A naturally occurring genetic variant in the human chorionic gonadotropin-beta gene 5 is assembly inefficient.

The hCGbeta gene family is composed of six homologous genes linked in tandem repeat on chromosome 19; the order of the genes is 7, 8, 5, 1, 2, and 3. Previous studies have shown that hCGbeta gene 5 is highly expressed during the first trimester of pregnancy. The purpose of our study was to identify naturally occurring polymorphisms in hCGbeta gene 5 and determine whether these alterations affected hCG function. The data presented here show that hCGbeta gene 5 was highly conserved in the 334 asymptomatic individuals and 41 infertile patients examined for polymorphisms using PCR followed by single stranded conformational polymorphism analysis. Most of the polymorphisms detected were either silent or located in intron regions. However, one genetic variant identified in beta gene 5 exon 3 was a G to A transition that changed the naturally occurring valine residue to methionine in codon 79 (V79M) in 4.2% of the random population studied. The V79M polymorphism was always linked to a silent C to T transition in codon 82 (tyrosine). To determine whether betaV79M hCG had biological properties that differed from those of wild-type hCG, a beta-subunit containing the V79M substitution was created by site-directed mutagenesis and was coexpressed with the glycoprotein hormone alpha-subunit in Chinese hamster ovary cells and 293T cells. When we examined betaV79M hCG biosynthesis, we detected atypical betaV79M hCG folding intermediates, including a betaV79M conformational variant that resulted in a beta-subunit with impaired ability to assemble with the alpha-subunit. The inefficient assembly of betaV79M hCG appeared to be independent of beta-subunit glycosylation or of the cell type studied, but, rather, was due to the inability of the betaV79M subunit to fold correctly. The majority of the V79M beta-subunit synthesized was secreted as unassembled free beta. Although the amount of alphabeta hCG heterodimer formed and secreted by betaV79M-producing cells was less than that by wild-type beta-producing cells, the hCG that was secreted as alphabeta V79M heterodimer exhibited biological activity indistinguishable from that of wild-type hCG.     

Diagnosis of hydatidiform mole and persistent trophoblastic disease: diagnostic accuracy of total human chorionic gonadotropin (hCG), free hCG {alpha}- and {beta}-subunits, and their ratios

OBJECTIVE: Human chorionic gonadotropin (hCG) is widely used in the management of hydatidiform mole and persistent trophoblastic disease (PTD). Predicting PTD after molar pregnancy might be beneficial since prophylactic chemotherapy reduces the incidence of PTD. DESIGN: A retrospective study based on blood specimens collected in the Dutch Registry for Hydatidiform Moles. A group of 165 patients with complete moles (of which 43 had PTD) and 39 patients with partial moles (of which 7 had PTD) were compared with 27 pregnant women with uneventful pregnancy. METHODS: Serum samples from patients with hydatidiform mole with or without PTD were assayed using specific (radio) immunoassays for free alpha-subunit (hCGalpha), free beta-subunit (hCGbeta) and 'total' hCG (hCG + hCGbeta). In addition, we calculated the ratios hCGalpha/hCG + hCGbeta, hCGbeta/hCG + hCGbeta, and hCGalpha/hCGbeta. Specificity and sensitivity were calculated and paired in receiver-operating characteristic (ROC) curve analysis, resulting in areas under the curves (AUCs). RESULTS: hCGbeta, hCGbeta/hCG + hCGbeta and hCGalpha/hCGbeta show AUCs ranging between 0.922 and 0.999 and, therefore, are excellent diagnostic tests to distinguish complete and partial moles from normal pregnancy. To distinguish partial from complete moles the analytes hCGbeta, hCG + hCGbeta and the ratio hCGalpha/hCGbeta have AUCs between 0.7 and 0.8. Although hCGalpha, hCGbeta and hCG + hCGbeta concentrations are significantly elevated in patients who will develop PTD compared with patients with spontaneous regression after evacuation of their moles, in predicting PTD, these analytes and parameters have AUCs <0.7. CONCLUSIONS: Distinction between hydatidiform mole and normal pregnancy is best shown by a single blood specimen with hCGbeta, but hCGbeta/hCG + hCGbeta and hCGalpha/hCGbeta are also excellent diagnostic parameters. To predict PTD, hCGalpha, hCGbeta, hCG + hCGbeta and hCGalpha/hCGbeta are moderately accurate tests, although they are not accurate enough to justify prophylactic chemotherapy treatment for prevention of PTD.     

