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INK-20, a synthetic phosphocholine lipid-AZT conjugate, was evaluated for antiviral activity against wild-type HIV-1, a matched pair of pre-AZT and post-AZT and multidrug resistant clinical isolates. In addition, it was tested for activity against viruses resistant to nucleoside (AZT, 3TC) and nonnucleoside (nevirapine) reverse transcriptase and protease (saquinavir) inhibitors using the syncytial plaque reduction assay for infectious virus multiplication. The EC50 values were 0.004, and 0.005 microM against wild-type HIV-1 for INK-20 and AZT, respectively. INK-20 showed little or no cytotoxicity when assayed in CEM-SS cells and four other cell types including PBMC. This resulted in a selective index of > 25,000 and > 20,000 for INK-20 and AZT, respectively. When tested against a matched pair of pre-AZT and post-AZT clinical isolates, the EC50 values were 0.01 and 0.03 microM for INK-20 and 0.0005 and 0.33 microM for AZT, respectively. INK-20 had moderate to good activity against two other AZT resistant variants and very good activity against a multi-drug resistant clinical isolate compared to marked resistance of these viruses to AZT alone. INK-20 retained significant activity against viruses resistant to 3TC, nevirapine, and saquinavir. The synthetic phosphocholine lipid-AZT conjugate INK-20 represents a novel class of anti-HIV compounds, which may provide new strategies for the treatment of HIV drug-resistant variants.

Phospholipid analogs are a new class of compounds with potent activity against HIV infection when used alone or conjugated with other therapeutic agents. When conjugated to the nucleoside analog AZT, the resulting phospholipid-AZT conjugate can double target the virus replication cycle by inhibiting the viral reverse transcriptase (by AZT) and inducing the production of defective virus particles that lack functional gp120 expression on the virus surface resulting in reduced capacity to bind to CD4+ cells and inhibition of infected cell-cell fusion (by phospholipid). Of great interest are data indicating that selected phospholipids are active against drug resistant variants, a current major problem in treating HIV/AIDS and controlling the epidemic occurring in various parts of the world. The purpose of this review is to provide current information on the design and synthesis of various types of phospholipids and phospholipid conjugates, in-vitro and in-vivo antiviral activity, tissue distribution, intracellular metabolism, and mechanism of action. The future development of this novel class of compounds offers an exciting approach for reducing the toxicity and enhancing the distribution of therapeutic drugs to the lymphatics and central nervous system and suppressing the emergence of drug resistant variants of HIV.

We previously synthesized a thioetherphospholipid-AZT conjugate (3'-azido-3'-deoxy-5'-(1-hexadecylthio-2-methoxypropyl)-phosphothymidine, CP-102) with potent anti-HIV-1 activity and significant reduction in cell cytotoxicity compared to AZT alone. To study the cellular metabolism of the conjugate compound we synthesized a double-tritium-labeled thioetherphospholipid-AZT conjugate (3'-azido-3'-deoxy-5'-(1-[9,10-3H]-S-octadecylthio-2-O-methoxypropyl)-phosphothymidine-[methyl-3H], [3H]CP-102). The intracellular radioactive metabolic products of [3H]CP-102 treated human lymphoblastoid CEM-SS cells were analyzed by HPLC and thin-layer chromatography. Results of this investigation provide evidence that a putative intracellular lipid cleavage enzyme metabolizes [3H]CP-102 to form a thioetherdiglyceride compound that migrates with an authentic 1-S-octadecyl-2-O-methyl-thioglycerol standard on TLC. The thioetherdiglyceride metabolite did not react with the ninhydrin reagent indicating it did not contain a primary amine such as that found on serine or ethanolamine containing phospholipids. Also, the product did not contain a phosphatidic acid group based on migration characteristics in the TLC plate. The other major hydrophilic metabolite was 3'-azido-3'-deoxythymidine-[methyl-3H]-monophosphate (AZT-MP) with lesser amounts of AZT, AZT-DP and AZT-TP. In summary, the best interpretation of these data is that the thioetherphospholipid-AZT conjugate, [3H]CP-102, is cleaved by a putative intracellular lipid cleavage enzyme to release a thioetherdiglyceride compound and AZT-MP. The resulting AZT-MP was either dephosphorylated to AZT or sequentially phosphorylated to AZT-DP and, ultimately, to AZT-TP, the known inhibitory metabolite against HIV-1 reverse transcriptase. Phospholipid-nucleoside conjugates may provide a unique approach for developing anti-HIV-1 prodrugs that do not have a strict requirement for a nucleoside kinase for initial activation of the prodrug to an antiviral form.

The oral route is the preferred route of delivery for a large number of drug molecules. However, the intestinal epithelium presents a formidable barrier for delivery of drugs into systemic circulation. Phospholipids are among compounds that enhance the absorption of drugs across the intestinal epithelium. In this paper, we describe structure-activity relationships for phospholipid derivatives as enhancers of paracellular permeability across Caco-2 cell monolayers. In a series of 2-alkoxy-3-alkylamidopropylphosphocholine derivatives, compounds with a long chain at C-3 (R3) and short chain at C-2 (R2) were potent in causing a decrease in transepithelial electrical resistance (TEER) and an increase in mannitol transport, but also showed significant cytotoxicity. Compounds with 9-11 carbons at C-3 and 6-10 carbons at C-2 provided good separation (up to 2.7-fold) between activity and cytotoxicity. Notably, a good correlation (r2 = 0.93) was observed between EC(50) (TEER) [concentration that caused a drop in TEER to 50% of its control (untreated) value] and EC10x (mannitol) [concentration that caused 10-fold increase in mannitol transport over the control (untreated) value], confirming that a decrease in TEER is associated with enhanced permeability of the hydrophilic compounds across Caco-2 cell monolayers. Compounds with medium to long carbon chains at C-2 and C-3, and the total carbons in the alkyl chains > 20, showed poor activity and no cytotoxicity.

