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Site-directed immunization was employed to identify sites on the envelope glycoprotein gp120 for antibody-mediated neutralization of human immunodeficiency virus type 1 (HIV-1). Antisera were raised in monkeys (Macaca fascicularis) against a series of 40 overlapping synthetic peptides covering the entire amino acid sequence of gp120 from the HTLV-IIIB strain of HIV-1. Immune sera against 12 of these peptides were reactive with gp120 by immunoblotting analysis, and antisera raised against 5 peptides, corresponding to amino acids (aa) 152-176, 193-218, 206-230, 248-269, and 307-330, were highly efficient in neutralizing HIV-1 (HTLV-IIIB) infectivity in vitro. Admixture of individual neutralizing anti-peptide monkey sera resulted in increment in neutralizing antibody titer. Antisera with reactivity to the relatively conserved regions defined by aa 152-176, 193-230, and 248-269 also neutralized to different extents the infectivity of the five Swedish clinical isolates of HIV-1 tested. Only a few HIV-1-infected people were found to make antibodies to these three conserved domains of gp120 as judged by ELISA using synthetic peptides as antigens. Three of the peptides (aa 152-176, 248-269, and 307-330) that induced neutralization antibodies also induced interleukin 2 production and lymphocyte proliferation when added to cultures of peripheral blood mononuclear cells from monkeys immunized with the corresponding peptides, indicating that these domains accommodate T-cell recognition sites. The results have obvious implications for the rational design of subunit vaccines against HIV-1 infection.

Synthetic peptides of 20-25 amino acids were employed in enzyme-linked immunosorbent assays to identify linear epitopes in the external glycoprotein gp46 and the transmembrane glycoprotein gp21 of human T-cell leukemia/lymphotropic viruses type I (HTLV-I) and II (HTLV-II). Ten linear epitopes were identified in the HTLV-I glycoproteins, seven in gp46 and three in gp21. Three major linear epitopes were identified in the gp46 of HTLV-II. Peptides representing linear epitopes of gp46 were found to be sensitive and specific for the detection of antibody and permit serological identification and differentiation of HTLV-I and HTLV-II infections.

Previous studies suggest that the Epstein-Barr virus nuclear antigen EBNA2 participates in the regulation of the expression of the viral latent membrane protein (LMP). We have used reporter plasmids containing DNA fragments of the 5' flanking region of the LMP gene in cotransfection experiments to analyze the effect of EBNA2 on the activity of the LMP promoter. The results show that the LMP promoter is controlled by positive and negative transcription elements in a DNA fragment that contains the LMP transcription initiation site and 634 base pairs of upstream sequences. The promoter is activated by EBNA2. The region between position -54 and +40 relative to the mRNA cap site contains a positive transcription element that is constitutively active in DG75 cells and independent of EBNA2. The -106 to -54 region contains a negative regulatory element that prevents adjacent positive elements from functioning in the absence of EBNA2. Regulatory sequences between -324 and -144 participate in maintaining a high level of transcription of the LMP promoter after induction with EBNA2. The regulatory elements in the -634 to -54 promoter region have the characteristics of an inducible enhancer, including orientation independence and ability to regulate a heterologous promoter.

Five distinct Epstein-Barr virus (EBV)-determined nuclear antigens (EBNA-1 to EBNA-5) were recently identified. Antibody responses to these antigens could conceivably differ, and thus prove of serodiagnostic value, in EBV-associated disease processes. As a first step, murine or human cell lines transfected with appropriate EBV DNA fragments and stably expressing either EBNA-1 or EBNA-2 were used to determine the frequency and time of emergence of antibodies to these two antigens in the course of acute and chronic infectious mononucleosis (IM) and to assess their titers in so-called chronic active EBV infections. Following IM, antibodies to EBNA-2 arose first and, after reaching peak titers, declined again in time to lower persistent or even nondetectable levels. Antibodies to EBNA-1 emerged several weeks or months after anti-EBNA-2 and gradually attained the titers at which they persisted indefinitely. The ratios between the anti-EBNA-1 and anti-EBNA-2 titers therefore were generally well below 1.0 during the first 6-12 months after IM and turned to well above 1.0 during the second year. In clear cases of chronic IM, the inversion of this ratio was delayed or prevented. In the less well-defined chronic EBV infections, low ratios were observed in only some of the patients. Because many of these illnesses were not ushered in by a proven IM and often showed EBV-specific antibody profiles within the normally expected range, a causal role of the virus in these cases remains doubtful.