Nonassembled human chorionic gonadotropin subunits and alphaalpha-homodimers use fast-track processing in the secretory pathway in contrast to alphabeta-heterodimers

In multimeric glycoproteins, like glycoprotein hormones, mutual subunit interactions are required for correct folding, assembly, and transport in the secretory pathway. However, character and time course of these interactions need further elucidation. The influence of the glycoprotein hormone alpha-subunit (GPHalpha) on the folding of the human chorionic gonadotropin (hCG) beta-subunit (hCGbeta) in hCG alphabeta-heterodimers was investigated in [(35)S]Met/Cys-labeled JEG-3 cells. Completeness of disulfide bridge formation during the time course of folding was estimated by labeling with [(3)H]N-ethylmaleinimide of free thiol groups not yet consumed. Subunit association took place between immature hCGbeta (high (3)H/(35)S ratio) and almost completely folded GPHalpha. Analysis revealed a highly dynamic maturation process comprising of at least eight main hCGbeta folding intermediates (molecular masses from 107 to 28 kDa) that could be micro-preparatively isolated and characterized. These hCGbeta variants developed while being associated with GPHalpha. The 107-kDa variant was identified as a complex with calnexin. In contrast to hCG alphabeta-heterodimers, free nonassociated hCGbeta, free large GPHalpha, and GPHalphaalpha homodimers showed a fast-track-like processing in the secretory pathway. At 10 min before hCG secretion, sialylation of these variants had already been completed in the late Golgi, whereas hCG alphabeta-heterodimers had still not arrived medial Golgi. This shows that the GPHalpha in the hCG alphabeta-heterodimers decelerates the maturation of the hCGbeta portion in the heterodimer complex. This results in a postponed approval of hCG alphabeta-heterodimers by the endoplasmic reticulum quality control unlike GPHalphaalpha homodimers, free hCGbeta, and GPHalpha subunits.     

Beta-core fragments are contaminants of the World Health Organization Reference Preparations of human choriogonadotrophin and its alpha-subunit

Highly purified preparations of human choriogonadotrophin (hCG), hCG alpha and hCG beta, including those preparations which are being distributed by the World Health Organization as International Standards, cross-reacted in a new radioimmunoassay with increased relative specificity for the beta-core fragment of hCG. A major portion of the beta-core immunoreactivity in the hCG and hCG alpha preparations eluted from Sephadex G-100 in a position (approximate apparent molecular size 15,000-18,000) corresponding to that of purified beta-core fragment prepared from pregnancy urine. However, in the case of hCG beta-subunit preparations, virtually all of the beta-core cross-reacting material eluted from Sephadex G-100 in the same fractions as the native hCG beta-subunit. Quantitatively, the cross-reacting beta-core material accounts for less than 1% (w/w) of the total hCG or subunit immunoreactivity, as measured by conventional radioimmunoassays. The presence of the beta-core fragments as discrete molecular components of the hCG and hCG alpha preparations should be borne in mind when these preparations are used to calibrate new radioimmunoassays for hCG-related molecules.     

Role of amino acid residues at the interface of alpha52asparginyl-N-glycosyl chain of human choriogonadotropin.

