Murine antibody-dependent cellular cytotoxicity to herpes simplex virus-infected target cells.

Freshly collected peritoneal cells (PC) and cultured spleen cells (SC) (but not fresh SC) from nonimmune mice could mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus (HSV)-infected cells in the presence of mouse or human sera containing antibody to HSV. PC also demonstrated variable natural killer cell cytotoxicity to infected cells. Both PC and cultured SC required high concentrations of antibody and high effector to target cell ratios for optimal ADCC. The time kinetics of the reaction appeared to depend on the state of activation of the effector cells. In both PC and SC populations, ADCC activity was limited to adherent cells, and was profoundly inhibited by particulate latex or silica. The murine effector cell found in PC and SC able to mediate ADCC to HSV-infected cells appears to be a macrophage.     

Human monocyte-macrophage-mediated antibody-dependent cytotoxicity to herpes simplex virus-infected cells.

Human peripheral blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus- (HSV) infected target cells consist of both adherent (MA) and nonadherent (MNA) effector cell populations. These two cell populations can be distinguished by their different phagocytic properties and morphologic appearance, their requirement for antibody in the ADCC reaction, and the rapidity with which they lyse target cells in the presence of immune serum. The MA cells are predominantly phagocytic and have the morphologic characteristics of monocyte-macrophages, whereas the MNA cells are nonphagocytic and appear to be small to medium-sized lymphocytes. Optimal expression of ADCC by MA cells requires higher concentrations of immune serum than does MNA cell-mediated ADCC. MA-mediated cell killing is first detectable by 8 hr and reaches completion after 24 hr of incubation. In contrast, MNA-mediated ADCC produces target cell damage by 2 hr and reaches completion at 8 hr of incubation. Unlike MNA effector cells, the MA effector cells are profoundly inhibited after preincubation with either latex or silica particles. The HSV immune status of the donor had no effect on the ability of either cell population to mediate ADCC. These data demonstrate the participation of both nonadherent mononuclear cells, presumably K cells, and monocyte-macrophages, in ADCC directed against HSV-infected target cells.     

Antibody-dependent cellular cytotoxicity and natural killer cytotoxicity of peritoneal cells from nude mice to herpes simplex virus-infected cells

Nude BALB/c mice (athymic) were more susceptible to fatal herpes simplex virus (HSV) than normal BALB/c mice (P = 0.002). The peritoneal cells of nude mice mediated levels of antibody-dependent cellular cytotoxicity (ADCC) of equal or greater magnitude than cells from normal BALB/c, heterozygote nu/+, or C57BL/6 mice. Unstimulated natural killer cytotoxicity of peritoneal cells from nude mice was higher (P less than 0.05) than that mediated by cells from C57BL/6 mice. Nude mice failed to make anti-HSV ADCC antibody 6 to 14 days post HSV inoculation, at times when nu/+, BALB/c, and C57BL/6 mice produced antibody. Passive reconstitution of nude mice with high titer intraperitoneal anti-HSV immune globulin provided circulating anti-HSV ADCC antibody and significant protection against lethal HSV infection.     

Attachment of human polymorphonuclear leukocytes to herpes simplex virus-infected fibroblasts mediated by antibody-independent complement activation

Herpes simplex virus (HSV)-infected cells can activate the human complement system without interference of specific anti-HSV antibodies. Analysis by flow cytometry showed that C3-like molecules were deposited on the membrane of the infected cell when incubated with human serum without specific antibodies. Depletion of calcium to block the classical pathway of the complement system had no effect on fluorescence intensity. The complement activation could be blocked by chelating both calcium and magnesium or by heating the serum. Furthermore, in the fluid phase C3 was converted to C3b by infected cells and not by uninfected cells. The antibody-independent activation did not lead to lysis of the virus-infected fibroblasts, indicating that the complement cascade is abrogated before formation of the membrane attack complex. This was also confirmed by measurement of the 50% hemolytic complement activities for total and alternative pathways. Polymorphonuclear leukocytes attached to infected fibroblasts after incubation of these fibroblasts with intact complement. This is most probably mediated by complement receptor binding of C3b and C3bi which is deposited on the membrane of the HSV-infected cell. Both type 1 and type 2 HSVs showed the same characteristics in complement activation and thereby mediated polymorphonuclear leukocyte adherence.     

