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.     

Reduced antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells of salivary polymorphonuclear leukocytes and inhibition of peripheral blood polymorphonuclear leukocyte cytotoxicity by saliva

Blood polymorphonuclear leukocytes (BPMN) have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) against HSV-infected cells. Although HSV infections are frequently found in the oral cavity, the ADCC capacity of salivary PMN (SPMN) has not been studied, mainly because methods to isolate SPMN were not available. We have recently developed a method to isolate SPMN, and in this study have evaluated their ADCC activity against HSV-infected cells. SPMN were obtained by repeated washings of the oral cavity, and separated from epithelial cells by nylon mesh filtration. ADCC was quantitatively determined by 51Cr release from HSV-infected Chang liver cells. SPMN in the presence of antibody were able to destroy HSV-infected cells, but SPMN were much less effective in mediating ADCC than BPMN (3.4% vs 40.7%, p less than 0.0001). In the presence of antiviral antibody, SPMN were able to adhere to HSV-infected cells, but less so than BPMN (34% vs 67%), and specific antibody-induced adherence was significantly lower in SPMN (p less than 0.04). The spontaneous adherence to HSV-infected cells was higher for SPMN than BPMN. SPMN demonstrated up-regulation of the adhesion glycoprotein CD18, but down-regulation of the FcRIII receptor. Incubation with saliva decreased ADCC capacity of BPMN, up-regulated CD18 expression, and down-regulated FcRIII expression.     

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.     

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.     

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.     

Herpes simplex virus type 1 Fc receptor protects infected cells from antibody-dependent cellular cytotoxicity.

Recent studies indicate that the herpes simplex virus type 1 (HSV-1) Fc receptor (FcR) can bind antiviral immunoglobulin G by participating in antibody bipolar bridging. This occurs when the Fab domain of an immunoglobulin G molecule binds to its antigenic target and the Fc domain binds to the HSV-1 FcR. In experiments comparing cells infected with wild-type HSV-1 (NS) and cells infected with an FcR-deficient mutant (ENS), we demonstrate that participation of the HSV-1 FcR in antibody bipolar bridging reduces the effectiveness of antibody-dependent cellular cytotoxicity.     

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.     

Polysome-associated proteins in herpes simplex virus-infected cells

In the cytoplasm of eucaryotic cells, mRNA is associated with proteins. These mRNA-protein complexes, termed messenger ribonucleoprotein (mRNP) particles, are divided into two functional classes. The first class contains free (non-ribosome-associated) mRNPs which have been termed informosomes by others. The second class of mRNPs, those associated with polysomes, are actively engaged in protein synthesis and are termed polysomal mRNPs. The experiments described in this paper examined the proteins associated with polyribosomes in uninfected and herpes simplex virus type 1-infected cells. The data indicate that after infection with herpes simplex virus type 1, specific changes occur in the proteins which normally are found associated with these polysomal mRNPs. These changes include both the appearance of new and possibly virus-specific proteins and the loss of normal host-specific proteins. The relationship of these changes to the patterns of protein synthesis in these cells is also discussed.     

Antibody-dependent cellular cytotoxicity to virus-infected target cells: role of nylon wool-adherent T cells as effectors

This study was designed to elucidate whether populations of human blood lymphocytes other than non-T, non-B, Fc receptor-positive K cells can mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells acutely infected with type 1 herpes simplex virus. With appropriate technical precautions, a subset of E rosette-positive, nylon wool-adherent T cells were found to be effective in killing antibody-coated target cells. Thus, there appears to be at least two relatively distinct populations of lymphocytes in human peripheral blood that function as effectors in ADCC: one that consists of E rosette-negative cells, the other of E rosette-positive cells. These findings suggest a possible relationship between “classical” E rosette-negative K cells and E rosette-positive T cells.     

Mapping of a herpes simplex virus type 2-encoded function that affects the susceptibility of herpes simplex virus-infected target cells to lysis by herpes simplex virus-specific cytotoxic T lymphocytes.