Detection of the products of the PCR reaction using nonisotopically labeled DNA molecules containing biotin, fluorescein, or digoxigenin has become a popular method for identification of specific products of polymerase chain reaction (PCR) (1,3). These labeled molecules are prepared either by PCR synthesis in the presence of labeled deoxyuridine triphosphate (1,3) or by hybridization of labeled probes to the unlabeled PCR product (1,2). Detection is then in a format very similar to enzyme-linked immunosorbent assays (ELISA) using similarly labeled antigens and antibodies, i.e., by capture on the receptor for one ligand (streptavidin or antibody) and using the other ligand for detection.

We have recently reported that certain ribosylated polyhalogenated benzimidazoles are potent and selective inhibitors of HCMV replication at noncytotoxic concentrations. To extend the structure-activity relationship beyond these first-generation compounds, we alkylated 5,6-dichloro-2-substituted-benzimidazoles with either a series of substituted benzyl halides or (2-bromoethyl)benzene to obtain five series of nonnucleoside analogues. Evaluation of these compounds for activity against herpes viruses revealed that the new compounds were less active than the benzimidazole ribonucleosides against human cytomegalovirus (HCMV) and inactive against herpes simplex virus type 1 (HSV-1). However, as part of our broader antiviral testing, we found that some of these compounds were active against HIV. Comparisons of the biological data revealed that a chloro or bromo group was required at the 2-position for the best separation of activity against HIV and cytotoxicity. Evaluation of the most active compounds against drug-resistant HIV suggested that they act by a mechanism other than inhibition of reverse transcriptase.

Our laboratories first reported two novel classes of complex synthetic lipids, including alkylamidophosphocholines (PC lipid; CP-51) and alkylamidophosphate ester-linked lipid-AZT conjugates (lipid-AZT conjugates; CP-92), with selective and potent activity against human immunodeficiency virus type 1 (HIV-1). To extend these observations, we synthesized additional PC lipids and lipid-AZT conjugates (INK and INK-AZT conjugate) to evaluate their structure-activity relationships by testing for selectivity against infectious wild-type (wt) and drug-resistant HIV-1 replication, virus fusogenic activity and toxicity for mouse bone marrow cells. PC lipid compounds with medium chain lengths at positions 1 and 2 gave an improved selective index (SI). INK-3, with 12 and 8 carbons and INK-15, with 10 and 12 carbons were among the most selective when evaluated in CEM-SS cells. INK-14, a lipid-AZT conjugate where AZT replaced the choline in PC lipid INK-3, gave the highest SI of > 1250 against both infectious wt HIV-1 replication in CEM-SS cells and a clinical isolate in peripheral blood leukocytes. Notably, the PC lipid compounds INK-3 and INK-15, but not the lipid-AZT conjugate INK-14, were potent inhibitors of matched pairs of AZT-sensitive and AZT-resistant HIV-1 clinical isolates. INK-3 also inhibited replication of HIV-2 and TIBO-resistant HIV-1, and inhibited HIV-1-mediated fusogenic activity by 78, 41 and 9% in a dose-dependent manner. The TC50 for mouse bone marrow cells was > 100 micrograms/ml for INK-3 compared to 9.15-14.17 micrograms/ml for CP-51 and 0.142-0.259 microgram/ml for AZT. These data suggest that optimum PC lipid compounds are significantly less toxic than AZT and have high potential as novel therapeutic agents for AIDS.

Invasive strains of Salmonella spp. cause both systemic and localized infections in humans. The ability to resist infection and some aspects of the tissue pathology associated with the presence of Salmonella in the gastrointestinal tract have been shown to be mediated in part by the induction of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine produced by activated macrophages and lymphocytes. Recent reports indicate that TNF-alpha is involved in the induction of human immunodeficiency virus replication by Salmonella in the latently infected human promonocytic cell line U1. In the present study, we investigated the effects of Salmonella on TNF-alpha production in U1 cells and a related cell line, U38. Unlike Escherichia coli or Yersinia enterocolitica, salmonellae rapidly induce TNF-alpha expression in these cells through a released factor(s). Time course experiments show that the kinetics of TNF-alpha production by U38 cells stimulated with Salmonella conditioned medium closely resemble those observed in response to live Salmonella. The observation that TNF-alpha levels are elevated by 60 min after exposure to either bacteria or their conditioned medium suggests that the soluble inducer is continuously released or shed by the bacteria and that the signal acts rapidly to increase TNF-alpha production. Furthermore, the ability to produce the TNF-alpha inducer is shared by at least four Salmonella serotypes and does not correlate with the abilities to invade and to survive within phagocytes. Treatment of active conditioned medium with trypsin, but not low pH, high temperature, or urea, significantly inhibits its TNF-alpha-inducing effect on U38 cells, a finding which points to a polypeptide product of Salmonella as the mediator of TNF-alpha production. Gel filtration chromatography of Salmonella conditioned medium reveals two peaks of activity, consistent with molecular masses of approximately 150 and 110 kDa.

