Herpes simplex virus 1 mutant deleted in the alpha 22 gene: growth and gene expression in permissive and restrictive cells and establishment of latency in mice.

R325-beta TK+, a herpes simplex virus 1 mutant carrying a 500-base-pair deletion in the alpha 22 gene and the wild-type (beta) thymidine kinase (TK) gene, was previously shown to grow efficiently in HEp-2 and Vero cell lines. We report that in rodent cell lines exemplified by the Rat-1 line, plating efficiency was reduced and growth was multiplicity dependent. A similar multiplicity dependence for growth and lack of virus spread at low multiplicity was seen in resting, confluent human embryonic lung (HEL) cells. The shutoff of synthesis of beta proteins was delayed and the duration of synthesis of gamma proteins was extended in R325-beta TK+-infected HEL cells relative to cells infected with the wild-type parent, but no significant differences were seen in the total accumulation of viral DNA. To quantify the effect on late (gamma 2) gene expression, a recombinant carrying the deletion in the alpha 22 gene and a gamma 2-TK gene (R325-gamma 2 TK) was constructed and compared with a wild-type virus (R3112) carrying a chimeric gamma 2-TK gene. In Vero cells, the gamma 2-TK gene of R325-gamma 2TK was expressed earlier than and at the same level as the gamma 2-TK gene of R3112. In the confluent resting HEL cells, the expression of the gamma 2-TK gene of the alpha 22- virus was grossly reduced relative to that of the alpha 22+ virus. Electron microscopic studies indicated that the number of intranuclear capsids of R325-beta TK+ virus was reduced relative to that of the parent virus in resting confluent HEL cells, but the number of DNA-containing capsids was higher. Notwithstanding the grossly reduced neurovirulence on intracerebral inoculation in mice, R325-beta TK+ virus was able to establish latency in mice. We conclude that (i) the alpha 22 gene affects late (gamma 2) gene expression, and (ii) a host cell factor complements that function of the alpha 22 gene to a greater extent in HEp-2 and Vero cells than in confluent, resting HEL cells.     

Tyrosine phosphorylation of the herpes simplex virus type 1 regulatory protein ICP22 and a cellular protein which shares antigenic determinants with ICP22.

At least eight herpes simplex virus type 1 (HSV-1) and five HSV-2 proteins were tyrosine phosphorylated in infected cells. The first viral tyrosine phosphoprotein identified was the HSV-1 regulatory protein ICP22. Also, two novel phosphotyrosine proteins were bound by anti-ICP22 antibodies. H(R22) is a cellular protein, while the F(R10) protein is observed only in HSV-1-infected cells.     

Similarities and differences in the Fc-binding glycoprotein (gE) of herpes simplex virus types 1 and 2 and tentative mapping of the viral gene for this glycoprotein.

We performed affinity chromatography and immunoprecipitation experiments to determine whether cells infected with herpes simplex virus type 2 (HSV-2) expressed a glycoprotein that was functionally and antigenically related to the HSV-1 Fc-binding glycoprotein designated gE. We found that a protein from extracts of HSV-2-infected HEp-2 cells bound specifically to an Fc affinity column and that the electrophoretic mobility of this protein in sodium dodecyl sulfate-acrylamide gels was slightly less than the mobility of HSV-1 gE. Immunoprecipitation experiments performed with an antiserum prepared against HSV-1 gE revealed that (i) extracts from HSV-2-infected cells contained a glycoprotein that was antigenically related to HSV-1 gE; (ii) the electrophoretic mobility of the HSV-2 gE was indistinguishable from the mobility of the HSV-2 Fc-binding protein; (iii) the antiserum reacted with both newly synthesized transient forms and stable fully processed forms of both HSV-1 gE and HSV-2 gE; and (iv) the transient and stable forms of HSV-2 gE all had lower electrophoretic mobilities than their HSV-1 counterparts. Electrophoretic analyses of gE precipitated from extracts of HEp-2 cells infected with two sets of HSV-1 x HSV-2 intertypic recombinant viruses suggested that the gene for gE is located at the right end of the HSV genome (0.85 to 0.97 map units) in the unique portion of the S component.     

Characterization of herpes simplex virus 1 alpha proteins 0, 4, and 27 with monoclonal antibodies.