Of all the four N-glycosyl chains present in hCG, only one of them at alpha52Asn is located at the alpha/beta subunit interface and is crucial for the biological function of the hormone. The other three are exposed on the surface of the molecule and play only a minor role in the function of the hormone. The alpha52Asn oligosaccharide interacts with five amino acid residues in the beta-subunit, Tyr59, Val62, Phe64, Ala83, and Thr97. The present studies were undertaken to determine the role of the residues at the alpha52Asn-oligosaccharide and the beta-subunit interface in the mechanism of subunit association and downstream signaling events. Ten mutants, two of the alpha-subunit by the replacement of Asn52 and Thr80 with Gln and eight of the beta-subunit by multiple or single amino acid mutations, were prepared. These mutants included, hCGbeta59,62,64,97Ala, hCGbeta59,62,64Ala, hCGbeta62,64Ala, hCGbeta59Phe, hCGbeta62Ala or Thr, hCGbeta83Ile and hCGbeta97Ala. The mutation of the Asn52 to Gln resulted in a drastic change in its conformation and as a consequence in its weak affinity with the wild type beta as compared with that of the wild type hCGalpha and hCGalpha80Gln. The mutants with mutations in the four or three amino acids as well as both mutants of hCGbeta62Val almost failed to combine with hCGalpha again as a result of conformational changes shown by circular dichroism (CD) analysis and not due to their direct involvement in the subunit association. The double mutant combined with hCGalpha and the heterodimer behaved more like the wild type hCG. The mutation of Tyr to Phe resulted in a drop of 20% in the receptor binding and cAMP stimulation although Tyr is considered to be involved directly in subunit association. HCGbeta with mutations in the other amino acids, Phe64, Ala83, and Thr97, combined with the alpha subunit forming heterodimers with biological activity comparable to that of the wild type hCG. Thus, it appears that among the five amino acids in the vicinity of alpha52Asn carbohydrate, only beta59Tyr and beta62Val may be involved directly or indirectly in the alpha/beta beta dimer formation.     

Inhibition of HIV type 1 replication in CD4+ and CD14+ cells purified from HIV type 1-infected individuals by the 2-5A agonist immunomodulator, 2-5A(N6B)

Two major interferon (IFN)-mediated antiviral defense enzymes are double-stranded (ds)RNA-dependent 2',5'-oligoadenylate (2-5A) synthetase (2-5OAS) and p68 kinase (PKR). When activated by dsRNA, 2-5OAS synthesizes 2-5A, which binds to and activates RNase L. Activated RNase L hydrolyzes single-stranded viral RNA, thereby inhibiting viral protein synthesis. HIV-1 inhibits the IFN-mediated intracellular antiviral pathways. We have reported the synthesis and characterization of a nuclease-resistant 2-5A agonist (2-5A(N6B)) that overcomes the HIV-1 induced blockades by restoring the 2-5OAS/RNase L antiviral pathway (Homan JW, et al., J Acquir Immune Defic Syndr 2002;30:9-20). The objective of this study was to test the effect of 2-5A(N6B) on chronically infected CD4(+) T lymphocytes and CD14(+) monocytes derived from HIV-1-seropositive individuals. Wild-type HIV-1 replication was effectively inhibited by 2-5A(N6B) in CD4(+) T lymphocytes and CD14(+) monocytes purified from HIV-1 seropositive individuals (n = 18) compared to untreated cells. We also assessed the cytotoxicity of 2-5A(N6B) and report that 2-5A(N6B) exerts its anti-HIV-1 activity with no evidence of cytotoxicity (IC(90) > 100,000 nM). Furthermore, 2-5A(N6B) did not alter the cellular RNA profile, affect CCR5 or CXCR4 coreceptor expression, or activate caspase-dependent apoptosis. Evidence is also provided to show that 2-5A(N6B), and naturally occurring 2-5A(4), act as ligands to activate human Toll-like receptor 4. These results indicate that the 2-5A agonist 2-5A(N6B) has the potential to enhance host cell innate and acquired immune defense mechanisms against HIV-1 infection.     

Peptides from conserved regions of paramyxovirus fusion (F) proteins are potent inhibitors of viral fusion.

The synthetic peptides DP-107 and DP-178 (T-20), derived from separate domains within the human immunodeficiency virus type 1 (HIV-1) transmembrane (TM) protein, gp4l, are stable and potent inhibitors of HIV-1 infection and fusion. Using a computer searching strategy (computerized antiviral searching technology, C.A.S.T.) based on the predicted secondary structure of DP-107 and DP-178 (T-20), we have identified conserved heptad repeat domains analogous to the DP-107 and DP-178 regions of HIV-1 gp41 within the glycoproteins of other fusogenic viruses. Here we report on antiviral peptides derived from three representative paramyxoviruses, respiratory syncytial virus (RSV), human parainfluenza virus type 3 (HPIV-3), and measles virus (MV). We screened crude preparations of synthetic 35-residue peptides, scanning the DP-178-like domains, in antiviral assays. Peptide preparations demonstrating antiviral activity were purified and tested for their ability to block syncytium formation. Representative DP-178-like peptides from each paramyxovirus blocked homologous virus-mediated syncytium formation and exhibited EC50 values in the range 0.015-0.250 microM. Moreover, these peptides were highly selective for the virus of origin. Identification of biologically active peptides derived from domains within paramyxovirus F1 proteins analogous to the DP-178 domain of HIV-1 gp4l is compelling evidence for equivalent structural and functional features between retroviral and paramyxoviral fusion proteins. These antiviral peptides provide a novel approach to the development of targeted therapies for paramyxovirus infections.     

