P24 Expression Research Articles

Expression of human immunodeficiency virus (HIV) in naturally infected peripheral blood mononuclear cells: comparison of a standard co-culture technique with a newly developed microculture method

Peripheral blood mononuclear cells (PBMCs) from 29 patients infected with human immunodeficiency virus (HIV) were cultured by two different methods. One was the standard co-culture technique, the other a newly developed microculture method. In this assay 10(6) PBMCs were cultivated in 250 microliters medium, no activating agents or allogeneic cells were present. P24 antigen production measured by this method was found in 7 out of 11 PBMC cultures of patients in the Walter Reed (WR) stage 1 or 2, whereas only 4 samples were positive by the co-culture procedure. Cultures from patients in the later stages of the disease (WR 5/6) showed a higher p24 production by the co-culture method than by the microculture assay. It is assumed that rapidly growing HIV strains can be better assessed by the co-culture method which may select for these strains. P24 expression can be more easily obtained by the microculture technique even in cases where slowly replicating strains may be present. In conclusion, results from the microculture procedure described may be a useful supplementation to findings observed by the co-culture method.

Characterization of a variant of human T-lymphotropic virus type I isolated from a healthy member of a remote, recently contacted group in Papua New Guinea.

We report the characterization of a variant of human T-lymphotropic virus type I (HTLV-I) isolated from an interleukin 2-dependent, CD8+ T-cell line derived from peripheral blood mononuclear cells of a healthy member of a remote, recently contacted hunter-horticulturalist group (Hagahai) in Madang province of Papua New Guinea. Antigenic characterization of this variant, designated PNG-1, by immunofluorescence, indicated no expression of gag-encoded proteins p19 and p24 (even after incubation with 5-bromo-2'-deoxyuridine), using monoclonal and polyclonal antibodies against HTLV-I gag gene products. Virus-specific proteins of 15, 19, 46, 53, and 61/68 kDa were demonstrated by Western blot analysis, using sera from patients with serologically and/or virologically confirmed HTLV-I myeloneuropathy, sera from HTLV-I-infected rabbits, and antibodies prepared against the C terminus of the major envelope glycoprotein gp46. Restriction endonuclease maps of PNG-1 proviral DNA differed from that of a prototype strain of HTLV-I (MT-2), but, as verified by polymerase chain reaction, PNG-1 was definitely HTLV-I, not HTLV-II. Nucleotide sequencing and further molecular genetic studies of this variant may provide insights into the origin and evolution of HTLV-I.

Dimethyl Sulfoxide Inhibits Human Immunodeficiency Virus Production in vitro

LDV/7, H9, and MOLT-4, three cell lines infectible by human immunodeficiency virus were incubated with dimethyl sulfoxide, an inducer of cell differentiation. It was shown that this is a powerful inhibitor of viral production, but its effect is transient: viral production resumes when the compound is removed from the culture medium. It does not inactivate the virus, and it fails to prevent viral infection or to inhibit expression of p24 on the surface of the infected cells.

Tumor factors predicting for prognosis in metastatic breast cancer: The presence of P24 predicts for response to treatment and duration of survival

Fifty-one patients with metastatic breast cancer were investigated to determine tumor parameters with prognostic significance. Investigations included determinations of P24 content by immunocytochemical means using a monoclonal antibody to P24 protein; immunocytochemical analysis of estrogen and progesterone receptors; ploidy analysis by flow cytometry, and histologic grading. There were significant correlations between the presence of P24 and estrogen receptor, between histologic grade and P24 expression, and between estrogen and progesterone receptors. Of the tumor factors investigated only P24 protein was, however, of prognostic significance. Patients with P24-positive tumors had a significantly higher rate of response to treatment as well as more prolonged duration of response and duration of survival from diagnosis of metastatic disease. None of the other variables investigated were significantly predictive of outcome. P24 protein may be a useful predictor of prognosis in metastatic breast cancer.