Interferon enhances the antibody-dependent cellular cytotoxicity (ADCC) of human polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMN) from healthy volunteers were tested for ADCC activity against both erythrocyte and tumor targets with and without the addition of human leukocyte interferon (IFN). It was demonstrated that IFN within 30 to 60 min enhanced the reaction in a dose-dependent manner with minimal IFN doses ranging from 1 to 100 units. Formal proof that the augmenting agent was IFN was obtained by using pure IFN proteins in combination with both mock-IFN preparations, which showed no enhancing activity, and anti-IFN antisera, which inhibited the action of the completely purified IFN proteins. In the light of data demonstrating that the IFN effect was most pronounced when the IgG antibodies in the ADCC reaction were present in suboptimal amounts, it is hypothesized that IFN may play a special role in the early nonspecific immune response against non-self antigens.     

Defective natural killer cytotoxicity and polymorphonuclear leukocyte antibody-dependent cellular cytotoxicity in patients with LFA-1/OKM-1 deficiency

Four children with an immunodeficiency involving the absence of leukocyte membrane glycoproteins reacting with anti-LFA-1 and OKM-1 monoclonal antibodies were unable to mediate adherence-dependent leukocyte functions. Even with normal Fc receptor function, their PMN-ADCC and MC-NKC were markedly deficient. Single cell analysis demonstrated deficient antibody-mediated PMN-target cell adherence. Monoclonal antibodies against LFA-1 and OKM-1 reproduced this immunodeficiency in leukocytes from normal adults. LFA-1/OKM-1 mediates a PMN-target cell adhesive step.     

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

Human recombinant interleukin-6 enhances antibody-dependent cellular cytotoxicity of human tumor cells mediated by human peripheral blood mononuclear cells

Summary The effects of human recombinant interleukin-6 (hrIL-6) on antibody-dependent cellular cytotoxicity (ADCC) activity mediated by human peripheral blood mononuclear cells (PMNC) were investigated. Human PMNC were preincubated for 24 h with various concentrations of hrIL-6 and were used as effector cells in a 4-h⁵¹Cr-release assay. The ability of hrIL-6 to augment ADCC was measured using anti-colorectal carcinoma mAbs D612, 17.1A and 31.1 (each directed against a distinct tumor antigen) and using three human colorectal carcinoma cell lines, LS-174T, WiDr and HT-29, as targets. A significant increase in ADCC activity was observed after PMNC were preincubated in 100–400 U/ml but not in lower concentrations of hrIL-6. Variations in activities of PMNC among donors were observed. Non-specific mAb showed no effect in augmenting ADCC activity. hrIL-6 treatment did not augment non-specific (non-mAb-mediated) cytotoxicity. The enhancement of ADCC activity was blocked by the addition of an antibody against hrIL-6 but not by an antibody to the IL-2 receptor (capable of blocking the induction of lymphokine-activated killer cell cytotoxicity by IL-2), suggesting that hrIL-6 augmentation of ADCC activity may not be mediated through IL-2. These results demonstrate that hrIL-6 augments ADCC activity of human PMNC using mAbs to human tumor antigens and human tumor cells as targets, suggesting a potential role for IL-6 in combination with anti-cancer antibodies for cancer immunotherapy.     