A biologically active single chain human chorionic gonadotropin analog with altered receptor binding properties

hCG is a heterodimer consisting of an alpha-subunit common among all members of the glycoprotein hormone family, LH, FSH, and TSH, and a unique beta-subunit responsible for receptor specificity. Biologically active single chain analogs of these hormones have been engineered in which the C-terminus of the beta-subunit was fused to the N-terminus of the alpha-subunit (N-beta-alpha-C) either with or without a linker such as the hCGbeta C-terminal peptide (CTP). This tandem order of subunits was chosen based on studies suggesting that the N-terminal region of hCGbeta and particularly the C-terminal region of the alpha-subunit are important in receptor binding and activation. Single chain hCG (YhCG1) can, in turn, be fused to the LH receptor to yield a hormone-receptor complex that is biologically active in transfected cells. Herein, we report the construction of a new single chain hCG analog (YhCG3) in which the C-terminus of the alpha-subunit is fused to the N-terminus of hCGbeta via a CTP (N-alpha-CTP-beta-C). Compared with YhCG1, this analog binds receptor with a 25- to 30-fold lower affinity, but, surprisingly, is capable of stimulating intracellular cAMP levels to the same extent. Furthermore, YhCG3 can be covalently linked to its receptor to produce a biologically active complex that results in elevated levels of basal cAMP in transfected cells. These results suggest that free N- and C-termini of hCGbeta and the alpha-subunit, respectively, are not essential for receptor binding and activation and that YhCG3 is in a more efficacious conformation for receptor activation than YhCG1.     

The heterogeneity of human chorionic gonadotropin (hCG). I. Characterization of peptide heterogeneity in 13 individual preparations of hCG

Peptide variations in the alpha-subunit (molecules starting at alpha 3 and alpha 4) and beta-subunit (missing linkages at beta 44-45 and beta 47-48) of hCG have been reported by several investigators. Studies, however, have been limited to standard hCG preparations (purified from large pools of urine) and other hCG samples from mixed urines. In this study we used chromatographic procedures to purify the total hCG content of 13 individual urines, 6 from patients with pregnancy and 7 from those with trophoblast disease (no hCG-containing fractions were excluded). Then, we examined for the first time the peptide variability among individual samples of hCG. We report 1) that individual hCG preparations have nicks (missing linkages) in the beta-subunit, primarily between residue 47-48 (11 of 13 samples) and, less commonly, at the linkage 44-45 or 46-47 (3 of 13 samples); 2) the extent of nicking varies greatly between individual preparations (range, 0-100% of molecules); 3) varying alpha-subunit N-terminal heterogeneity (N-terminus starting at alpha 3 or alpha 4) was also present (range, 0-28% of molecules), but was confined to preparations from individuals with trophoblast disease (6 of 7 samples from trophoblast disease urine, 0 of 6 from pregnancy urine); 4) hCG missing the beta-subunit C-terminal region was also detected (2 of 13 hCG preparations); and 5) 1 of 13 preparations was nicked on the hCG alpha-subunit, between residues 70 and 71. Thus, 12 of 13 individual hCG samples demonstrated at least 1 of 4 different forms of peptide heterogeneity. We conclude that individual hCG samples vary widely in the type and extent of peptide heterogeneity, an observation that is not appreciated when pools of hCG are studied.     