A function(s) involved in the altered susceptibility of herpes simplex virus type 2 (HSV-2)-infected cells to specific lysis by cytotoxic T lymphocytes was mapped in the S component of HSV-2 DNA by using HSV-1 X HSV-2 intertypic recombinants (RH1G44, RS1G25, R50BG10, A7D, and C4D) and HSV-1 MP. Target cells infected with R50BG10, A7D, and C4D exhibited reduced levels of cytolysis, as did HSV-2-infected cells, whereas RH1G44 and RS1G25 recombinant-infected and HSV-1 MP-infected cells showed levels of lysis equal to that of HSV-1 KOS-infected cells. The intertypic recombinants R50BG10, RS1G25, RH1G44, and HSV-1 MP induced cross-reactive cytotoxic T lymphocytes. Coinfection of cells with HSV-1 KOS and either HSV-2 186 or R50BG10 recombinant also resulted in a decrease in the level of specific lysis by anti-HSV cytotoxic T lymphocytes.     

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity. I. General requirements of the reaction and temporal relationship between lethal hits and cytolysis.

The effect of various physical and chemical parameters on the cytotoxic reaction was studied in a ⁵¹Cr-release assay in order to analyze the mechanism by which human blood mononuclear cells (MC) damage antibody-sensitized target cells infected with herpes simplex virus. Centrifugation of the target cell-MC mixture consistently increased the velocity of the reaction. In addition, uncentrifuged target cell-MC cultures showed a sigmoidal kinetic curve of ⁵¹Cr release with an initial lag phase of at least 10 min, whereas ⁵¹Cr release in centrifuged cultures followed a linear pattern with time without an initial lag. These findings indicate that direct contact between target and effector cells is necessary for cytotoxicity to occur. The reaction as a whole was temperature dependent, proceeding well at 37 °C and not at all at 4 °C. Incubation of the MC at 46 °C for 10 min abolished their cytotoxic potential without affecting their viability; similar heating of the target cells did not affect their background isotope release or sensitivity to the lytic process. Heating target cell-MC mixtures at 46 °C for 10 min thus provided a tool by which the temporal relationship between the mounting of “lethal hits” and specific isotope release, or cell lysis, could be studied. Using this technique, we observed virtually simultaneous occurrence of lethal hits and cell lysis, measured at various intervals between 10 and 360 min postincubation. Likewise, we were unable to demonstrate a transient period of increased osmotic fragility in target cells after contact with MC but before actual cell lysis. Taken together, these findings imply either that cell lysis, as indicated by ⁵¹Cr release, results from a sudden nonosmotic injury to the target cell membrane or, alternatively, osmotic damage leading to ⁵¹Cr release occurs too rapidly to be detected by the methods employed in this study. These findings imply either a qualitative or a quantitative difference between antibody-dependent cellular cytotoxicity (ADCC) mediated by K cells and cytotoxicity mediated by sensitized T cells.The cytotoxic reaction was completely inhibited by 10 mM EDTA and did not occur in a Ca²⁺- and Mg²⁺-free medium. Neither Ca²⁺ nor Mg²⁺ alone produced as much cytotoxicity as the two cations in tandem; in addition, when added to the culture medium in suboptimal amounts, the two cations were either additive or synergistic. These observations suggest that both cations are necessary in ADCC and also that there may be separate Ca²⁺- and Mg²⁺-dependent events in the lytic pathway.     

Antibody-dependent cell-mediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus.

The phenomenon of antibody-dependent cell-mediated cytoxicity (ADCC) has been extended to include target cells acutely infected with herpes simplex type 1 virus (HSV-1) or herpes simplex type 2 virus (HSV-2) in an in vitro system that employs immune human serum and human blood mononuclear cells. The cytotoxic reaction was detectable after 1 hr of incubation and was complete between 4 and 8 hr. The amount of ADCC noted was directly proportional to the logarithm(10) of the effector: target cell ratio (E:T), and ADCC was noted at E:T as low as 1:1. The mononuclear effector cell was present in the blood of both HSV immune and non-immune individuals. The immune serum factor was demonstrated to be an antibody with specificity for HSV membrane antigen(s) and was reactive with target cells infected with either of the two HSV types. The antibody rendered the mononuclear cell cytotoxic by sensitization of the target cell rather than by direct attachment to or "arming" of the mononuclear cell. The physiochemical properties of the antibody as well as its presence in cord blood demonstrated that it is an immunoglobulin on the IgG class.     

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.     

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.     