Within peripheral blood mononuclear cells, antibody-dependent cellular cytotoxicity of rituximab-opsonized Daudi cells is promoted by NK cells and inhibited by monocytes due to shaving

Treatment of chronic lymphocytic leukemia patients with anti-CD20 mAb rituximab (RTX) leads to substantial CD20 loss on circulating malignant B cells soon after completion of the RTX infusion. This CD20 loss, which we term shaving, can compromise the therapeutic efficacy of RTX, and in vitro models reveal that shaving is mediated by effector cells which express Fc gammaRI. THP-1 monocytes and PBMC promote shaving, but PBMC also kill antibody-opsonized cells by antibody-dependent cellular cytotoxicity (ADCC), a reaction generally considered to be due to NK cells. We hypothesized that within PBMC, monocytes and NK cells would have substantially different and competing activities with respect ADCC or shaving, thereby either enhancing or inhibiting the therapeutic action of RTX. We measured ADCC and RTX removal from RTX-opsonized Daudi cells promoted by PBMC, or mediated by NK cells and monocytes. NK cells take up RTX and CD20 from RTX-opsonized B cells, and mediate ADCC. PBMC depleted of NK cells show little ADCC activity, whereas PBMC depleted of monocytes have greater ADCC than the PBMC. Pre-treatment of RTX-opsonized B cells with THP-1 cells or monocytes suppresses NK cell-mediated ADCC, and blockade of Fc gammaRI on monocytes or THP-1 cells abrogates their ability to suppress ADCC. Our results indicate NK cells are the principal cells in PBMC that kill RTX-opsonized B cells, and that monocytes can suppress ADCC by promoting shaving. These results suggest that RTX-based immunotherapy of cancer may be enhanced based on paradigms which include infusion of compatible NK cells and inhibition of monocyte shaving activity.     

Protection of neonatal mice against herpes simplex virus infection: probable in vivo antibody-dependent cellular cytotoxicity

Infant mice are extremely susceptible to fatal Herpes simplex virus (HSV) infection. They are unable to produce antibody to HSV, and their leukocytes cannot mediate antibody-dependent cellular cytotoxicity (ADCC) to HSV-infected cells. In order to avoid H-2-dependent effector mechanisms and instead analyze possible in vivo ADCC, a murine model employing adoptive transfer of antibody and human leukocytes was developed. Administration of either human immune globulin or leukocytes i.p. from HSV immune or nonimmune humans could not protect infant C57BL/6 mice from fatal HSV infection. In contrast, a combination of a subneutralizing dilution of globulin and leukocytes from nonimmune or immune human donors, given one day before inoculation, was highly protective against lethal HSV infection. The cells involved included lymphocytes or monocyte-macrophages. At least 5 X 10(6) viable leukocytes (or 1 X 10(6) monocyte-macrophages) and immune serum globulin concentrations as low as 10(-8) were protective. Infected cell monolayer adsorption and DEAE column fractionation demonstrated that the protection by globulin was due to specific antiviral IgG antibody. Protection was n ot seen in animals receiving virus before immune transfer. Protection did not involve synergistic viral neutralization by antibody and cells, as shown by in vitro experiments. Animals receiving globulin and cells, unlike normal infant mice, had circulating antiviral antibody and peritoneal leukocytes able to mediate ADCC to HSV-infected cells. This is the first in vivo evidence for the role of human ADCC. This model also allows for the in vivo evaluation of the ability of cells from immunocompromised humans to curb viral infection.     

Requirements for the induction of interleukin-6 by herpes simplex virus-infected leukocytes

Cytokines play important roles in the clearance of herpes simplex virus (HSV) infections and in virus-induced immunopathology. One cytokine known to contribute to resistance against HSV is interleukin-6 (IL-6). Here we have investigated virus-cell interactions responsible for IL-6 induction by HSV in leukocytes. Both HSV type 1 and type 2 are potent inducers of IL-6, and this phenomenon is augmented in the presence of gamma interferon. The ability to induce IL-6 is dependent on de novo protein synthesis and is sensitive to UV irradiation of the virus. Virus mutants lacking the virion-transactivating protein VP16 or any of the immediate-early proteins ICP0, ICP4, or ICP27 displayed unaltered capacities to induce IL-6. However, wild-type virus was unable to induce IL-6 in a macrophage cell line overexpressing a mutant of double-stranded RNA-activated protein kinase (PKR). This suggests a role for PKR in HSV-induced IL-6 expression. HSV infection led to enhanced binding to the kappaB, CRE, and AP-1 sites of the IL-6 promoter, and inhibitors against NF-kappaB and the p38 kinase strongly reduced accumulation of IL-6 mRNA in infected cells. Moreover, macrophage cell lines expressing dominant negative mutants of IkappaBalpha and p38 responded to HSV-1 infection with reduced IL-6 expression compared to the control-vector-transfected cell line. The results show that induction of IL-6 by HSV in leukocytes is dependent on PKR and cellular signaling through NF-kappaB and a p38-dependent pathway.     