Characterization of a factor(s) from partially purified human gonadotrophin preparations which inhibit(s) the binding of radiolabelled human LH and human chorionic gonadotrophin to Candida albicans

We have shown previously that partially purified human chorionic gonadotrophin (hCG) preparations inhibited the specific binding of 125I-labelled hLH or hCG to Candida albicans membranes at much lower concentrations than did highly purified hLH or hCG preparations. We now describe the characterization and partial purification of a heat-labile glycoprotein from commercially available gonadotrophin preparations. The factor strongly inhibited LH binding to Candida membranes, but not to sheep or pig luteal LH receptors. This material had a molecular weight of 16,000-21,000 daltons, bound strongly to CM-Sepharose at physiological pH, and could be resolved completely from hCG and from epidermal growth factor-like factors present in commercial gonadotrophin preparations. Its activity was not attenuated by a range of inhibitors specific for the four major classes of proteolytic enzymes, nor did it inhibit hormone binding by causing degradation of 125I-labelled hLH or hCG tracers. Factors which inhibited hLH binding to Candida membranes were also present in partially purified human urinary and equine serum gonadotrophin preparations and in placental extracts, but were not detected in highly purified CG of hLH preparations. The properties of this factor were similar to those described for beta-core protein, a cleavage product of the beta subunit of hCG which is a contaminant of commercial gonadotrophin preparations. Highly purified beta-core protein inhibited 125I-labelled hLH binding to Candida membranes, but not to sheep luteal binding sites. Preparations of hCG depleted of inhibitor activity could stimulate adenylate cyclase activity in Candida membranes almost five fold. In contrast, partially purified inhibitor preparations strongly inhibited basal adenylate cyclase activity (to 18% of control levels). These observations suggest that endogenous LH-like factors, perhaps similar to beta-core proteins of hCG, may play a role in the regulation of morphogenesis in Candida species.     

The beta-subunit of human chorionic gonadotrophin exists as a homodimer

The free beta-subunit of human chorionic gonadotrophin (hCGbeta) is well recognised as a product of many epithelial tumours. Recently, it has been shown that this ectopic production may have a functional relationship to tumour growth. The growth-promoting activity of hCGbeta may be explained by its structural similarity to a family of growth factors which all contain the same distinct topological fold known as the cystine-knot motif. Since the other members of this family all exhibit their activities as homo- and heterodimers, it is possible that the same may be true for hCGbeta. Using size-exclusion chromatography, low stringency SDS-PAGE and matrix assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) mass spectrometry (MS) we have shown that pure preparations of hCGbeta contain hCGbetabeta homodimers. Size-exclusion chromatography revealed asymmetric elution profiles with a forward peak corresponding to the size-exclusion characteristic of a globular protein with an approximate mass of 44-54 kDa and a late shoulder centered around an elution position expected for a globular protein of approximately 29 kDa. Two immunoreactive hCGbeta species, of approximately 32 and 64 kDa, were clearly resolved by SDS-PAGE and Western blotting. When analysed by MALDI-TOF MS a |mf23 kDa monomer and a |mf46 kDa dimer were identified. Formation of hCGbetabeta homodimers is consistent with the behaviour of other cystine-knot growth factors and strengthens the inclusion of the glycoprotein hormones within this superfamily. It has yet to be determined whether it is this dimeric molecular species that is responsible for growth-promoting activity of hCGbeta preparations in tumours.     

Human chorionic gonadotropin inhibits Kaposi's sarcoma associated angiogenesis,matrix metalloprotease activity,and tumor growth

Kaposi's sarcoma is a highly angiogenic, AIDS-associated neoplasm that is more frequent in male than in female patients. Cases of spontaneous regression during pregnancy have been reported and the pregnancy hormone human chorionic gonadotropin (hCG) has shown anti-Kaposi's sarcoma activity in several, but not all, clinical trials. Antiproliferative and proapoptotic activities specific for Kaposi's sarcoma (KS) cells have been shown. We report here further analyses of the anti-KS activity of the hormone and show that urinary hCG, the hCG beta-subunit, the hCG beta-core, and to a lesser extent a recombinant hCG, directly inhibit the activity of matrix metalloproteases of different origin. The hCG hormone also inhibited angiogenesis in vivo in the matrigel sponge assay as well as growth of KS cell xenografts in nude mice. The effect of the pure recombinant hormone dimer on xenograft growth was transient, indicating that the activity of intact hCG alone is not sufficient to overcome the growth potential of this tumor and suggesting that active hCG fragments or other anti-KS activities contribute to the activity of urinary hCG.     