Recombinant vectors carrying DNA fragments from the BamHI E region of the B95-8 Epstein-Barr virus (EBV) genome were transfected into COS-1 cells, and the transient expression of EBV-encoded nuclear antigens (EBNAs) was analyzed by using polyvalent human antisera and rabbit antibodies to synthetic peptides. Vector DNA containing two rightward open reading frames in the BamHI E fragment, BERF2a and BERF2b, induced the expression of a nuclear antigen identical serologically and with respect to size to the larger of the two polypeptides previously designated as EBNA4 in B95-8 cells. An antigen corresponding to the smaller polypeptide was induced in cells transfected with constructs that contained two neighboring reading frames, BERF3 and BERF4. This antigen also reacted with a rabbit antiserum to the synthetic peptide 203, deduced from BERF4. Thus, the findings show that the two components of the EBNA4 doublet in B95-8 cells are encoded by separate genes. The antigen encoded by BERF2a and/or BERF2b has been designated as EBNA4 and the antigen encoded by BERF3 and/or BERF4 has been designated as EBNA6. Polyvalent human antisera detected EBNA4 and EBNA6 in 9 of 11 lymphoid cell lines carrying independent EBV isolates. In the remaining two lines, either EBNA4 or EBNA6 was not detectable.

The intracellular Epstein-Barr virus (EBV) DNA present in virus-transformed cells was partly purified from 23 cell lines or biopsies of Burkitt lymphoma, nasopharyngeal carcinoma, infectious mononucleosis, or healthy carrier origin. Such DNA was cleaved in fragments (A-K) of molecular weights between 1 x 10(6) and 30 x 10(6) with restriction enzyme EcoRI, and these fragments were analyzed by standard methods involving agarose gel electrophoresis, transfer to nitrocellulose filters, and hybridization with radioactive EBV DNA or complementary RNA. Sequence variability among different EBV DNA isolates was largely confined to the A, C, and I fragments. These results are discussed in relation to the linkage map of the EcoRI fragments of EBV DNA. The EcoRI cleavage pattern of intracellular viral DNA of an EBV-like virus from baboon cells, Herpesvirus papio, was entirely different from that of human EBV isolates.

Synthesis of Rous sarcoma virus RNA was examined in vitro with a new assay for radioactive virus-specific RNA. Nuclei from infected and uninfected cells were incubated with ribonucleoside [alpha-(32)P]triphosphates, Mn(++), Mg(++) and (NH(4))(2)SO(4). Incorporation into total and viral RNA proceeded with similar kinetics for up to 25 min at 37 degrees . About 0.5% of the RNA synthesized by the infected system was scored as virus-specific, compared to 0.03% of the RNA from the uninfected system and 0.005% of the RNA synthesized by monkey kidney cell nuclei. Preincubation with DNase or actinomycin D completely suppressed total and virus-specific RNA synthesis. alpha-Amanitin, a specific inhibitor of eukaryotic RNA polymerase II, completely inhibited virus-specific RNA synthesis, while reducing total RNA synthesis by only 50%. We conclude that tumor virus-specific RNA is synthesized on a DNA template, most probably by the host's RNA polymerase II.

Large Epstein-Barr virus (EBV) DNA restriction fragments corresponding to regions transcribed in transformed, proliferating cells were cloned in a cosmid derivative of the dominant-acting selection vector pSV2-gpt. Recombinant vectors carrying the EcoRI A fragment of EBV DNA were modified in the region corresponding to the deletion of the virion DNA in the non-transforming viral substrain P3HR-1, to create a series of recombinants lacking parts of this region. The recombinant vectors were introduced into 3T3 mouse fibroblasts under selective conditions, and resistant clones shown to contain EBV DNA sequences were analyzed for the expression of EBV-related antigens detectable by direct, indirect, and anticomplement immunofluorescence techniques. Cells that contained the BamHI K fragment expressed the EBV-determined nuclear antigen (EBNA) as expected. Cells transfected with recombinant vectors containing the BamHI W, Y, and H fragment part of the EcoRI A fragment also express a nuclear antigen detectable with certain anti-EBNA-positive human sera in anticomplement immunofluorescence tests. The BamHI WYH-induced EBNA polypeptide is similar in size to the EBNA2 polypeptide in Raji cells, as shown by gel electrophoresis and immunoblotting. The antigen is not detected in cells transfected with EcoRI A-derived vectors in which the BamHI H fragment has been deleted or in cells transformed with vectors carrying the BamHI H fragment alone. Direct and indirect immunofluorescence did not reveal the presence of antigens associated with productive infection in any of the EBV DNA-transfected fibroblast clones.