Fas-independent cytotoxicity mediated by human CD4+ CTL directed against herpes simplex virus-infected cells

The present study was undertaken to clarify the mechanisms of cytotoxicity mediated by virus-specific human CD4+ CTLs using the lymphocytes of family members with a Fas gene mutation. CD4+ CTL bulk lines and clones directed against HSV-infected cells were established from lymphocytes of a patient with a homozygous Fas gene mutation and of the patient's mother. HSV-specific CD4+ CTLs generated from lymphocytes of the patient and her mother exerted cytotoxicity against HSV-infected cells from the patient (Fas-/-) and from her mother (Fas+/-) to almost the same degree in an HLA class II-restricted manner. mRNAs for the major mediators of CTL cytotoxicity, Fas ligand, perforin, and granzyme B, were detected in these CD4+ CTLs using the RT-PCR and flow cytometry. The cytotoxicity of the HSV-specific CD4+ CTLs appeared to be Ca2+-dependent and was almost completely inhibited by concanamycin A, a potent inhibitor of the perforin-based cytotoxic pathway. Although the Fas/Fas ligand system has been reported to be the most important mechanism for CD4+ CTL-mediated cytotoxicity in the murine system, the present findings strongly suggest that granule exocytosis, not the Fas/Fas ligand system, is the main pathway for the cytotoxicity mediated by HSV-specific human CD4+ CTLs.     

Interaction of polymorphonuclear leukocytes and viruses in humans: adherence of polymorphonuclear leukocytes to respiratory syncytial virus-infected cells

The nature of neutrophil-respiratory syncytial virus (RSV) interaction was investigated by assessing factors that influence neutrophil adherence to RSV-infected tissue culture monolayers. The adherence of neutrophils to infected cells was directly proportional to the degree of RSV replication as evidenced by infectious virus production, cytopathological changes, or viral antigen appearance. Sixty-one percent of the neutrophils adhered to the RSV-infected cells as compared with 52.7% on noninfected monolayers (P less than 0.05). The addition of RSV-specific antibody markedly increased polymorphonuclear leukocyte adherence to 88.5% (P less than 0.001). Complement in the absence of antibody augmented polymorphonuclear leukocyte adherence, but to a lesser degree, 69.0% (P less than 0.025). Arachidonic acid metabolism appeared to play a critical role in the adherence process; thromboxane was the single most important arachidonic acid metabolite. Inhibition of thromboxane synthesis reduced antibody-dependent polymorphonuclear leukocyte adherence on RSV-infected cells to 52.3% (P less than 0.025). These observations suggest a role for neutrophils in RSV infection. It is proposed that neutrophils may participate in RSV infection at the site of viral replication through the attachment to infected cells and the subsequent release of mediators of inflammation.     

The herpes simplex virus 1 IgG fc receptor blocks antibody-mediated complement activation and antibody-dependent cellular cytotoxicity in vivo

