnel viral glycoproteins and viral particles to a limited region of the plasma membrane of the polykaryocytes rather than directing exocytic flow in a more dispersed fashion as seen insyn+virus-infected cells exhibiting fragmented and dispersed Golgi. (iii) In most polykaryocytes, the microtubules formed parallel bundles extending along the axis of recruitment of new cells. (iv) Fragmentation of the microtubules at the periphery of the cell near the plasma membrane was observed in untreated or cycloheximide-treated cells 2 h after infection withsyn−virus HSV-1(MP) orsyn+HSV-1(mP) but not in mock-infected cells. These observations suggest that peripheral depolymerization is initiated at the time of infection and that a factor which determines thesyn−orsyn+phenotype is whether the microtubular network regenerates concomitant with cell fusion or reorganizes to form a collapsed network surrounding nuclei ofsyn+infected cells.

The expression of herpes simplex virus 1 gamma (late) genes requires functional alpha proteins (gamma1 genes) and the onset of viral DNA synthesis (gamma2 genes). We report that late in infection after the onset of viral DNA synthesis, cell nuclei exhibit defined structures which contain two viral regulatory proteins (infected cell proteins 4 and 22) required for gamma gene expression, RNA polymerase II, a host nucleolar protein (EAP or L22) known to be associated with ribosomes and to bind small RNAs, including the Epstein-Barr virus small nuclear RNAs, and newly synthesized progeny DNA. The formation of these complexes required the onset of viral DNA synthesis. The association of infected cell protein 22, a highly posttranslationally processed protein, with these structures did not occur in cells infected with a viral mutant deleted in the genes U(L)13 and U(S)3, each of which specifies a protein kinase known to phosphorylate the protein.

The products, RNA or proteins, of the herpes simplex virus 1 open reading frame U(L)43 have not been previously identified. The expression of an open reading frame antisense to U(L)43, U(L)43.5 (P. L. Ward, D. E. Barker, and B. Roizman, J. Virol. 70:2684-2690, 1996), has been reported. We report the existence of a transcript corresponding to the domain of the U(L)43 open reading frame extending approximately 30 bp from the predicted TATA box to the predicted polyadenylation signal. The RNA of the U(L)43 open reading frame accumulates to higher levels in the presence of phosphonoacetic acid, an inhibitor of viral DNA synthesis, than in its absence, whereas the U(L)43.5 transcript accumulates in larger amounts in the absence of phosphonoacetic acid. The open reading frame tagged with a sequence encoding a 20-amino-acid epitope yielded a protein with an apparent Mr of 32,000, i.e., considerably lower than that predicted from the size of the open reading frame. The discovery of a pair of antisense genes expressed during productive infection raises the possibilities that additional antisense genes exist and that the antisense arrangement provides still another mechanism for regulation of gene expression.

An open reading frame mapping antisense to the UL43 gene of herpes simplex virus 1 encodes a protein with an apparent Mr of 38,000. The protein was detected in wild-type-infected cells with rabbit monospecific polyclonal antibody directed against a fusion protein containing all of the sequences encoded by the open reading frame. The antibody did not react with mutants from which the open reading frame was deleted. Expression of this gene, designated UL43.5, was grossly decreased or abolished in infected cells incubated in medium containing inhibitory concentrations of phosphonoacetic acid, suggesting that it is regulated as a gamma gene. UL43.5 is dispensable in cell culture. UL43.5 protein colocalized with the major capsid protein (infected cell protein 5) and the capsid scaffolding proteins (infected cell protein 35) in nuclear structures situated at the periphery of the nucleus. The predicted amino acid sequence indicates that the UL43.5 protein is a highly hydrophilic protein. The colocalization of UL43.5 protein with capsid proteins in discrete nuclear structures suggests that the former may be involved in assembly of viral particles in an accessory role in cells in culture.

In cells infected with herpes simplex virus 1 (HSV-1), the viral proteins ICP5 (infected-cell protein 5) and VP19c (the product of UL38) are associated with mature capsids, whereas the same proteins, along with ICP35, are components of immature capsids. Here we report that ICP35, ICP5, and UL38 (VP19c) coalesce at late times postinfection and form antigenically dense structures located at the periphery of nuclei, close to but not abutting nuclear membranes. These structures were formed in cells infected with a virus carrying a temperature-sensitive mutation in the UL15 gene at nonpermissive temperatures. Since at these temperatures viral DNA is made but not packaged, these structures must contain the proteins for immature-capsid assembly and were therefore designated assemblons. These assemblons are located at the periphery of a diffuse structure composed of proteins involved in DNA synthesis. This structure overlaps only minimally with the assemblons. In contrast, tegument proteins were located in asymmetrically distributed structures also partially overlapping with assemblons but frequently located nearer to nuclear membranes. Of particular interest is the finding that the UL15 protein colocalized with the proteins associated with viral DNA synthesis rather than with assemblons, suggesting that the association with DNA may take place during its synthesis and precedes the involvement of this protein in packaging of the viral DNA into capsids. The formation of three different compartments consisting of proteins involved in viral DNA synthesis, the capsid proteins, and tegument proteins suggests that there exists a viral machinery which enables aggregation and coalescence of specific viral protein groups on the basis of their function.