Two series of thymidine analogs with a hydroxyalkylammonium(amine) moiety have been synthesized and evaluated for antitumor and antiviral activities. The hydroxyalkylammonium-(amine) group was introduced at the 5' position of the 2'-deoxyribose residue of thymidine or at a corresponding position in acyclic thymidine analogs. In order to increase the lipophilicity of these compounds and potentially enable them to cross the cell membrane, the free hydroxy group also was esterified with a long hydrocarbon chain. The hexadecanoyl analogs (compounds 1c, 1d, 7c, and 7d) showed moderate antitumor cytotoxicity against SV-28 and KB cell lines (IC50 approximately 20 microM). Compound 1d showed moderate anti-HIV activity (EC50 = 6.8 microM), while compound 5 showed weak anti-HIV activity (EC50 = 55 microM). None of the compounds showed antiherpes simplex virus activity.

The glycosylation of 3,4-dicyano-2-[(ethoxymethylene)amino]pyrrole (7) with 2-deoxy-2-fluoro-alpha-D-erythro-pentofuranosyl bromide (2) furnished an anomeric mixture of nucleosides (8a,b). This mixture was separated, and the individual anomers were treated with methanolic ammonia to effect a concomitant deblocking and ring closure. This furnished both anomers of 2'-deoxy-2'-fluoro-ara-toyocamycin (9a,b). The cyano moiety of 9b was converted to the carboxamide moiety to furnish 2'-deoxy-2'-fluoro-ara-sangivamycin (10) and to the thiocarboxamide moiety to furnish 2'-deoxy-2'-fluoro-ara-thiosangivamycin (11). The target compounds 10 and 11 showed similar antiproliferative activity against L1210 cells in vitro, with IC50's of 3 and 5 microM. Antiviral evaluation revealed a somewhat different pattern of activity. All analogs, both alpha and beta anomers, were active against human cytomegalovirus (HCMV), albeit the beta anomers were most active. The beta anomers also were active against herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus (HIV). Compound 10 was most active in the series, ca. 10-fold more potent than 11; IC50's for 10 ranged from 4 to 25 nM for HCMV, HIV, and varicella zoster virus (VZV) and from 30 to 500 nM for HSV-1. Even though compound 10 was cytotoxic, which will probably preclude its use as an antiviral drug (IC50's = 0.2-5.5 microM), the difference between cytotoxicity and activity against HCMV, HIV, and VZV was sufficient to indicate specific activity against a viral target.

Membrane-interactive phospholipids (PLs), previously evaluated for activity against HIV-1 in vitro, are known to affect late steps in viral replication. Studies were done to determine the effects of PL analogs on post-translational processing of HIV-1 proteins, binding of viral surface gp160/gp120 to CD4 receptor, and HIV-1-induced cell fusion. Results of this investigation indicated that PL alone (1-octadecanamido-2-ethoxypropyl-rac-3-phosphocholine, CP-51) and PL-AZT conjugate (1-octadecanamido-2-ethoxypropyl-rac-3-phospho-3'- azido-3'-deoxythymidine, CP-92) have no effect on HIV-1-induced syntheses or processing of gp160/gp120, pr51, p24, or p17 (including myristoylation) in infected cells. Progeny HIV-1 particles made in CP-92-treated H9IIIB cells contained gp120, pr51, and p24; however, these virus particles had reduced capacity to bind to CD4+ cells. Both CP-51 and CP-92 inhibited syncytium (cell fusion) formation between treated HIV-1-infected cells and uninfected CD4+ cells, and, they reduced HIV-1 gp160/gp120 binding to CD4+ cells and monoclonal antibody. These results suggest that anti-HIV-1 activity of PL compounds involves alteration of cell surface membranes and viral envelopes. Phospholipid compounds are a novel class of membrane interactive compounds with potential use in blocking the spread of HIV-1 infection and pathogenesis in AIDS.

Salmonellae possess the ability to adhere to and invade macrophages and in so doing trigger a number of intracellular events that are associated with cellular activation. As an initial approach to defining the mechanisms by which invasive salmonellae alter macrophage function, we have explored the impact of Salmonella infection on the production of human immunodeficiency virus (HIV) in U1 cells, a promonocytic cell line latently infected with the virus. Infection of U1 cells with a pathogenic strain of Salmonella enteritidis resulted in a marked induction of macrophage activation and HIV production. The stimulatory effect of salmonellae was mediated by signals other than lipopolysaccharide. Salmonella mutants with specific defects in invasion or intracellular survival were markedly less effective in the induction of HIV production. In contrast to S. enteritidis, strains of Yersinia enterocolitica, Legionella pneumophila, and Escherichia coli did not induce HIV production. However, all of these bacteria induced comparable levels of gene expression mediated by the HIV long terminal repeat. The results of this study are consistent with the notion that invasive salmonellae possess the ability to activate the macrophage by at least one mechanism that is not shared with several other species of gram-negative bacteria. Furthermore, the expression of this unique property is maximal with Salmonella strains that are not only invasive but also capable of prolonged survival within the macrophage. Our results indicate that the U1 cell line may be a very useful model system with which to examine the biochemical pathways by which internalized salmonellae modulate the activation state of the macrophage.