Identification of target antigen for antibody-dependent cellular cytotoxicity on cells carrying Epstein-Barr virus genome

The target antigen for antibody-dependent-cellular cytotoxicity (ADCC) on Epstein-Barr virus-(EBV) carrying lymphoblastoid cells expressing EBV-specific membrane antigen (MA) were examined with human serum antibody and adult human peripheral lymphocytes as effector cells. These studies confirmed that anti-MA-positive but not MA-negative sera were reactive in the ADCC. The ADCC reaction was positive with cells in which the MA consisted of late (LMA) and early (EMA) components. These included 1) MA-positive cells prepared by EBV antigen-adsorption, 2) cells carrying de novo-synthesized MA without adsorbed MA, and 3) EBV-producer cells expressing MA spontaneously. In all these preparations, the target cells were lysed roughly in parallel with the frequency of MA-positive cells. Inhibition of LMA synthesis in EBV-superinfected cells by phosphonoacetate (PA) reduced ADCC sensitivity significantly and to a far greater extent than MA synthesis as measured by immunofluorescence. This suggests that a target for ADCC is the PA-sensitive LMA. No ADCC reaction occurred with the cell preparation comprised of a high percentage of MA-positive cells induced by 5-iodo-2'-deoxyuridine, which is believed to be EMA only. These results strongly suggest that the target antigen for ADCC in EBV-positive cells is a late MA but not early MA.     

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.     

Polymorphonuclear leukocytes in antibody-dependent cellular cytotoxicity.

Human polymorphonuclear leukocytes (PMN) lyse antibody-coated target cells in an immunologically specific fashion--antibody-dependent cellular cytotoxicity (ADCC). PMN-mediated cytolysis is independent of complement, de novo protein synthesis, and DNA replication. Cytolysis is rapid, detectable at low PMN:target cell ratios, and exceeds lymphocyte-mediated ADCC. The interaction appears to be mediated via an Fc receptor and is inhibited by aggregated gamma-globulin. A role for PMN in host tumor cell immunity is discussed.     

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity. II. Reversibility of the heart inactivation of K cell killing and relationship of Fc receptor capping to lysis.

The heat inactivation of human blood mononuclear cells active in antibody-dependent cellular cytotoxicity (ADCC) is largely reversed after 24 hr in culture at 37 °C. The reactivation process is inhibited by actinomycin D, cycloheximide, and emetine but not by mitomycin-C, indicating that recovery requires RNA and protein synthesis but not DNA synthesis. The ability of lymphocytes to cap surface immunoglobulin (SIg) and IgG-Fc receptors (FcR) was also studied. As with ADCC effector cell activity, both SIg and FcR capping were abolished by heating, and the kinetics of inactivation was similar to that of the inactivation of ADCC effector activity. In addition, the heat inactivation of capping was reversible in culture and followed kinetics of reactivation similar to that of K cell reactivation. These results suggest the participation of heat-labile proteins at or near the surface of the effector cell, which are also apparently involved in the capping of surface receptors. Presumably these heat-labile proteins are membrane-associated enzymes, but they may also be cytoskeletal structures such as microfilaments or microtubules whose heat-sensitivity is currently unknown. The mounting of lethal hits may involve the same membrane machinery which is responsible for capping or a capping process itself.     

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.     

Characterization of VP22 in herpes simplex virus-infected cells

We examine biochemical characteristics of the herpes simplex virus (HSV) tegument protein VP22 by gel filtration, glycerol sedimentation, and chemical cross-linking experiments and use time course radiolabeling and immunoprecipitation assays to analyze its synthesis and interaction with other infected-cell proteins. VP22 was expressed as a delayed early protein with optimal synthesis requiring DNA replication. In immunoprecipitation assays, VP22 was found in association with several additional proteins including VP16 and a kinase activity likely to be that of UL13. Furthermore, in sizing chromatography experiments, VP22 was present in several higher-order complexes in infected cells. From gel filtration analysis the major form of VP22 migrated with a molecular mass of approximately 160 kDa, consistent with its presence as a tetramer, or a dimer complexed with other proteins, with a fraction of the protein migrating at larger molecular mass. In vitro-synthesized VP22 sedimented in a size range consistent with a mixture of tetramers and dimers. Short N- or C-terminal deletions resulted in migration almost exclusively as dimers, indicating that VP22, in the absence of additional virus-encoded proteins, could form higher-order assemblies, most likely tetramers, but that both N-and C-terminal determinants were required for stabilizing such assemblies. Consistent with this we found that isolated proteins encompassing either the N-terminal or C-terminal region of VP22 sedimented as dimers, and that the purified C-terminal domain could be cross-linked into dimeric structures. These results are discussed with regard to possible virus and host interactions involved in VP22 recruitment into virus particles.