Earlier studies have shown that the Golgi apparatus was fragmented and dispersed in herpes simplex virus 1-infected Vero and HEp-2 cells but not in human 143TK(-) cells, that the fragmentation and dispersal required viral functions expressed concurrently with or after the onset of DNA synthesis (G. Campadelli-Fiume, R. Brandimarti, C. Di Lazzaro, P. L. Ward, B. Roizman, and M. R Torrisi, Proc. Natl. Acad. Sci. USA 90:2798-2802, 1993), and that in 143TK(-) cells, but not Vero or HEp-2 cells, infected with viral mutants lacking the U(L)20 gene virions were glycosylated and transported to extracellular space (J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman, J. Virol. 65:6414-6424, 1991; E. Avitabile, P. L. Ward, C. Di Lazzaro, M. R Torrisi, B. Roizman, and G, Campadelli-Fiume, J. Virol. 68:7397-7405, 1994). Experiments designed to elucidate the role of the microtubules and of intact or fragmented Golgi apparatus in the exocytosis of virions showed the following (i) In all cell lines tested (Vero, 143TK(-), BHK, and Hep-2) microtubules underwent fragmentation particularly evident at the cell periphery and then reorganized into bundles which circumvent the nucleus, This event was not affected by inhibitors of viral DNA synthesis. We conclude that redistribution of microtubules may be required but is not sufficient for the fragmentation and dispersal of the Golgi apparatus. (ii) In all infected cell lines tested, nocodazole caused fragmentation and dispersal of the Golgi and a far more extensive depolymerization of the microtubules than was seen in untreated, infected Vero or HEp-2 cells. Taxol precluded the depolymerization of the microtubules and fragmentation of the Golgi in both infected cell lines. Neither nocodazole nor taxol affected the exocytosis of infectious virus from Vero, HEp-2, or 143TK(-) cells infected with wild-type virus. We conclude that the effects of nocodazole or of taxol are dominant over the effects of viral infection in the cell lines tested and that viral exocytosis is independent of the organization of microtubules or of the integrity of the Golgi apparatus. Lastly, the data suggest that herpes simplex viruses have evolved an exocytic pathway for which the U(L)20 protein is a component required in some cells but not others and in which this protein does not merely compensate for the fragmentation and dispersal of the Golgi apparatus.

The Herpes simplex virus genome encodes 75 proteins. Of these, only 37 are required for growth of the virus is culture. These essential genes encode functions related to entry of virus into cells, regulation of gene expression and replication and packaging of viral DNA into virious. The genes that are not essential for replication in culture play a key role in multiplication of the virus and its transfer from cell to cell, in complementing cellular functions lost as a consequence of viral replication, in fine-tuning viral gene expression and in overcoming the host's response to infection. No virally encoded functions are required for establishment of the latent state, but a full complement of viral genes is essential for efficient reactivation of the virus from the latent state.

The Golgi apparatus is fragmented and dispersed in Vero cells but not in human 143TK- cells infected with wild-type herpes simplex virus 1. Moreover, a recombinant virus lacking the gene encoding the membrane protein UL20 (UL20- virus) accumulates in the space between the inner and outer nuclear membranes of Vero cells but is exported and spreads from cell to cell in 143TK- cell cultures. Here we report that in Vero cells infected with UL20- virus, the virion envelope glycoproteins were of the immature type, whereas the viral glycoproteins associated with cell membranes were fully processed up to the addition of sialic acid, a trans-Golgi function. Moreover, the amounts of viral glycoproteins accumulating in the plasma membranes were considerably smaller than those detected on the surface of Vero cells infected with wild-type virus. In contrast, the amounts of viral glycoproteins present on the plasma membranes of 143TK- cells infected with wild-type or UL20- virus were nearly identical. We conclude that (i) in Vero cells infected with UL20- virus the block in the export of virions is at the entry into the exocytic pathway, and a second block in the exocytosis of viral glycoproteins associated with cytoplasmic membranes is due to an impairment of transport beyond Golgi fragments containing trans-Golgi enzymes and not to a failure of the Golgi oligosaccharide-processing functions; (ii) these defects are manifested in cells in which the Golgi apparatus is fragmented; and (iii) the UL20 protein compensates for these defects by enabling transport to and from the fragmented Golgi apparatus.