Triciribine (TCN) and its 5'-monophosphate (TCN-P) are novel tricyclic compounds with known antitumor activity; TCN-P is currently in phase II human clinical trials. We now report that these compounds have potent and selective activity against HIV-1 and HIV-2. Using a syncytial plaque assay, TCN and TCN-P were active against HIV-1 at 0.01-0.02 microM and had differential selectivities of 2250 and 1900, respectively, compared to 1850 for AZT. In contrast, TCN and TCN-P had minimal selectivity against human cytomegalovirus (50 and 27, respectively). TCN and TCN-P markedly inhibited HIV-1-induced p24 core antigen production, reverse transcriptase, and infectious virus production in a dose-dependent manner using HIV-1 acutely infected CEM-SS, H9, and persistently infected H9IIIB and U1 cells. In acutely infected PBL cells, TCN and TCN-P inhibited reverse transcriptase and infectious virus production but not p24 core antigen production. Using a microtiter XTT assay, TCN and TCN-P were active against a panel of HIV-1 and HIV-2 strains at IC50 values ranging from 0.02 to 0.46 microM. Evaluation of matched pairs of predrug and postdrug therapy HIV-1 isolates established that AZT-resistant and TIBO-resistant variants of HIV-1 were sensitive to TCN or TCN-P. Furthermore, unlike AZT and other fraudulent nucleosides, neither TCN, TCN-P, nor TCN-TP inhibited the viral reverse transcriptase. Thus, even though triciribine is a nucleoside chemically, it does not act biologically by classic nucleoside modalities but rather by a unique mechanism yet to be elucidated.

The effect of herpes simplex virus type 2 (HSV-2) infection on the oxidative response in infant and adult rabbit alveolar macrophages (AM) was studied using either phorbol myristate acetate (0.5 microgram PMA/ml) or latex (250 micrograms/ml) as eliciting agents in a chemiluminescence (CL) assay. Results indicated that uninfected infant AM responded to a latex-elicited but not PMA-elicited CL response. HSV-2 infection (moi = 1.0) of infant AM for 2 hr at 37 degrees C did not alter the PMA or latex-elicited CL responses. In contrast, uninfected adult AM exhibited a markedly increased CL response when elicited with either PMA or latex. HSV-2 infection (moi = 1) of adult AM for 2 hr further increased both PMA- and latex-elicited CL responses. Increasing the moi to 10 inhibited both PMA- and latex-elicited CL responses. Incubation of uninfected control and HSV-2 infected adult AM for 18 hr at 37 degrees C resulted in spontaneous priming of the cells for increased CL responses. In the absence of PMA HSV-2 alone failed to elicit a CL response in adult AM. Infection with heat-inactivated HSV-2 (moi = 1.0 before heat inactivation) did not prime adult AM for enhanced CL responses. AM from BCG immunized adult rabbit produced a considerably higher level CL response that nonimmunized AM; however, HSV-2 infection of these cells did not further enhance the response. In summary, these data indicate that adult AM but not infant AM can be primed by active HSV-2 infection for an increased CL response elicited by either PMA or latex.

Combinations of an amidoalkylphosphocholine, 8, and AZT have been found to cause an apparent synergistic action in suppressing infectious HIV-1 replication. In addition, amidoalkyl, oxyalkyl, and thioalkyl ether lipids have been chemically linked to anti-HIV-1 nucleosides (AZT and DDI) through phosphate and phosphonate linkages. These conjugates have shown promising in vitro anti-HIV-1 activity. Also, the conjugates have a 5-10-fold reduction in cell cytotoxicity compared to AZT alone. The most active compound, an amidoalkyl ether lipid-AZT conjugates, 4A, was found to have a differential selectivity of 1793 in a syncytial plaque assay. In comparison, AZT alone has a value of 1281.

Experiments were designed to determine whether HIV-1 and herpes simplex virus type 2 (HSV-2) coinfection leads to simultaneous replication of both viruses in the same human CD4+ cell (MT-4 cell line) and the possible effects of coinfection on infectious virus production. Results from transmission electron microscopy analysis revealed replication of typical HSV-2 nucleocapsids in the nucleus and budding of HIV-1 particles through the plasma membrane and through intracytoplasmic vacuoles containing enveloped HSV-2 particles in the same coinfected cell. Coinfection of HIV-1 persistently infected H9IIIB or promonocytic U1 cells with HSV-2 did not alter total production of infectious HSV-2 or the percentage of HSV-2 infectious centers compared with control H9 and U937 cells infected with HSV-2 alone. However, in coinfected promonocytic U1 cells HSV-2 induced infectious HIV-1 production measured by syncytial plaque assay. In summary, both HIV-1 and HSV-2 can coinfect and simultaneously replicate in the same human CD4+ cell. Interactions between HIV-1 and HSV-2 appear to be unidirectional, resulting in accelerated replication of HIV-1 as reported by Albrecht et al. (J Virol 1989;63:1861-1868), but not HSV-2 as shown by us.

A new class of membrane-active ether lipid (EL) analogs of platelet-activating factor were studied for in vitro anti-HIV-1 activity. Human T-cell (CEM-ss) monolayers or suspension cultures were used to determine effects of structural modifications of Type A phosphorus-containing and Type B nonphosphorus EL analogs on (a) the inhibitory concentration50 (IC50) for HIV-1 syncytial plaque formation and cell growth, and, (b) virus budding at the cell plasma membrane. Results indicate that representative Type A and Type B EL inhibit HIV-1 but not herpes simplex virus type 2 plaque formation when added before or up to 2 days after viral infection. Anti-HIV-1 activity does not involve direct inactivation of virus infectivity. Type A EL (IC50 range = 0.2-1.4 microM) with alkyoxy, alkylthio, or alkyamido substitution at glycerol position 1 and ethoxy or methoxy substitution at position 2, and Type B compounds (IC50 range = 0.33-0.63 microM) with an inverse choline or nitrogen heterocyclic substitution at position 3 have selective activity against HIV-1-infected T-cells. EL treatment of HIV-1-infected cells is associated with subsequent release of reverse transcriptase activity, but infectious virus production is inhibited with time after infection. Electron microscopic examination of HIV-1-infected and EL-treated cells revealed absence of detectable budding virus at the plasma membrane but presence of intracytoplasmic vacuolar virus particles. In summary, these data suggest that EL analogs are a novel class of agents that induce defective intracytoplasmic vacuolar HIV-1 formation in T-cells. Being membrane interactive, EL are ideally suited for combination chemotherapy with DNA-interactive anti-HIV nucleoside analogs.