Analyses of the reactivity and patterns of synthesis of infected cell polypeptides (ICPs) specified by herpes simplex virus (HSV) 1 and 2 and by HSV-1 X HSV-2 recombinants indicated that monoclonal antibody H1183 reacted with HSV-1 alpha ICP0, whereas monoclonal antibody H1113 reacted with both HSV-1 and HSV-2 alpha ICP27. H1083 and H1113 and a monoclonal antibody to ICP4 (H640) similar to one previously described (D. K. Braun et al., J. Virol. 46:103-112.) were then used to study the properties of these alpha proteins. The results were as follows: alpha ICP0, ICP4, and ICP27 accumulated primarily in the nuclei of infected cells. ICP4 and ICP27 were poorly soluble in nondenaturing buffer solutions. ICP0 was considerably more soluble than ICP4 and ICP27. ICP0, ICP4, and ICP27 were readily partially purified by immunoaffinity chromatography from lysates of infected cells solubilized with denaturing agents such as sodium dodecyl sulfate. ICP0 and ICP27 were phosphorylated in cells overlaid with medium containing 32P early (1 to 3 h) or late (18 to 20 h) postinfection. A fraction, but not all, 32P that was incorporated early was chased in the presence of unlabeled phosphate. ICP0, ICP4, and ICP27 labeled with either 32P or [35S]methionine yielded multiple spots upon two-dimensional separations. However, ICP4 quantitatively precipitated at the origin when the migration in the first dimension was from acid to base, and both ICP4 and ICP27 partially precipitated at the origin when the direction of migration was reversed.     

Herpes simplex virus 1 ICP22 regulates the accumulation of a shorter mRNA and of a truncated US3 protein kinase that exhibits altered functions

The U(S)3 open reading frame of herpes simplex virus 1 (HSV-1) was reported to encode two mRNAs each directing the synthesis of the same protein. We report that the U(S)3 gene encodes two proteins. The predominant U(S)3 protein is made in wild-type HSV-1-infected cells. The truncated mRNA and a truncated protein designated U(S)3.5 and initiating from methionine 77 were preeminent in cells infected with a mutant lacking the gene encoding ICP22. Both the wild-type and truncated proteins also accumulated in cells transduced with a baculovirus carrying the entire U(S)3 open reading frame. The U(S)3.5 protein accumulating in cells infected with the mutant lacking the gene encoding ICP22 mediated the phosphorylation of histone deacetylase 1, a function of U(S)3 protein, but failed to block apoptosis of the infected cells. The U(S)3.5 and U(S)3 proteins differ with respect to the range of functions they exhibit.     

Type-Common CP-1 Antigen of Herpes Simplex Virus Is Associated with a 59,000-Molecular-Weight Envelope Glycoprotein

The CP-1 antigen of herpes simplex virus type 1 (HSV-1) is a glycoprotein found in the soluble portion of infected cells, in detergent extracts of infected cell membranes, and in the envelope of purified virus. Antisera were prepared against a further purified form of CP-1 prepared from HSV soluble antigen mix; a glycoprotein, gp52, isolated from detergent-treated infected cells; and detergent extracts of purified virus. Each of the antisera reacted with CP-1 to give a single immunoprecipitin band of identity, and each antiserum neutralized the infectivity of HSV-1 and HSV-2. Our results suggested that the type-common determinants involved in the stimulation of neutralizing antibody resided on a 52,000-molecular-weight (52K) glycoprotein. The envelope of HSV contains several glycoproteins: one component at 59K and a complex of two or three components at 130K, none of which corresponds in molecular weight to gp52. Using the antisera as immunological probes, we performed pulse-chase experiments with [(35)S]methionine-labeled HSV-1-infected cells and followed the disposition of the glycoproteins during the infectious cycle. Each antiserum immunoprecipitated a (35)S-labeled 52K protein from lysates of cells pulse-labeled at 5 h after infection. By 10 h, the label was chased into a 59K protein also precipitable by each of the three antisera. The results suggest that gp52 is a precursor of gp59 and that the latter corresponds in molecular weight to one of the major glycoproteins of the virion envelope.     

Temporal regulation of herpes simplex virus type 2 VP22 expression and phosphorylation

The VP22 protein of herpes simplex virus type 2 (HSV-2) is a major component of the virion tegument. Previous work with HSV-1 indicated that VP22 is phosphorylated during infection, and phosphorylation may play a role in modulating VP22 localization in infected cells. It is not clear, however, when phosphorylation occurs in infected cells or how it is regulated. Less is known about the synthesis and phosphorylation of HSV-2 VP22. To study the complete biosynthetic history of HSV-2 VP22, we generated a monoclonal antibody to the carboxy terminus of VP22. Using immunoprecipitation and Western blot analyses, we show that HSV-2 VP22 can be found in three distinct isoforms in infected cells, two of which are phosphorylated. Like HSV-1 VP22, HSV-2 VP22 is synthesized ca. 4 h after infection, and the isoform later incorporated into virions is hypophosphorylated. In addition, we demonstrate for the first time (i) that newly synthesized VP22 is phosphorylated rapidly after synthesis, (ii) that this phosphorylation occurs in a virus-dependent manner, (iii) that the HSV-2 kinase UL13 is capable of inducing phosphorylation of VP22 in the absence of other viral proteins, (iv) that phosphorylated VP22 is very stable in infected cells, (v) that phosphorylated isoforms of VP22 are gradually dephosphorylated late in infection to produce the virion tegument form, and (vi) that this dephosphorylation occurs independently of viral DNA replication or virion assembly. These results indicate that HSV-2 VP22 is a stable protein that undergoes highly regulated, virus-dependent phosphorylation events in infected cells.     