Long-Term Combined rIFN-Alpha-2A and Zidovudine Therapy for HIV-Associated Kaposi's Sarcoma: Clinical Consequences and Side Effects

Interferon alpha (IFN-alpha) has been shown to be effective in treating HIV-associated KS in at least 30% of patients, and Zidovudine has proved beneficial for AIDS patients. Moreover, both drugs have demonstrated an inhibitory effect on HIV replication. Based on the above, we combined IFN-alpha and zidovudine for treatment of HIV-associated KS in order to evaluate tolerance and clinical efficacy. Twenty-one homosexual men with histologically proved HIV-associated KS were treated in an open trial with rIFN-alpha-2a 18 X 10(6) IU every second day and zidovudine 800-1200 mg/d. Treatment was discontinued within the first month in six patients: three of them developed subjective intolerance, and three others contracted severe opportunistic infections or HIV-cachexia. Fifteen evaluable patients received combination treatment over a period of 2-20 months (average 10 months). The dosage was reduced as required based on drug-induced cytotoxicity. Complete remission was observed in four patients, partial remission in three, stable disease in two, and progression in six, resulting in an overall response rate of 46%. Negative p24 expression prior to treatment was a positive predictor. Although extracutaneous involvement had a negative influence on tumor remission, even patients with a mean initial T-helper cell count below 100 mm3 responded positively. In conclusion, combination therapy of rIFN-alpha-2a with AZT may effectively control HIV-related Kaposi's sarcoma in more than 40% of patients. In contrast to monotherapy with IFN-alpha, patients with severely reduced immune systems will also benefit from combined treatment.

Expression and purification of p24, the core protein of HIV, using a baculovirus-insect cell expression system

The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been genetically manipulated to yield a recombinant virus capable of expressing p24, the major core protein of HIV-1, in insect cell culture. The expressed product is a p24 protein flanked by short regions of p17 at the amino terminus and p12 at the carboxy terminus. It has been identified and characterized using monoclonal antibodies on Western blots and by amino-terminal sequence analysis. The presence of p24 in the soluble fraction of infected cells following lysis by detergent or sonication, combined with a high level of expression (in excess of 50 mg/l of culture) facilitates the enrichment of large quantities of recombinant HIV antigen in a simple two-step procedure involving ammonium sulphate fractionation and gel filtration. p24 antigen purified in this way is shown to be an efficient diagnostic reagent.

MAP 30: a new inhibitor of HIV-1 infection and replication

A new inhibitor of human immunodeficiency virus (HIV) has been isolated and purified to homogeneity from the seeds and fruits of the Momordica charantia. This compound, MAP 30 (Momordica Anti-HIV Protein), is a basic protein of about 30 kDa. It exhibits dose-dependent inhibition of cell-free HIV-1 infection and replication as measured by: (i) quantitative focal syncytium formation on CEM-ss monolayers; (II) viral core protein p24 expression; and (iii) viral-associated reverse transcriptase (RT) activity in HIV-1 infected H9 cells. The doses required for 50% inhibition (ID 50) in these assays were 0.83, 0.22 and 0.33 nM, respectively. No cytotoxic or cytostatic effects were found under the assay conditions. These data suggest that MAP 30 may be a useful therapeutic agent in the treatment of HIV-1 infections. The sequence of the N-terminal 44 amino acids of MAP 30 has been determined.

Interactive Laser Cytometric Analysis of Retroviral Protein Expression in HIV-Infected Lymphocytic Cell Lines

We have used interactive laser cytometry to investigate the expression of human immunodeficiency virus (HIV) envelope glycoproteins gp160, gp41, gp120, and the core protein p24 in the HIV-infected human lymphocyte cell lines H-9, CEM-SS, and C8166. This method allowed for the ultrasensitive detection of fluorescence signals at the single cell level and, when combined with specific anti-HIV antibodies, permitted unique quantitative detection of HIV antigens. Indirect immunofluorescence assays with monoclonal antibodies directed against gp120 revealed that a large proportion of lymphocytic cells expressed increased gp120-associated fluorescence consistent with HTLV-IIIRF infection. Certain monoclonal and polyclonal antibodies were also effective in quantifying gp160, gp41, and p24 expression. Expression of these antigens was found to vary significantly within 48 h. Significant loss (greater than or equal to 50%) of gp120 expression was observed when cells were treated with 1.0 microM AZT. The expression of the HIV-associated protein markers gp160, gp41, and p24 was detectable 24 h after infection of C8166, a cord blood lymphocytic cell line. C8166 cells expressed an additional 6- to 10-fold increase in gp120 in 48 h as well as a 3- to 4-fold increase in gp160, gp41, and p24. AZT (0.01 and 0.1 microM) decreased the expression of gp120, gp160, and p24 in a dose-dependent fashion. This new application of interactive laser cytometry permits early, sensitive, and statistically based distinctions in the expression of HIV-associated antigens in infected target cells at the single-cell level, and allows detection of important changes in HIV-associated antigen expression and the kinectics thereof.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of P24 protein in human breast cancer: influence of receptor status and oestrogen exposure