Experiments were designed to identify herpes simplex virus type 2 (HSV-2)-specific functions expressed during stimulation of human embryo fibroblast DNA synthesis. Cultures were partially arrested in DNA synthesis by pretreatment with 5-fluorouracil and maintenance in low-serum (0.2%) medium during virus infection. Results showed that continuous [methyl-(3)H]thymidine uptake into cellular DNA was ninefold greater in HSV-2-infected than in mock-infected cultures measured after 24 h of incubation at 42 degrees C. Shifting mock-infected cultures from low- to high-serum (10%) medium also caused some stimulation, but [methyl-(3)H]thymidine uptake was only twofold greater than in cells maintained with low serum. Plating efficiencies of both HSV-2-infected and mock-infected cells at 42 degrees C were essentially the same and ranged from 37 to 76% between zero time and 72 h of incubation. De novo RNA and protein syntheses were continuously required for HSV-2 stimulation of cellular DNA synthesis. HSV-2 infection markedly enhanced transport, phosphorylation, and rate of incorporation of [methyl-(3)H]thymidine into cellular DNA, starting at 3 h and reaching a maximum by 12 h; after 12 h, these processes gradually declined to low levels. In mock-infected cells these processes remained at low levels throughout the observation period. Pretreatment of cells with interferon or addition of arabinofuranosylthymine at the time of virus infection inhibited stimulation caused by HSV-2. 5-Bromodeoxyuridine density-labeled experiments revealed that HSV-2 stimulates predominantly semiconservative DNA replication and some DNA repair. Stimulation of [methyl-(3)H]thymidine into cellular DNA correlated with detection of virus-specific thymidine kinase activity. In conclusion, HSV-2 stimulation of cellular DNA synthesis appeared to involve at least four virus-specific functions: induction of thymidine transport, HSV-2 thymidine kinase activity, semiconservative replication, and repair of cellular DNA.

Rat embryo fibroblasts (REF) morphologically transformed by herpes simplex virus type 2 (HSV-2) and tumor-derived cells were tested for ability to grow in the presence of 9-(2-hydroxyethoxymethyl) guanine (acyclovir). Results indicated that the effective dose of acyclovir (ACV) required to inhibit HSV-2-transformed and tumor-derived cell growth by 50% (ED50) compared to mock-treated control cells averaged 15 to 75 micrograms/ml. In contrast, the ED50 of acyclovir was more than HEp-2 cells. HSV-2-transformed and tumor-derived cells after both low (less than 30) and high (greater than 30) serial passages expressed detectable levels of the virus-coded thymidine kinase (TK) measured in cell extracts by serum neutralization assay. HSV-2-transformed or tumor-derived cells converted two- to ten-fold more acyclovir to phosphorylated forms than nontransformed REF cells. Preliminary data showed that the drug inhibited tumor development in newborn syngeneic rats inoculated with HSV-2-transformed cells. The inhibitory activity of acyclovir and presence of low levels of HSV-2 TK activity appeared to correlate.

This study was undertaken to define the age-related alterations in lipid composition that resident rabbit alveolar macrophages (AM) undergo during postnatal development. The eventual goal is to correlate these changes with the functional maturation of these cells. The number of AM recorded from total lung lavages rose markedly during the first 14 days of life, from 4.9 X 10(5) to 1.1 X 10(7). Adult lungs yielded 1.1 X 10(8) AM. A gradual but significant increase in fluorescence polarization (P) was observed during development when purified AM plasma membranes were tagged with the probe 1,6-diphenyl-1,3,5 hexatriene trimethyl ammonium. The rise ranged from a mean P value of less than or equal to 0.22 to 0.24 (p less than 0.001) for AM plasma membranes from rabbits 1- or 7-day-old to 30- or 150-day-old rabbits, respectively. This finding suggests that the fluidity of the AM plasma membrane decreased during postnatal development. Palmitic, stearic, oleic, and linoleic acids were the most prevalent fatty acids found in the neutral lipid fraction of the AM plasma membrane throughout development. The content of stearic acid rose from 10 to 16%, arachidonic acid rose from 2.8 to 9%, myristic acid decreased from 3.2 to 1.3%, palmitic acid decreased from 42 to 36%, and oleic and linoleic acids changed relatively little during the first 30 days of life. The levels of docosatetraenoic and docosapentaenoic increased gradually during the first 14 days of life, and by 30 days of life the levels declined to that observed at birth. The sum of these changes resulted in an increase in the ratio of unsaturated to saturated fatty acids (1 to 1.15) in the neutral lipid fraction. During the first month of life, the neutral lipid fatty acid pool in the total lipid fraction of AM plasma membrane increased from 12 to 18 mole %, cholesterol increased from 7 to 14 mole %, and total phospholipids decreased from 81 to 67 mole %. These changes resulted in increasing the cholesterol to phospholipid ratio from 0.09 at birth to 0.23 by 150 days of life. The levels of all three major lipid fractions were comparable at 30 days and 150 days of life. Adult levels of choline phosphoglycerides, the predominant phospholipid, were observed by 7 days of life to have decreased from 47 to 34.5 mole %, and the levels of ethanolamine phosphoglycerides and sphingomyelin increased from 17.5 to 25 mole % and from 9 to 13 mole %, respectively. Adult levels of lyso-bis-phosphatidic acid were reached by 30 days of life increasing from 8.2 to 17.8 mole %.(ABSTRACT TRUNCATED AT 400 WORDS)