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells.     

Comparative study of rabies virus persistence in human and hamster cell lines.

Persistent infections by rabies virus in BHK-21/13S and HEp-2 cells were studied comparatively. No evidence of interferon production, selection of virus-resistant cells, or integration of the viral genome could be found. Persisting viruses replicated efficiently at 34, 36, and 40 degrees C. Both persistently infected cultures released defective interfering virus particles. A cyclical pattern of infection, which was not characteristic of the persistently infected HEp-2 system, was observed in persistently infected BHK cultures. The virus from persistently infected BHK cultures lost its virulence for mice, whereas the virus from persistently infected HEp-2 cultures retained mouse-killing capacity for more than 3 years.     

Detection of herpes simplex virus type 2 glycoproteins expressed in virus-transformed rat cells.

Rat embryo fibroblasts transformed by herpes simplex virus type 2 (HSV-2) were assayed for the expression of certain virus-specific glycoproteins on the surface membranes. Monospecific antisera to HSV-2-specific glycoproteins, designated gAgB, gC, and gX, were used in membrane immunofluorescence studies with HSV-2-transformed cell lines tREF-G-1, tREF-G-2, and a tumor-derived rat fibrosarcoma cells line produced in syngeneic rats inoculated with tREF-G-1 cells. Analysis of the three HSV-2-transformed cell lines showed that antisera to the gAgB and gX glycoproteins were reactive with these cells. In contrast, no significant reactivity was observed when anti-gC serum was reacted with the HSV-2-transformed cell lines. All three antiglycoprotein sera reacted positively with rat cells productively infected with HSV-2. Additionally, the HSV-2-transformed and tumor-derived cell lines showed positive internal immunofluorescence after reaction with antiserum to an early, nonstructural viral protein designated VP143 (molecular weight, 143,000). Infectivity of HSV-2 in standard plaque assays was neutralized by hyperimmune rat antisera to tREF-G-2 or rat fibrosarcoma cells and to HSV-2 virions and by sera from rats bearing the fibrosarcoma. Adsorption of rat-anti-HSV-2 serum with tREF-G-2 or rat fibrosarcoma cells reduced neutralizing activity to 10 and 12%, respectively, compared with 90% neutralization by antiserum adsorbed with nontransformed rat embryo fibroblast cells and 100% neutralization with unadsorbed antiserum. In summary, HSV-2-transformed rat cells retained and expressed genetic information necessary for the production of HSV-2 glycoproteins and a nonstructural protein after high passage in tissue culture or in the syngeneic host.     

Nuclear localizations of the herpes simplex virus type 1 tegument proteins VP13/14, vhs, and VP16 precede VP22-dependent microtubule reorganization and VP22 nuclear import

Herpes simplex virus type 1 (HSV-1) induces microtubule reorganization beginning at approximately 9 h postinfection (hpi), and this correlates with the nuclear localization of the tegument protein VP22. Thus, the active retention of this major virion component by cytoskeletal structures may function to regulate its subcellular localization (A. Kotsakis, L. E. Pomeranz, A. Blouin, and J. A. Blaho, J. Virol. 75:8697-8711, 2001). The goal of this study was to determine whether the subcellular localization patterns of other HSV-1 tegument proteins are similar to that observed with VP22. To address this, we performed a series of indirect immunofluorescence analyses using synchronously infected cells. We observed that tegument proteins VP13/14, vhs, and VP16 localized to the nucleus as early as 5 hpi and were concentrated in nuclei by 9 hpi, which differed from that seen with VP22. Microtubule reorganization was delayed during infection with HSV-1(RF177), a recombinant virus that does not produce full-length VP22. These infected cells did not begin to lose microtubule-organizing centers until 13 hpi. Repair of the unique long 49 (UL49) locus in HSV-1(RF177) yielded HSV-1(RF177R). Microtubule reorganization in HSV-1(RF177R)-infected cells occurred with the same kinetics as HSV-1(F). Acetylated tubulin remained unchanged during infection with either HSV-1(F) or HSV-1(RF177). Thus, while alpha-tubulin reorganized during infection, acetylated tubulin was stable, and the absence of full-length VP22 did not affect this stability. Our findings indicate that the nuclear localizations of tegument proteins VP13/14, VP16, and vhs do not appear to require HSV-1-induced microtubule reorganization. We conclude that full-length VP22 is needed for optimal microtubule reorganization during infection. This implies that VP22 mainly functions to reorganize microtubules later, rather than earlier, in infection. That acetylated tubulin does not undergo restructuring during VP22-dependent, virus-induced microtubule reorganization suggests that it plays a role in stabilizing the infected cells. Our results emphasize that VP22 likely plays a key role in cellular cytopathology during HSV-1 infection.     