The expression of oestrogen regulated protein, P24, was investigated in 69 breast cancers. At initial evaluation P24 protein was detected significantly more frequently and was present in significantly higher concentration in oestrogen receptor positive than in receptor negative tumours. There was, however, no correlation between P24 staining and progesterone receptor, tumour ploidy or proliferative index. Nineteen patients received a short course of treatment with diethylstilboestrol. Following treatment with oestrogen, P24 staining became positive in 7/13 tumours previously negative for P24, including six tumours which were oestrogen receptor negative. Oestrogen administration also caused an increase of the proliferation index in 12/19 tumours, including 5/7 that were oestrogen receptor positive and 7/12 that were oestrogen receptor negative. In some instances oestrogenic stimulation of proliferation occurred together with increased P24 expression; in other instances proliferation index increased without induction of P24 synthesis. The in vivo effects of oestrogen in clinical breast cancer thus appear to show dissociation between enhancement of protein synthesis and cellular proliferation.

Inhibition of human immunodeficiency virus (HIV-1) infection by diphenylhydantoin (dilantin) implicates role of cellular calcium in virus life cycle

Details of the molecular interactions between human immunodeficiency virus (HIV-1) and its host cell during the infection process are not entirely clear. Building on recent reports by Lehr and Zimmer (1986, DMW 111, 1001–1002) that the membrane-reactive, anti-epileptic drug diphenylhydantoin (dilantin or phenytoin) (PHT) inhibited binding of HIV to lymphocytes, we hypothesized that understanding the relevant effects of this drug on cells may shed light on aspects of HIV-1 infection. We found that PHT inhibited, in a dose-dependent manner, de novo infection of various T-cell lines as well as a monocytic cell line. Moderate inhibition of HIV-1 infection was observed with drug concentrations that are therapeutic in vivo for epilepsy (∼20 μg/ml), and no concentrations used induced deleterious effects on cell growth or viability. Surprisingly, treatment of chronically infected H9 cells reduced HIV p24 expression within 1–6 weeks according to dose. This apparent induction into latency was not inhibited by cotreatment of the chronically infected cells with 5-azacytidine, which indicated that PHT was not inducing latency by induction of methylation of the viral DNA. Flow cytometric analysis demonstrated that PHT did not significantly reduce cell-surface expression of CD4. The possibility remained that the drug inhibited HIV infection due to its known effects on calcium-dependent cellular processes. Subsequent measurements of intracellular calcium demonstrated that an increase of [Ca 2] i occurred at least 24 hr postinfection, prior to synthesis of detectable viral structural protein p24, and that this virus-induced increase in [Ca 2+] i was not due to binding of HIV to the cell. This HIV-induced rise in [Ca 2+] i was significantly inhibited by PHT. PHT demonstrated variable inhibitory effects on infection of normal PHA-stimulated PBLs cultured in vitro, but it was synergistic to low-dose AZT (0.01 μg/ml) in inhibiting infection of cell lines. Because of the known inhibitory effects of PHT on calcium-dependent biochemical processes in the cell, inhibition of HIV-1 infection by PHT suggests that calcium may play a role in HIV infection and maintenance. The drug may also be a candidate therapy for individuals infected with HIV.

Cholesteryl-conjugated oligonucleotides: synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture.

A family of oligonucleotides and phosphorothioate oligonucleotide analogues was synthesized with a cholesteryl group tethered at the 3'-terminal internucleoside link. This modification, introduced to enhance interaction of the polyanions with cell membranes, significantly increases the antiviral activity of the oligomers, as judged by inhibition of syncytia formation and expression of viral proteins p17, p24, and reverse transcriptase for human immunodeficiency virus 1 in Molt-3 cells. In the most favorable case, with a 20-mer cholesteryl-phosphorothioate derivative, complete inhibition by all assays was obtained with an oligomer concentration of 0.2 microM. Even decamers were active, and some antiviral activity was observed for a heptanucleotide cholesteryl-phosphorothioate derivative, which binds very poorly to complementary oligonucleotides. These facts, and the finding that the activity of the phosphorothioate decamers does not correlate with a specific sequence, suggests that a mechanism other than "antisense inhibition" may be operative in these systems.