The UL20 protein of herpes simplex virus 1, an intrinsic membrane protein, is required in infected Vero cells in which the Golgi apparatus is fragmented for the transport of virions from the space between the inner and outer nuclear membranes and for the transport of fully processed cell membrane-associated glycoproteins from the trans-Golgi to the plasma membrane. It is not required in the human 143TK- cell line, in which the Golgi apparatus remains intact. We report the following. (i) The UL20 protein was detected in infected cells beginning at 6 h postinfection and was regulated as a gamma 1 gene. (ii) Pulse-chase experiments revealed no detectable alteration in the mobility of the UL20 protein in polyacrylamide gels. (iii) In both infected Vero and infected 143TK- cells, the UL20 protein was detected by immunofluorescence in association with nuclear membranes and in the cytoplasm. Some of the cytoplasmic fluorescence colocalized with beta-COP, a protein associated with Golgi-derived transport vesicles. UL20 protein was present in virions purified from the extracellular space but could not be detected in the plasma membrane. These results are consistent with the hypothesis that UL20 is a component of virion envelopes and membranes of virion transport vesicles and is selectively retained from the latter in a Golgi compartment.

In Vero monkey cells and HEp-2 human epidermoid carcinoma cells infected with herpes simplex virus 1 the proteins beta-COP, galactosyltransferase, and alpha-mannosidase II associated with the Golgi apparatus appear to be associated with numerous smaller structures dispersed throughout the cytoplasm. Concomitantly, the intracytoplasmic ligands of lectins normally associated wholly (Helix pomatia or Ricinus communis agglutinin) or in part (wheat germ agglutinin) with the Golgi apparatus increased in amount and became dispersed. This phenomenon was seen in some of the baby hamster kidney cells analyzed but not in others and not in the human 143TK- cells. The fragmentation and dispersal of the Golgi apparatus was a late event in the reproductive cycle coinciding with virion assembly, processing of viral glycoproteins, and exocytosis from infected cells. The fragmentation of the Golgi apparatus is morphologically different from that seen with brefeldin A and may reflect disequilibration between the anterograde and retrograde Golgi transport caused by the huge influx of viral glycoproteins contained in virions and membranes flowing through the exocytic pathway.

Studies involving tumor escape from host immune surveillance have focused heavily on loss of major histocompatibility class I antigens as well as loss of tumour-associated antigens as possible mechanisms by which tumors escape recognition and lysis by cytolytic T cells. Examples of both phenomena are found in murine tumors induced by viruses, chemical mutagens, a spontaneous tumor mutagenized in vitro and some u.v.-induced tumors. However, evidence also exists for the escape of tumors from immune destruction without loss of major histocompatibility class I molecules or tumor antigens and additional mechanisms undoubtedly are involved in the complex phenomena of tumor progression.

A recombinant virus from which the start codon and 53% of the UL20 open reading frame had been deleted was constructed and characterized. We report the following: (i) The UL20- mutant formed small plaques in 143 tk- cells but failed to form plaques in Vero cells. Virus yields were approximately 10- to 100-fold lower than those of wild-type virus in all cell lines tested. (ii) Electron microscopic examination of Vero cells infected with the UL20- mutant revealed that enveloped and unenveloped capsids accumulated in the cytoplasm, possibly in the space between the inner and outer lamellae of the nuclear membrane, and that virtually no virus was present in the extracellular space. (iii) Glycoproteins B, C, D, E, H, and I recovered from lysates of cells infected with the UL20- mutant could not be differentiated from those present in lysates of cells infected with the wild-type parent virus with respect to the electrophoretic mobility of mature and precursor forms. (iv) Repair of the deleted sequences restored the wild-type phenotype. (v) The gene product of the UL20 gene was shown to be associated with cellular membranes and to possess characteristics of integral membrane proteins. We conclude that the UL20 gene encodes an integral membrane protein with a hitherto unrecognized function in that it enables the transit of virions to the extracellular space. The function of the UL20 gene product is complemented by some cell lines but not by Vero cells. The vesicles which serve to transport virions may have an origin different from those associated with transport of normal cellular proteins.