Herpes simplex virus 1 (HSV-1) glycoprotein E (gE) mediates cell-to-cell spread and functions as an IgG Fc receptor (FcγR) that blocks the Fc domain of antibody targeting the virus or infected cell. Efforts to assess the functions of the HSV-1 FcγR in vivo have been hampered by difficulties in preparing an FcγR-negative strain that is relatively intact for spread. Here we report the FcγR and spread phenotypes of NS-gE264, which is a mutant strain that has four amino acids inserted after gE residue 264. The virus is defective in IgG Fc binding yet causes zosteriform disease in the mouse flank model that is only minimally reduced compared with wild-type and the rescue strains. The presence of zosteriform disease suggests that NS-gE264 spread functions are well maintained. The HSV-1 FcγR binds the Fc domain of human, but not murine IgG; therefore, to assess FcγR functions in vivo, mice were passively immunized with human IgG antibody to HSV. When antibody was inoculated intraperitoneally 20 h prior to infection or shortly after virus reached the dorsal root ganglia, disease severity was significantly reduced in mice infected with NS-gE264, but not in mice infected with wild-type or rescue virus. Studies of C3 knockout mice and natural killer cell-depleted mice demonstrated that the HSV-1 FcγR blocked both IgG Fc-mediated complement activation and antibody-dependent cellular cytotoxicity. Therefore, the HSV-1 FcγR promotes immune evasion from IgG Fc-mediated activities and likely contributes to virulence at times when antibody is present, such as during recurrent infections.     

Development of immunomonitoring of antibody-dependent cellular cytotoxicity against neuroblastoma cells using whole blood

Neuroblastoma, a childhood tumour of neuroectodermal origin, accounts for 15 % of paediatric cancer deaths, which is often metastatic at diagnosis and despite aggressive therapies, it has poor long-term prognosis with high risk of recurrence. Monoclonal antibody (mAb) therapy targeting GD2, a disialoganglioside expressed on neuroblastoma, has shown promise in recent trials with natural killer cell (NK)-mediated antibody-dependent cellular cytotoxicity (ADCC) thought to be central to efficacy, although other immune effectors may be important. To further enhance therapy, immunomonitoring of patients is essential to elucidate the in vivo mechanisms of action and provides surrogate end points of efficacy for future clinical trials. Our aim was to establish a ‘real-time’ ex vivo whole-blood (WB) immunomonitoring strategy to perform within the logistical constraints such as limited sample volumes, anticoagulant effects, sample stability and shipping time. A fluorescent dye release assay measuring target cell lysis was coupled with flow cytometry to monitor specific effector response. Significant target cell lysis with anti-GD2 antibody (p < 0.05) was abrogated following NK depletion. NK up-regulation of CD107a and CD69 positively correlated with target cell lysis (r > 0.6). The ADCC activity of WB correlated with peripheral blood mononuclear cells (r > 0.95), although WB showed overall greater target cell lysis attributed to the combination of NK-mediated ADCC, CD16+ granulocyte degranulation and complement-dependent cytotoxicity. Response was maintained in heparinised samples stored for 24 h at room temperature, but not 4 °C. Critically, the assay showed good reproducibility (mean % CV < 6.4) and was successfully applied to primary neuroblastoma samples. As such, WB provides a resourceful analysis of multiple mechanisms for efficient end point monitoring to correlate immune modulation with clinical outcome.     

The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. I. Identification of the population of effector cells.

Mononuclear cells (MC) from human blood were fractionated by a variety of physical and immunologic techniques, and the cellular subpopulations generated were assessed for their capacity to lyse herpes simplex virus (HSV)-infected target cells in the presence of IgG antibody to HSV. Latex phagocytosis and surface marker studies were performed in parallel in order to identify the major effector cells by their phagocytic properties and their possession of surface immunoglobulin and receptors for either sheep erythrocytes, C3, or the Fc fragment of IgG. Cytotoxic effector cell activity was unaffected or slightly enhanced after the removal of plastic-adherent or carbonyl iron-adherent MC, indicating that the major effector cell is not a classical monocyte. Similar results were obtained after removal of more than 90% of the T cells by depletion of rosette-forming cells. Likewise, effector cell activity was generally unchanged when more than 95% of the B cells were removed by filtering MC on nylon wool columns. Effector cell function was also found to be normal in three patients with B cell-deficient X-linked agammaglobulinemia. These observations strongly suggest that the effector cells are not T cells or B cells. A 4- to 5-fold enrichment in effector cells, however, was consistently found in a subpopulation, consisting of 5% of the unfractionated MC, that was dramatically enriched both for nonphagocytic cells with only Fc receptor (K cells) and for nonphagocytic cells with no detectable surface markers (null cells). Since, as is demonstrated in the accompanying report, effector surface Fc receptors play a critical role in the mediation of antibody-dependent cellular cytotoxicity directed at HSV-infected target cells, the major mononuclear effector cell in human blood is a K cell.     