CD4 antigen-based antireceptor peptides inhibit infectivity of human immunodeficiency virus in vitro at multiple stages of the viral life cycle.

Benzylated derivatives of peptides corresponding to residues 81 through 92 of the CD4 molecule [CD4-(81-92)] inhibit human immunodeficiency virus 1 (HIV-1)-induced cell fusion and infection in vitro. If such peptides are to be considered as candidates in the therapy of HIV infection, it is crucial to know if the anti-HIV efficacy of CD4-based peptides is limited to blockade of infection and virus-induced cell fusion or if other stages of the viral life cycle are affected by these compounds. Accordingly, an in vitro quantitative microassay for acute HIV infection was divided into two kinetic phases corresponding to the two general stages of the viral life cycle: (i) viral infection and (ii) transmission of virus and viral protein products through cell contact or release of free virions. CEM-SS cell cultures were treated with peptide during either the infection or the transmission phase of the assay. When peptides were present during the infection phase, inhibition of syncytium formation correlated with decreased expression of viral core protein p24 and lack of infectious cell centers when cells exposed to virus were washed and replated onto fresh uninfected indicator cells. These data are consistent with complete inhibition of viral infection when peptide is present only during initial exposure to virus. Unexpectedly, parallel inhibition of syncytium formation, decreased p24 levels, and inhibition of infectious cell center formation were also seen even when peptides were added as late as 48 hr after inoculation, during the transmission period of the assay. Since viral binding and penetration are completed well before 48 hr in this assay system, CD4-(81-92) peptide derivatives appear to exert a virostatic effect on cultures already infected with HIV-1, decreasing p24 production, cytopathicity, and cell-mediated infectivity.

High yield synthesis of the bovine leukemia virus (BLV) p24 major internal protein in Saccharomyces cerevisiae

Bovine leukemia virus (BLV) p24 gene was expressed in Saccharomyces cerevisiae under the control of the PHO5 (encoding repressible acid phosphatase, rAPase) promoter. Yeast cells were transformed by a yeast- E. coli shuttle vector carrying the PH05 promoter, the p24 gene and the CYC1 transcription terminator. After low inorganic phosphate (P i) induction of the PHO5 promoter, p24 accumulated in the producing cells up to a concentration representing 10% of total soluble proteins. The expression level of p24 gene was not increased by insertion of the positive regulatory gene PHO4 on the p24 expression vector. The p24 produced in this system and incubated in crude yeast extract showed a remarkably high resistance to proteolytic degradation, a feature that presumably correlates with the compact globular conformation of the protein combined to the stabilizing effect of the N-terminal residue.

Expression of human immunodeficiency virus type 1 (HIV-1) gag antigens on the surface of a cell line persistently infected with HIV-1 that highly expresses HIV-1 antigens

MT-4 cells persistently infected with human immunodeficiency virus type 1 (HIV-1) (MT-4/HIV-1) were recently isolated (K. Ikuta, C. Morita, M. Nakai, N. Yamamoto, and S. Kato, Japan. J. Cancer Res. (Gann), 79, 418–423, 1988). Mouse hybridoma cell clones producing monoclonal antibodies (MoAbs) to HIV-1 gag p24 and pl8, and pol reverse transcriptase (RT) were isolated by using this MT-4/HIV-1 cell line for the screening of MoAb production by the immunofluorescence (IF) test. By indirect IF tests of acetone-fixed cells with these MoAbs, the IF intensities in MT-4/HIV-1 cells were found to be higher than those in the other HIV-1 infected cells, such as MOLT-4/HIV-1, HL-60/HIV-1, and U937/HIV-1 cells. Cell surface expression of the HIV-1 gag p24 and p18 antigens examined by IF and radioimmune techniques with these MoAbs revealed the p24 and pl8 antigens to be expressed strongly on the cell surface of MT4/HIV-1 cells and faintly on the cell surface of MOLT-4/HIV-1 cells, respectively. However, monoclonal antibody isolated in the present study failed to detect pol RT antigen on the surface of MT-4/HIV-1 cells. These results indicate that the gag p24 and p18 antigens are expressed, at least in part, on the surface of HIV-1-infected cells.

Frequent lack of antibodies against human T-cell leukemia virus type I gag proteins p24 or p19 in sera of patients with adult T-cell leukemia.