Unstimulated human leukemia T-cell lines (MOLT-4, MT-4) were tested for their susceptibility to herpes simplex virus type 2 (HSV-2) infection. Permissive infection of MT-4 cells was demonstrated by growth curve and infectious center assays. In growth curve experiments new progeny virus replication was detected by 24 hrs and maximum titers of HSV-2 replication were measured by 72 hrs after infection of MT-4 cells, whereas, MOLT-4 cells did not produce detectable infectious HSV-2 in growth curve experiments. It may be that a T-cell subset is involved with infectious HSV-2 production, since 5.7% of MT-4 cells were scored as infectious centers after HSV-2 infection compared to only 0.06% of MOLT-4 cells. Furthermore, HSV-2 infected MT-4 (45% of cells) and MOLT-4 cells (30% of cells) expressed viral induced antigen(s) detected by immunofluorescence assays. These data provide the first evidence of infectious HSV-2 replication in T-cells not prestimulated in vitro with mitogens, pharmacologic agents or growth factors. The establishment of T-cell systems that permit rapid and efficient replication of HSV-2 could greatly facilitate studies on interactions between human herpesviruses and AIDS retroviruses since recent published evidence indicates possible synergistic interactions between these virus groups.

The effect of herpes simplex virus type 2 infection on the synthesis of phospholipids in human embryo fibroblasts was determined at temperatures permissive (35 C) or nonpermissive (42 C) for virus replication. Incorporation of [32P]i was decreased by herpes simplex virus type 2 infection after 6 hr, which corresponds to the time of initiation of progeny virus production. No differences were observed in the relative incorporation of [32P]i phospholipid classes. In another series of experiments cells were labeled with [3H]ethanolamine before infection and with [14C]ethanolamine after infection. The incorporation of [14C]ethanolamine was also decreased after 6 hr of infection. When choline was substituted for ethanolamine, a similar, although less pronounced, decrease in incorporation was seen in infected cells compared to mock-infected cells. During abortive infections at 42 C, incorporation of [3H]thymidine into cellular DNA was stimulated, but the incorporation of phospholipid precursors was decreased. Total phospholipid composition and phospholipid acyl group composition were not changed appreciably during abortive or productive infection, regardless of whether the cells were labeled before or after infection. In conclusion, these data indicated that, during herpes simplex virus type 2 infection, the incorporation of lipid precursors into phospholipid was decreased. The stimulation of cellular DNA synthesis previously observed during abortive infection at 42 C was not paralleled by a detectable stimulation of total phospholipid synthesis. Neither productive nor abortive infection resulted in significant phospholipid compositional changes in the host cell; however, both resulted in a marked inhibition of phospholipid synthesis.

Infected cell protein 10 (ICP10) or antigen 4 (Ag4) and infected cell-specific protein 11/12 (ICSP11/12) have been suggested as specific antigenic markers for cervical carcinoma. Experiments were designed to determine whether ICP10 and ICSP11/12 are distinct antigens and to determine the cellular localization of ICP10. Results indicate that an apparent 160 kdalton (kDa) protein analyzed by 8.5% polyacrylamide gels (= 144 kDa protein analyzed by 7.0% polyacrylamide gels) was detected in HSV-2-infected but not mock-infected extracts. This protein is an early virus-induced protein appearing 2-4 h after HSV-2 infection, and it was synthesized in the presence of successive blocks with cycloheximide and actinomycin D. These properties are characteristic for ICP10 (Ag4), thus establishing the identity of the 160 kDa/144 kDa protein as ICP10. Furthermore, Western blot analyses indicated that ICP10 and ICSP11/12 are distinct antigens recognized by antibodies in sera from immune rabbit or human cervical carcinoma patients. In addition, monoclonal antibodies to the HSV-2-induced ribonucleotide reductase were reactive with ICP10. Antibodies in sera from rabbits immunized against ICP10 and monoclonal antibodies to the HSV-2-induced ribonucleotide reductase were reactive with antigens on the plasma membrane surface of HSV-2-infected cells. Also, the reactivity of monoclonal antibodies with these antigens was blocked by the rabbit antibodies based on immunofluorescence analyses. These data provide evidence that ICP10 is antigenically distinct from ICSP11/12, and that ICP10 is present on the plasma membrane of HSV-2-infected cells. Also, our data confirm and extend the tentative identification of ICP10 with the HSV-2-induced ribonucleotide reductase recently suggested by Bacchetti et al. (J. Virol. 49, 591-593, 1984).

Polyhedral cytoplasmic deoxyribovirus virions contain a DNA-dependent RNA polymerase which catalyzes the incorporation of ribonucleotides into an acid-precipitable product. Treatment of virions with sodium deoxycholate and dithiothreitol resulted in the formation of subviral particles which could be separated from virions by rate zonal centrifugation in sucrose gradients. Subviral particles were RNA polymerase-positive and more active per unit mass of protein than virions. In vitro enzyme activity associated with subviral particles required addition of ribonucleotides, Mg(2+), and exogenous denatured DNA template. Optimal enzyme activity occurred over a broad pH (7.2 to 8.8) and Mg(2+) concentration (2 to 10 mumol) range. The specific activity of the RNA polymerase was maximal at 37 C. Addition of DNase or actinomycin D to the reaction mixture reduced the incorporation of [(3)H]UMP into an acid-precipitable product. The product of the reaction was sensitive to degradation by RNase but not to DNase or Pronase. These data suggest that the enzyme copies DNA into RNA.