Common structural antigen of papovaviruses of the simian virus 40-polyoma subgroup.

An antigenic determinant common to the major capsid polypeptide (VP1) of simian virus 40 (SV40) and polyoma virus is described. Antisera prepared against intact viral particles reacted only with cells infected with the homologous virus by immunofluorescence tests (IF). However, antisera prepared against disrupted SV40 particles reacted in IF with both polyoma- and SV40-infected permissive cells. The cross-reaction with polyoma was localized to VP1 by the following evidence. (i) The IF cross-reaction was inhibited by preincubation of the antiserum with purified SV40 VP1; (ii) purified radiolabeled polyoma VP1 was precipitated by the cross-reactive serum, and this reaction was inhibited by unlabeled SV40 VP1; (iii) other antisera prepared against purified SV40 VP1 or polyoma VP1 reacted in IF with both SV40- and polyma-infected permissive cells. These cross-reacting antisera also reacted in IF with permissive cells infected with BK virus, rabbit kidney vacuolating virus, and the stumptailed macaque virus, suggesting that all members of the polyoma-SV40 subgroup share a common antigenic determinant located in their major capsid polypeptides.     

Accumulation of herpes simplex virus type 1 early and leaky-late proteins correlates with apoptosis prevention in infected human HEp-2 cells

We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803-2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5, d1-2, M11, M15, M16, n504R, n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3, d3-4, d4-5, d5-6, and d6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (gamma(2)) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (beta) and leaky-late (gamma(1)) proteins correlates with the prevention of apoptosis in infected HEp-2 cells.     

A novel cellular protein, p60, interacting with both herpes simplex virus 1 regulatory proteins ICP22 and ICP0 is modified in a cell-type-specific manner and Is recruited to the nucleus after infection

Herpes simplex virus 1 encodes two multifunctional regulatory proteins, infected-cell proteins 22 and 0 (ICP22 and ICP0). ICP0 is a promiscuous transactivator, whereas ICP22 is required in vivo and for efficient replication and expression of a subset of late (gamma2) genes in rodent or rabbit cell lines and in primary human cell strains (restrictive cells) but not in HEp-2 or Vero (permissive) cells. We report the identification in the yeast two-hybrid system of a cellular protein designated p60 that interacts with ICP22. This protein (apparent Mr of 60,000) has not been previously described and has no known motifs. Analyses of p60 revealed the following. (i) p60 bound fast-migrating, underprocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not ICP22 lacking the carboxyl-terminal 40 amino acids, whereas the previously identified cellular protein p78 (R. Bruni and B. Roizman, J. Virol. 72:8525-8531, 1998) bound all forms of ICP22. The interaction of p60 with only one isoform of ICP22 supports that hypothesis that each isoform of herpes simplex virus proteins performs a specific function that may be different from that of other isoforms. (ii) p60 also bound ICP0; the binding of ICP0 was independent of that of ICP22. (iii) p60 localized in uninfected rabbit skin cells in both nuclei and cytoplasm. In rabbit skin cells infected with wild-type virus, p60 was posttranslationally processed to a higher apparent Mr but was not redistributed. Posttranslational processing required the presence of the genes encoding ICP22 and UL13 protein kinase. (iv) In uninfected HEp-2 cells, p60 localized primarily in nuclei. Soon after infection with wild-type virus, the p60 localized in discrete small nuclear structures with ICP0. Late in infection, both ICP0 and p60 tended to disperse but p60 did not change in apparent Mr. The localization of p60 was independent of ICP22, but p60 tended to be more localized in small nuclear structures and less dispersed in cells infected with mutants lacking the genes encoding the UL13 or US3 protein kinases. The results suggest that posttranslational modification of p60 is mediated either by ICP0 (permissive cells) or by ICP22 and UL13 protein kinase (restrictive rabbit skin cells) and that the restrictive phenotype of rabbit skin cells may be related to the failure to process p60 by mutants lacking the genes encoding UL13 or ICP22.     