Specificities of antibodies against human T-cell leukemia virus type I (HTLV-I) in sera of 27 patients with adult T-cell leukemia (ATL) were studied. All sera were positive for HTLV-I antigens by immunofluorescence assay using cells expressing HTLV-I antigens; sera from five ATL patients, however, did not react or hardly reacted with p19 or p24 gag proteins of HTLV-I upon immunoprecipitation assay. Therefore, the relationships among antibody specificities against HTLV-I, the proviral structures of HTLV-I genomes in leukemic cells, and the expression of viral antigens by leukemic cells after cultivation in vitro for a few days were examined. Analyses of the genomic structures of the proviruses revealed deletions in at least seven cases. However, we could not detect deletions in the proviral genomes of four out of the five ATL patients who lacked antibodies against gag proteins. Furthermore, expression of p19 and p24 was detected in these patients' peripheral blood lymphocytes (PBL) cultured in vitro for a few days. Thus, some ATL patients could not or could hardly raise antibodies against gag proteins, although they harbored complete HTLV-I genomes and their PBL expressed gag proteins in vitro. All patients harboring deleted proviruses, so far tested, raised antibodies not only against viral proteins that should be encoded by the integrated proviruses, but also against viral proteins that should be encoded by the deleted regions. Antibodies against viral proteins were detected also in sera of ATL patients whose PBL did not express viral proteins after in vitro cultivation. Specificities of antibodies against viral proteins in ATL patients could not be predicted by the structures of proviruses in leukemic cells or by expression of viral proteins in vitro. Immune responses to HTLV-I antigens were weak or lost in some ATL patients.

Inhibitory effect of papaverine on HIV replication in vitro.

The ability of papaverine to inhibit human immunodeficiency virus (HIV) replication in H9 cell line and in peripheral blood mononuclear cell (PBMC) culture was examined. HIV-infected H9 cells were exposed to different concentrations of papaverine for 20 days. Reverse transcriptase (RT) activity and the presence of p24 in the supernatant were determined to assess the level of viral replication in treated and control cultures. The most effective concentration of papaverine in the culture medium was 10 micrograms/ml, a dose that did not significantly affect cell proliferation. At this drug concentration the treatment resulted in no RT activity or p24 expression in the supernatant and no virus antigen detection at the cellular level as demonstrated by Western blot (WB) analysis. The activity of the drug occurred in a short period of time (60 hours) as shown by radioimmunoprecipitation (RIP) assay and affected the synthesis of the env precursor protein gp160. The drug was also effective in inhibiting HIV replication in PBMC cultures and influenced specific viral markers, namely, RT and p24. Evidence of the efficacy of papaverine treatment was enforced by the finding in the treated PBMC cultures, compared with the untreated ones, of a reduced percentage of cells forming syncitia and of the inhibition of the virus-induced decrease in the number of cells. When an equal number of virus-infected H9 cells exposed or unexposed to papaverine was analyzed for HIV-specific proteins, a marked decrease in the expression of the viral proteins was observed in the treated cultures. At the same time, one cellular protein of molecular weight 69,000 was not inhibited by papaverine. This may indicate that, at least for one protein, synthesis may not be affected by the drug. Our data suggest that papaverine merits attention as a possible nontoxic candidate for the treatment of HIV infection.

The p24 leucocyte membrane antigen: modulation associated with lymphocyte activation and differentiation.

Monoclonal antibodies of the CD9 cluster recognize a 24 kD protein (p24) found on platelets and endothelium, and expressed by lymphocytes at restricted stages of maturation and activation. In this study, we explore the possibility that p24 is involved in the response of lymphocytes to signals delivered by lymphokines. p24 is expressed only very weakly by resting B lymphocytes. We found no increase in expression when cells were activated with anti-immunoglobulin together with interleukin-4, or induced to proliferate by low-molecular weight B cell growth factor (LMW-BCGF). Culture of activated B cells with B cell differentiation factor was associated with an increased mean expression of p24. In cells from a patient with chronic lymphocytic leukaemia (CLL), culture with LMW-BCGF up-regulated p24 expression. Resting T cells (p24-negative) were induced to express p24 strongly when activated with antibody against CD3. CD9 antibody did not modulate B or T cell responses to activation stimuli. The results suggest that the p24 molecule is not involved in the primary interaction of cells with lymphokine, but rather may be involved in a secondary reaction, such as ion flux, which follows as a consequence of the action of lymphokines on cells.