In this report we describe results showing that both arachidonic acid (20:4) release and prostaglandin (PG) synthesis in herpes simplex virus type 2 (HSV-2)-transformed tumor-derived rat fibrosarcoma (RFS) cells are inhibited by indomethacin (cyclooxygenase inhibitor). These data suggest that cyclooxygenase (prostaglandin endoperoxide synthetase, PES) and phospholipase are coupled in their regulation in RFS cells. Data obtained using a cell-free assay to measure directly cyclooxygenase specific activity in the absence of phospholipase activity indicate that constitutive cyclooxygenase activity is very low in non-transformed REF and is not induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, after an initial degradation of constitutive enzyme, cyclooxygenase is synthesized de novo following TPA treatment of HSV-2-transformed RFS clonal cells. At least a 5-fold induction of cyclooxygenase occurs by 6 h post-treatment of HSV-2-transformed RFS clonal cells. This induction is inhibited by pretreatment of cells with cycloheximide (protein synthesis inhibitor) 1 h prior to TPA treatment. Results obtained following delayed addition of cycloheximide to cells after TPA treatment indicate that the newly synthesized cyclooxygenase has a half-life of approximately 5 min. In addition, HSV-2-transformed RFS clonal cells at low passage (less than 10) show over a 3-fold greater increase in cyclooxygenase specific activity after TPA treatment compared to that in TPA-treated high passage (greater than 20) cells, suggesting that induction of high levels of PG synthesis is transient in these cells. These data indicate that HSV-2 transformation of REF cells is associated with alteration of cyclooxygenase regulation.

The effect of selected monosaccharides on the random migration of normal adult rabbit alveolar macrophages (AM) was investigated. It was observed that 10 mM of L-fucose, L-galactose, or D-mannose stimulated AM migration 1.5-2.0 times. In addition, derivatives of L-fucose and D-mannose occupying the carbon-6 position such as L-fucosyl-lactose, D-mannose-6-phosphate, D-mannitol, and mannan enhanced the migration of AM, whereas derivatives of L-fucose and D-mannose in the carbon-1 position produced no migration enhancement. Macrophage migration enhancement activity that was produced spontaneously by spleen cell cultures from normal young rabbits was destroyed by treatment with L-fucosidase. Accordingly, the migration enhancement factor (MEF) found in spleen cell culture supernatants appeared to depend on L-fucose conjugated to some protein carrier because MEF was non-dialyzable. When normal adult AM were treated with L-fucosidase, they lost their responsiveness to migration inhibitory factor (MIF) but retained their responsiveness to MEF. We have interpreted this to mean that the MIF and MEF receptors are distinct. Synthetic MEFs were prepared by conjugating L-fucose, D-mannose, of L-galactose to bovine serum albumin (BSA). It was noted that these sugar-BSA conjugates were about 200 times more effective than the corresponding free sugars in producing migration enhancement. In addition, these sugar-BSA conjugates neutralized MIF activity in a migration inhibition test.

The consequences of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) interaction with clonal HSV-2-transformed nontumorigenic and weakly tumorigenic cells were studied. Enhancement of anchorage-independent growth (cloning efficiency) occurred in a dose-dependent manner when transformed but not nontransformed cells were pretreated with TPA. All subclonal nontumorigenic cell lines progressed to become tumorigenic after 20 serial passages in the presence of TPA. Also, subclonal, weakly tumorigenic cell lines were enhanced in tumorigenic potential (measured by reduced latent period for tumor formation) after transient treatment with TPA. In summary, TPA irreversibly enhanced the cloning efficiency and tumorigenic potential of transformed cells long after initiation by the virus.

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.

The consequences of herpes simplex virus type 2 (HSV-2) and human embryonic fibroblast cell interaction at different temperatures (37, 40, and 42 degrees C) were investigated. Incubation at 37 or 40 degrees C was permissive for HSV-2 inhibition of host DNA synthesis, induction of virus-specific DNA replication, and infectious virus production. The amount of [methyl-3H]thymidine incorporated into viral DNA and the final yield of new infectious virus were significantly reduced at 40 degrees C compared to 37 degrees C. At 42 degrees C, detectable virus-specific DNA synthesis was totally blocked. Maximum stimulation of host cell DNA synthesis at 42 degrees C was measured after a multiplicity of infection of 0.5 to 1.0 PFU/cell. By autoradiography, data indicated that HSV-2 stimulates host cell chromosomal DNA synthesis. Stimulation of thymidine kinase activity with thermostability properties in common with a virus enzyme was detected during the first 24 h of infection at 42 degrees C, after 24 h the enhanced thymidine kinase activity had properties in common with host cell isozymes. The data obtained during this investigation indicated that stimulation of host cell DNA synthesis does not require viral DNA synthesis.

Experiments were done to determine if cloned transforming sequences from herpes simplex virus type 2 (HSV-2) DNA confer upon transfected Rat-2 cells the capacity to be stimulated in phospholipase and cyclooxygenase activities following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Tumor-derived Rat-2 cells transformed with sub-fragments (BamHI-E, HindIII/HpaI-ED, or PstI-C) overlapping the right-hand end of the BglII-C transforming region of HSV-2 DNA were stimulated by TPA in both phospholipase activity, measured by deacylation of arachidonic acid, and cyclooxygenase activity, measured by prostaglandin synthesis. Non-transfected Rat-2 control cells showed no increase in these enzyme activities following TPA treatment. To our knowledge, this is the first evidence that cloned HSV-2 DNA has the capacity to induce cellular functions (phospholipase and cyclooxygenase) in transformed mammalian cells.