Expression and characterization of transforming growth factor alpha precursor protein in transfected mammalian cells.

Analysis of a cDNA clone derived from retrovirus-transformed rat fibroblasts has recently suggested that the mature 50-amino-acid form of transforming growth factor alpha (TGF alpha) is derived from a 159-amino-acid transmembrane precursor by proteolytic cleavage. To understand the processing of the TGF alpha precursor molecule in more detail, we have expressed this protein in baby hamster kidney (BHK) fibroblasts under control of the metal-ion-inducible metallothionein promoter and characterized the expressed precursor with site-specific antipeptide antibodies. One of the BHK transfectants, termed 5:2, expressed the TGF alpha mRNA in a cadmium- and zinc-inducible manner. The TGF alpha precursor protein was detected by immunoprecipitation analysis of radiolabeled cell cultures. In the induced 5:2 cells, a polypeptide of Mr 13,000 to 17,000 was readily identified by peptide antisera made to three different regions of the TGF alpha precursor protein. No such protein species were observed in BHK cells treated with cadmium and zinc or in uninduced 5:2 cells. However, two cell lines known to produce TGF alpha naturally, Leydig testicular tumor cells and Snyder-Theilan feline sarcoma virus-transformed Fisher rat embryo fibroblasts, possessed detectable levels of immunologically related Mr 13,000 to 17,000 proteins. Cell fractionation studies indicate that the Mr 13,000 to 17,000 species expressed in induced 5:2 cells is membrane associated, consistent with predictions based on the cDNA sequence of the TGF alpha precursor. Media conditioned by induced 5:2 cells contained epidermal growth factor receptor-competing activity, which, upon size fractionation, was similar in size to the mature processed form of TGF alpha. These data show that these nontransformed BHK cells possess the ability to process the TGF alpha precursor molecule into its native form.     

Baculovirus expression of mammalian G protein alpha subunits.

Complementary DNAs encoding three subtypes of the alpha subunit (alpha i-1, alpha o and alpha s) of rat guanyl nucleotide regulatory proteins were used to construct recombinant baculoviruses which direct high-level expression of the corresponding proteins in cultured Sf9 insect cells. The expressed proteins were recognized by polyclonal antisera specific for the different alpha chains, and co-migrated with the native proteins from rat brain membranes in immunoblotting analyses. Soluble and particulate forms of all three immunoreactive alpha chains were observed following ultracentrifugation of cell lysates. Biosynthetic radiolabelling of infected cells with [35S]methionine or [3H]myristate showed that both soluble and particulate forms of alpha i-1 and alpha o were myristoylated; in contrast, alpha s did not incorporate myristate. The soluble fractions from cells expressing alpha chains showed high levels of GTP-binding activity over that observed in uninfected cells, or in cells infected with wild-type virus. The peak expression levels observed at 72 h post-infection were highest for alpha o at ca. 400 pmol of GTP-gamma-35S/mg protein, or roughly 2% of the total soluble protein. The results of this work show that the baculovirus system can be employed for high-level production of mammalian G protein alpha chains which retain GTP-binding activity and are appropriately modified by myristoylation.     

Flow cytometric detection of herpes simplex virus type 2 infected and transformed cells by immunoenzymatic and by indirect immunofluorescence staining

The glucose oxidase antiglucose oxidase (GAG) immunoenzymatic staining procedure has been used to detect herpes simplex virus (HSV) antigens microscopically. In this study, the GAG procedure was adapted to cells in suspension, and its potential usefulness in flow cytometry was examined. HSV-2 infected monkey kidney and HSV-2 transformed mouse cells were stained using antisera to HSV-2 or to an HSV-2 specific protein with a molecular weight of 38 Kd, respectively, with the GAG procedure. Flow cytometric analysis of the GAG stained cells was then performed by the measurement of scattered light intensity in the angular intervals 1 degree-2 degrees, 2.5 degrees-19 degrees, and 3 degrees-6 degrees. The greatest scattered light intensity decrement caused by staining occurred in the 3 degrees-6 degrees angular interval, as predicted by previous work. In infected cells, which stain intensely by immunofluorescence, the difference between positively and negatively stained cells was adequate for detecting infected cells using the GAG method; however, this was not the case for the lightly staining transformed cells. The indirect immunofluorescence method of analysis of the same populations was superior to the scattered light method of analysis of the GAG stained infected and transformed cells.