Specific polypeptide differences in normal versus malignant breast tissue by two-dimensional electrophoresis.

High resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in combination with computer-assisted densitometry was used to analyze 800-1000 silver stained postmitochrondrial and 600-800 cytosolic polypeptides extracted from malignant and nonmalignant human breast tissues. The 2D-PAGE patterns of polypeptides from malignant and normal tissues were similar, although both qualitative and quantitative polypeptide differences were noted. Six cytosolic polypeptides (pI/molecular mass x 10(-3), 5.20/80, 5.75/43, 6.20/40, 5.43/35, 5.46/34.5, and 5.50/34 were detected exclusively in malignant tissues. One constitutive polypeptide, p52 (7.25/52), was not detected in tumor samples. Marked quantitative differences in spot density were noted in polypeptides localized mainly in the molecular weight ranges of 22-40 kDa and pI of 5.65-7.00. An overall increase in polypeptide expression was noted in this region of 2D-PAGE gels of malignant tissues as compared to normal. Twenty-two acidic and 19 polypeptides separated under nonequilibrium isoelectric focusing conditions were significantly increased in tumor samples while one polypeptide was decreased. One polypeptide, p24 (6.15/24), was expressed in greatest concentrations in tumors which also expressed the greatest estrogen receptor content. Expression of p24 was markedly reduced in normal tissue and in malignant tissues expressing low levels of estrogen and progesterone receptors. No significant differences in the expression of the Yb and Ya subunits of glutathione-S-transferases (GST)-A, -B and ligandin were observed between normal and malignant breast tissue. None of the Yp subunits of the placental isoform of GST were detected in either normal or malignant breast tissues.

Influence of methisoprinol (lsoprinosine) on hiv‐infected human lymphocytes: In vitro immunological, virological, and ultrastructural studies

Methisoprinol (isoprinosine) is a synthetic compound with reported antiviral and immunomodulating properties. Results of the present study showed that methisoprinol at concentrations greater than or equal to 200 micrograms/ml reduces the p24 and gp120 viral antigen expression on the surface of human immunodeficiency virus (HIV)-infected lymphocytes and the reverse transcriptase levels. In addition, cell viability, the number of the CD4+ cells, and the CD4+/CD8+ cell ratio are higher in methisoprinol-pretreated cell suspensions than in untreated HIV-infected cell cultures. A quantitative freeze-fracture study on the density of the intramembranous particles (IMP) present on both fracture faces of the plasma membrane of lymphocytes has shown that pretreatment with methisoprinol induces a different molecular organization resulting in a nearly three times increase of IMP density.

Human immunodeficiency virus (HIV-1) cytotoxicity: Perturbation of the cell membrane and depression of phospholipid synthesis

The molecular mechanism(s) by which human immunodeficiency virus (HIV-1) injures a T-cell line was studied. A pathological role for viral env proteins, which are inserted into the plasma membrane, has been previously demonstrated for HIV as well as other retroviruses which are cytopathic. We therefore initiated studies examining whether perturbations of the cell membrane or membrane-associated biochemical events may be occurring in cells acutely infected with HIV and whether such perturbations, if present, may be responsible for cytopathology. A human T-cell line (ERIC), which is sensitive to the cytopathic effects of HIVs, was infected with HTLV-III B and its membrane permeability to cations and its lipid metabolism were studied coincident with the peak expression of viral p24 and with the first sign of cytopathology (slowing of cell division) 72 to 96 hr after infection. It was found that the rate of influx of Ca 2+ into the cell increased over that of uninfected cells and that phospholipid synthesis, primarily phosphatidylcholine, became depressed. Diacylglycerol, which serves both as an intermediate for synthesis of phospholipids and as a second-messenger for lymphocyte activation, was also greatly reduced. However, triglyceride synthesis was enhanced, indicating that not all lipid metabolic pathways were being shut down. This decreased membrane-synthetic ability and reduced second-messenger for cell division are likely to be important causes of HIV-1 cytopathology in ERIC cells. This hypothesis was supported by our finding that HIV cytopathology of ERIC cells could be partially prevented by treatment with compounds (diacylglyceride or PMA and transiently by oleic acid) which either replenish diacylglycerol in the infected cell and/or activate protein kinase C or phosphocholine cytidyltransferase, the latter being the rate-limiting step in synthesis of the major structural phospholipid in most cells.