The purpose of this investigation was to determine whether cells transformed by herpes simplex virus type 2 (HSV-2) can be stimulated to synthesize prostaglandins (PG). Stimulation was determined by measuring the release of PG into overlay fluids from cell monolayers prelabeled with [3H]arachidonic acid. Results showed that Ca2+ ionophore A-23187 markedly stimulated arachidonic acid release starting 30 min after treatment of HSV-2-transformed and nontransformed rat embryo fibroblast cells. However, only HSV-2-transformed cells were stimulated in production of PG. HSV-2-transformed, nontumorigenic, rat embryo fibroblast, line G, clone 2.0 cells synthesize nearly equal amounts of prostaglandin E2 (PGE2) and prostaglandin F2 alpha, while tumor (rat fibrosarcoma) cells synthesize primarily PGE2. Stimulation of PGE2 synthesis by Ca2+ ionophore A-23187 or 12-O-tetradecanoyl-phorbol-13-acetate decreased as rat fibrosarcoma cells were serially passaged in tissue culture. At low passage of parental rat fibrosarcoma cells, four distinct morphological clonal cell lines were isolated, which varied markedly in their capacity to be stimulated in PG synthesis by 12-O-tetradecanoyl-phorbol-13-acetate. There was correlation between the capacity of clone 1 cells to be stimulated in PGE2 synthesis by serum alone and capacity of the tumors produced by the clone 1 cells to metastasize to the lungs of syngeneic tumor-bearing rats. In summary, cell transformation by HSV-2 appears to be essential for stimulation of PG synthesis in cells. The capacity to be stimulated in arachidonic acid metabolism and PG synthesis may be important in the process of carcinogenesis by a putative human cancer virus.

Infection of human embryonic fibroblast cell monolayers with neutral red and light-inactivated herpes simplex virus, type 2 (HSV-2) at supra-optimal temperature (42 degrees C) resulted in persistence of viable cells in suspension culture at 37 degrees C which have properties in common with virus transformed cells: formation of cell aggregates, HSV-2-specific antigens and colony formation in soft methyl cellulose medium. These data are consistent with the idea that photodynamic inactivated HSV-2 has potential oncogenic activity.

The effect of herpes simplex virus type 2 (HSV-2) infection on the synthesis of DNA in human embryonic fibroblast cells was determined at temperatures permissive (37 C) and nonpermissive (42 C) for virus multiplication. During incubation of HSV-2 infected cultures at 42 C for 2 to 4 days or after shift-down from 42 to 37 C, incorporation of (3H)TdR into total DNA was increased 2-to 30-fold as compared with mock-infected cultures. Analysis of the (3H)DNA suggested that host cell DNA synthesis was induced by HSV-2 infection. Induction of host cell DNA synthesis by HSV-2 also occurred in cells arrested in DNA replication by low serum concentration. The three strains of HSV-2 tested were capable of stimulating cellular DNA synthesis. Virus inactivated by UV irradiation, heat, or neutral red dye and light did not induce cellular DNA synthesis, suggesting that an active viral genome is necessary for induction.

Increased cytosolic phospholipid-sensitive, Ca2+-dependent protein kinase C (PK-C) activity is correlated with the highly tumorigenic potential of rat embryo fibroblasts transformed by herpes simplex virus type 2 (HSV-2). Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a decrease in the cytosolic PK-C with a concomitant increase in PK-C recovered in the membrane fraction. Translocation of the PK-C was dependent upon length of exposure to the phorbol diester. PK-C activity in the cytosolic fraction could be stimulated by TPA without the addition of phosphatidylserine and diacylglycerol. It is tempting to speculate that HSV-2 induction of cellular PK-C activity may be important in phosphorylation of proteins needed for promotion of HSV-2-induced carcinogenesis.

Two classes of aminoacyl fucosides termed FL3 and FL4 were studied as possible markers of tumorigenic and metastatic potential in herpes simplex virus type 2 transformed rat cells. In the present study, clonal cell lines of transformed highly tumorigenic and metastatic (t-REF-G-1.1), weakly tumorigenic and nonmetastatic (t-REF-G-2.1), nontumorigenic (t-REF-G-2.0), and secondary nontransformed rat embryo fibroblast cells were labeled with [3H]fucose, and cell extracts were analyzed for ratio of radioactivity incorporated into FL3 and FL4. Results indicated that, in extracts from t-REF-G-2.0 and nontransformed rat embryo fibroblast cells, the ratios of FL4/FL3 were 5.78 and 5.71, respectively. In contrast, t-REF-G-2.1 cells exhibited a FL4/FL3 ratio of 1.45, while t-REF-G-1.1 cells exhibited a FL4/FL3 ratio of 0.74. In subclonal cell lines isolated from TPA-treated and mock-treated t-REF-G-2.1 cells, the FL4/FL3 ratios correlated with the tumorigenic and metastatic potential of these subclones in newborn syngeneic White Buffalo rats. These data suggested that alterations in fucose-labeled components can be used to predict the tumorigenic and metastatic potential of herpes simplex virus type 2-transformed rat cells.

Resistance of frog virus multiplication to rifampin suggests that components peculiar to cytoplasmic deoxyribonucleic acid replicating viruses (e.g., poxvirus) are not equally sensitive to rifampin.