Inhibition of antibody-dependent cellular cytotoxicity by autologous lymph node cells.

A population of lymph node cells that lack the usual T, B, or K cell markers was found to inhibit autologous spleen cells from mediating antibody-dependent cellular cytotoxicity (ADCC) to antibody-coated chicken erythrocytes. Inhibitor cells were not susceptible to treatment with anti-Thy 1.2 or anti-Ig and C; they did not adhere to Sephadex G-10, to nylon wool, or to monolayers of sheep erythrocytes (E) or erythrocytes plus 7S antibody (EA). After a brief (4-min) exposure to 45 degrees C, the ability to inhibit was lost whereas other cellular responses remained intact. ADCC mediated by nonadherent splenic effector cells (presumptive K cells) was highly susceptible to inhibition. Possible mechanisms for and implications of lymphocyte-mediated inhibition of ADCC are discussed.     

The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. II. Identification as a K cell.

Studies were carried out to determine whether the mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity (ADCC) to herpes simplex virus (HSV)-infected target cells has surface Fc receptors which participate in the reaction. The F (ab')2 fragment of human IgG antibody was inactive both in ADCC and in complement-mediated cytolysis, but retained the capacity to neutralize infectious virus, to agglutinate erythrocytes coated with viral antigens, and to bind to the surface of virus-infected cells. Treatment of sensitized virus-infected target cells with staphylococcus protein A, which has affinity for the Fc epitope of IgG, strongly reduced their susceptibility to lysis by ADCC in a dose-dependent relationship. These findings indicate that the Fc portion of IgG antibody to the virus is necessary for cytotoxicity. Treatment of blood mononuclear cells with either heat-aggregated gamma-globulin or HSV immune complexes inhibited effector cell activity. The presence of "third party" cellular immune complexes also strongly inhibited ADCC. Adsorption of mononuclear cells to plastic surfaces coated with soluble third party immune complexes resulted in a significant reduction in effector cell activity. These findings demonstrate that the ADCC effector cell possesses surface Fc receptors which are utilized in the ADCC reaction. The presence of Fc receptors on the surface of the effector cell indicates that it is a K cell rather than a null cell.     

Analysis of HCF, the cellular cofactor of VP16, in herpes simplex virus-infected cells

Herpes simplex virus (HSV) immediate-early (IE) gene expression is initiated via the recruitment of the structural protein VP16 onto specific sites upstream of each IE gene promoter in a multicomponent complex (TRF.C) that also includes the cellular proteins Oct-1 and HCF. In vitro results have shown that HCF binds directly to VP16 and stabilizes TRF.C. Results from transfection assays have also indicated that HCF is involved in the nuclear import of VP16. However, there have been no reports on the role or the fate of HCF during HSV type 1 (HSV-1) infection. Here we show that the intracellular distribution of HCF is dramatically altered during HSV-1 infection and that the protein interacts with and colocalizes with VP16. Moreover, viral protein synthesis and replication were significantly reduced after infection of a BHK-21-derived temperature-sensitive cell line (tsBN67) which contains a mutant HCF unable to associate with VP16 at the nonpermissive temperature. Intracellular distribution of HCF and of newly synthesized VP16 in tsBN67-infected cells was similar to that observed in Vero cells, suggesting that late in infection the trafficking of both proteins was not dependent on their association. We constructed a stable cell line (tsBN67r) in which the temperature-sensitive phenotype was rescued by using an epitope-tagged wild-type HCF. In HSV-1-infected tsBN67r cells at the nonpermissive temperature, direct binding of HCF to VP16 was observed, but virus protein synthesis and replication were not restored to levels observed at the permissive temperature or in wild-type BHK cells. Together these results indicate that the factors involved in compartmentalization of VP16 alter during infection and that late in infection, VP16 and HCF may have additional roles reflected in their colocalization in replication compartments.