Occurrence of fragmentation of free and combined forms of the beta-subunit of human chorionic gonadotropin.

In an attempt to further study various fragments of free and combined forms of hCG beta present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG beta-(111-116) portion (monoclonal antibody FB12) antiserum to the hCG beta(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG beta nicked subunit (beta NS) [hCG beta-(48-145)], three other fragments of mol wt 18,000 (F1), 16,500 (F2), and 12,000 (F3) were detectable after cleavage of disulfide bonds. Both the immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG beta-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the beta NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free beta-subunit contained beta NS. Furthermore, free hCG beta present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its beta-subunit by a two-step chromatography procedure, identification of hCG and hCG beta immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of beta NS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the beta-subunit of hCG is modified by multiple fragmentations.     

The thyrotropin in hydatidiform moles is human chorionic gonadotropin.

Thyrotropic activity (TSH), measured by the McKenzie mouse bioassay, has been correlated with human chorionic gonadotropin (hCG) activity, measured by radioimmunoassay, in serum and tissue samples from 11 patients with hydatidiform mole and in partially and highly purified preparations of urinary hCG. Serum samples, taken at various times before and after removal of the moles, gave a ratio of 0.42 plus or minus 0.24 muU TSH/U hCG (mean plus or minus SD) (N)=43). In all cases where hCG activity fell below 150-175 U/ml (n=49), thyroid stimulating activity was undetectable (smaller than 40 muU/ml). We extracted lyophylized molar tissue by a modification of the Bates alcohol-saline method and purified the resultant extract by a combination of gel chromatography, affinity chromatography using Concanavalin A coupled to Sepharose, and isoelectrofocusing. Following extraction, an approximately 20-fold purification was achieved without significant alteration of the ratio of the two activities. Using results from all phases of purification the ratio of muU TSH/U hCG was 0.51 plus or minus 0.35(n = 23).Both activities were in the same position on disc gel electrophoresis. Activity ratios were less constant when partially purified preparations of urinary hCG were assayed for both thyrotropic and hCG activities. The presence of an hCG immunoreactive species, presumably hCG-beta subunit, which contains no thyrotropic activity but has an approximately 10-fold greater activity on a weight basis than intact hCG, may be a partial explanation for this observation. Isoelectrofocusing of a urinary hCG preparation showed that all hCG immunoreactive species with pl's between 3. 5 and 5.0 contained thyrotropic activity in proportion to their hCG content. Seven highly purified hCG preparations had thyrotropic activity with a ratio of 0.48 plus or minus 0.18 muU TSH/U hCG. These results indicate that hCG has intrinsic thyrotropic activity. On a molecular basis it is calculated that hCG contains approximately 1/4000 the thyrotropic activity of human pituitary TSH. In conditions of grossly elevated serum hCG levels, such as hydatidiform mole, this thyrotropic activity can be sufficient to produce hyperthyroidism.     

Ribonucleases in HIV type 1 inhibition: effect of recombinant RNases on infection of primary T cells and immune activation-induced RNase gene and protein expression

Ribonucleases (RNases) have therapeutic potential against cancer and viral diseases and have been reported to inhibit replication of the human immunodeficiency virus type 1 (HIV-1) in chronically infected cell lines. The ribonuclease eosinophil-derived neurotoxin (EDN) is responsible for the anti-HIV-1 activity of a soluble factor produced in response to human alloantigens (ASF). Four recombinant RNases (EDN; a four amino acid extension of the N-terminus EDN, -4EDN; RNase A; and angiogenin) were tested for inhibition of HIV-1 replication in PHA blasts. All RNases showed anti-HIV-1 activity, irrespective of whether the RNases were added before, during, or 2 h after infection. Polyclonal antibodies against the four RNases blocked the antiviral activity. ASF inhibited HIV-1 replication in vitro if added up to 4 h after infection. We demonstrated that allostimulation induced EDN, RNase A, and angiogenin mRNA expression in peripheral blood mononuclear cells (PBMCs), although only EDN protein was detected. We identified monocytes and dendritic cells, but not macrophages or T cells, as EDN-producing cells. These findings raise the possibilities that multiple naturally occurring RNases may contribute to protection against HIV-1 infection and could be considered for utilization in HIV-1 therapy.