The rejection of murine UV-induced skin cancers by normal mice is a striking example of powerful immune surveillance of the normal host against malignant cells. In this study, we show that UV-induced regressor tumors regularly grew progressively and killed mice that were depleted of CD8+ T-cells. Depletion of CD4+ T-cells had no effect, suggesting that CD8+ but not CD4+ T-cells were required for this immune surveillance. To determine whether change in major histocompatibility complex (MHC) class I expression was a frequent event that caused low immunogenicity of tumors or facilitated escape from immune destruction, recently isolated murine tumors of varying degrees of immunogenicity, including highly immunogenic UV-induced regressor, less immunogenic UV-induced progressor, and poorly immunogenic spontaneous progressor tumors, were compared. There was no correlation between the ability of a tumor to grow progressively in a normal immunocompetent host and the level of constitutive class I expression or the level of expression induced in vitro by gamma interferon. (Only 1 of more than 20 progressor tumors analyzed showed complete loss of a MHC class I molecule.) Some progressor variants showed loss of a unique tumor-specific cytotoxic T-lymphocyte-defined antigen, consistent with earlier evidence of antigen loss providing a mechanism for tumor escape. However, most of the host-selected progressor variants retained both MHC class I antigens and the unique tumor antigens that we could detect with cytotoxic T-lymphocyte clones, suggesting that mechanisms other than loss of MHC class I or of the unique target antigen may be involved in escape of some tumors from a highly effective CD8-dependent host surveillance.

We have isolated UV light-induced and spontaneous tumors along with nonmalignant cells and tissues from each host. CD8+ CTL clones generated to a number of highly immunogenic UV-induced tumors did not react with autologous normal fibroblasts nor with autologous second tumors. Using up to 25 independently induced tumors as targets, these CTL clones were found to be uniquely specific for the particular tumor used for immunization even when multiple tumors isolated from the same animals were used as targets. In addition to this extensive antigenic diversity of independently induced tumors, we found that a single cancer cell can express multiple independent antigens that were uniquely expressed on the tumor but were not detectable on autologous nonmalignant fibroblasts. A poorly immunogenic spontaneous tumor was also found to express an antigen that was uniquely specific for the immunizing tumor in that it was absent from any of 25 other tumors tested. This antigen was recognized by a mAb and not detected on autologous nonmalignant fibroblasts or on an autologous second spontaneous tumor. These findings demonstrate that syngeneic CTL clones or mAbs can define unique antigens on UV-induced or spontaneous tumors. The use of autologous nonmalignant fibroblast targets made it unlikely that these antigens were widely expressed on normal cells. The availability of cloned immunological probes to antigens on tumors isolated with autologous normal cells will allow a reliable identification of the genetic origins of unique antigens on experimentally induced and spontaneous tumors and permit a decisive answer to whether these unique antigens are encoded by normal genes or by genes that have undergone somatic mutations; i.e., whether these antigens are truly tumor specific.

The modern era of cancer immunology began with the discovery that inbred mice could be immunized against cancers that had been induced by chemical carcinogens such as the polycyclic hydrocarbon methylcholanthrene (MCA) [1–4]. Particularly, studies of Prehn and Main in 1957 made it highly unlikely that the antigens on the cancers were also widely expressed on normal tissue. It was shown that normal tissue of the host from which the tumor had been isolated did not immunize the recipient to reject the tumor challenge; furthermore, mice immunized against the tumor still accepted normal skin grafts from the mouse of tumor origin. Thus, these antigens were seemingly tumor specific. Another important aim of the experiments using MCA-induced murine tumors was the search for antigens that were tumor specific as well as shared among different independently induced cancers.

Exhausted CD8 T (Tex) cells are a distinct cell lineage that arise during chronic infections and cancers in animal models and humans. Tex cells are characterized by progressive loss of effector functions, high and sustained inhibitory receptor expression, ...Read More

We have studied the components of a complex of tumor-specific antigens to determine if all of the components of the complex were lost during progression from a rather benign regressor tumor to a highly malignant (HM) cancer. We find that the HM tumor cells have lost antigens recognized by CTL but retained antigens recognized by Th cells. Immunization with variants expressing Th-defined antigens induced tumor-specific immunity to challenge with a parental variant that expressed a CTL-recognized target antigen, but did not induce immunity to challenge with the variant that expressed the Th-defined antigen alone. Together, these findings suggested that Th cells fail to exert direct selective pressure upon the tumor, resulting in retention of "lineage-specific," Th-recognized antigens by highly immunoselected variants. Possible advantage could be taken of this fact for the development of specific immunotherapy.