Biomedical subjects

G Franchini

Publications and source records attributed to G Franchini.

At least 127 records · Page 7Linked to original sources

PubMed1988

The human immunodeficiency virus type 2 (HIV-2) contains a novel gene encoding a 16 kD protein associated with mature virions.

The HIV-2 genome contains an open reading frame (designated X-orf) that does not have a counterpart in HIV-1. To establish whether X-orf is a gene, we studied its expression in HIV-2-infected individuals and in infected cells in vitro. An HIV-2 proviral DNA fragment containing the X-orf was expressed in E. coli, and the recombinant protein was used in an immunoblot assay. The X-orf protein was recognized specifically by the sera of HIV-2-infected people but not by the sera of SIV-infected monkeys or HIV-1-infected humans. A rabbit antiserum raised against the recombinant X-orf protein recognized a 16 kD protein in HIV-2-infected cells. The native X-orf protein was not glycosylated or phosphorylated, was localized in the cytoplasm of HIV-2-infected cells, and appeared to be associated with mature virions.

Acquired Immunodeficiency Syndrome↗

PubMed1988

The simian immunodeficiency virus envelope open reading frame located after the termination codon is expressed in vivo in infected animals.

Genetic comparison of SIVmac to the human retroviruses generally associated with AIDS revealed a closer relationship to HIV-2 than to HIV-1. A common feature differentiating SIV and HIV-2 from HIV-1 is the size of the transmembrane portion of the envelope, which is smaller (gp32) in SIVmac and HIV-2 than in HIV-1 (gp41). The presence of this truncated form of the transmembrane glycoprotein in SIVmac and HIV-2 virions is apparently related to the presence of a translation termination codon in the env gene of all SIV proviruses analyzed as well as in one HIV-2 provirus. Since the carboxy terminus of the envelope transmembrane protein has been implicated in the cytopathic effect of HIV-1 in vitro, we decided to investigate whether putative expression of the open reading frame located after the termination codon correlates with the pathogenicity of SIVmac in vivo. We generated two synthetic peptides from the inferred amino acid sequence of SIVmac and tested their reactivity by Western blot against the sera of naturally and experimentally infected monkeys as well as against sera of HIV-2-infected individuals. Our results indicate that the protein synthesized from this open reading frame is expressed in vivo, since an immunoresponse can be detected against the synthetic peptides in two of three experimentally SIVmac-infected animals. However, no correlation can be found between its expression and disease progression at this time. Furthermore, a rabbit immune serum raised against the synthetic peptide failed to identify any specific protein in SIVmac-infected cells.

Amino Acid Sequence↗

PubMed1987

Long latency precedes overt seroconversion in sexually transmitted human-immunodeficiency-virus infection.

Signs of latent HIV infection were sought in stored serum samples collected before overt seroconversion, confirmed by enzyme-linked immunosorbent assay (ELISA), from 9 subjects with human-immunodeficiency-virus (HIV) infection, in serum from 25 seronegative sexual partners of HIV-seropositive men and from 23 other seronegative, homosexually active men. Free HIV antigen and/or low-titre antibodies to recombinant structural (core, env) or non-structural (3' orf, sor, tat) proteins were seen 6-14 months before seroconversion in all 9 subjects who seroconverted. Antibodies against core proteins detected by western blot were usually the first sign of latent HIV infection. 5 of the 25 ELISA-negative exposed partners have shown HIV antigenaemia and antibodies against core proteins for 16-34 months. By in-situ hybridisation, HIV-specific RNA was detected in peripheral-blood non-lymphoid mononuclear cells in some of the latently infected partners. All subjects with latent HIV infection had normal numbers of T4 lymphocytes but half of them lost their in-vitro proliferative T-cell response to a recall antigen (purified protein derivative of tuberculin). Early HIV infection, characterised by a low-level and restricted antibody response towards HIV core and regulatory proteins, seems mainly to affect antigen-presenting cells.

Acquired Immunodeficiency Syndrome↗

PubMed1987

Genetic analysis of a new subgroup of human and simian T-lymphotropic retroviruses: HTLV-IV, LAV-2, SBL-6669, and STLV-IIIAGM.

A new primate retrovirus, STLV-IIIAGM, has been recently isolated from healthy African green monkeys and is apparently nonpathogenic in its natural host. However, spontaneous infection as well as inoculation of STLV-IIIAGM into macaques induces a disease with clinical features that resemble human AIDS. Independent isolates of human retroviruses, serologically closely related to STLV-IIIAGM, have been obtained from healthy individuals (HTLV-IV) and patients with immunodeficiency (LAV-2FG and SBL 6669) from West Africa. The latter have also been referred to as HIV-2 because, like HTLV-III/HIV-1, they may be associated with immune deficiency, or as West African human retroviruses because of their prevalence and probable origin from that region. We have molecularly cloned the STLV-IIIAGM genome and have generated probes from the gag-pol and envelope genes to analyze the genetic relatedness of these simian and human retroviruses. Our results indicate that all these retroviruses are genetically closely related to each other, HTLV-IV and STLV-IIIAGM differing only by a few restriction enzyme sites while LAV-2FG and SBL 6669 exhibit greater polymorphism from HTLV-IV/STLV-IIIAGM. These data mirror the variable degree of relatedness among members of the first subgroup of human retroviruses, HTLV-III/HIV.

Animals↗

PubMed1987

Structure of the long terminal repeat of simian lymphotropic virus type III (African green monkey) and its relatedness to that of HIV.

The simian T-lymphotropic virus type III (STLV-III[AGM]) is a retrovirus in wild African green monkeys which is serologically related to the human T-lymphotropic virus type III (HTLV-III/LAV-1/HIV) and other related human retroviruses. The long terminal repeats (LTR) contained in clones of viral DNA of (STLV-III[AGM]) were subcloned in M13 and their DNA sequence was determined and compared with that of HIV (HTLV-III[BH10]). The STLV-III(AGM) LTR is considerably larger than that of HTLV-III(BH10) (800 bp vs 634 bp) and contains a 498 bp U3 region, a 176 bp R region, and a 126 bp U5 region. These two LTR sequences share regions of significant homology. Regions of greatest homology include the 5' portion of U3, a core enhancer sequence in U3, sequences including and surrounding the TATAA promoter box in U3 and the AATAAA polyadenylation/termination signal in R, and the 3'-most region of U5. The relatively larger size of the STLV-III LTR is due to the presence in all three parts of the LTR of sequences which have no apparent homolog in the HIV LTR. Overall, the two LTRs are 47% homologous. Even greater homology (75%) is evident with a 300 bp segment including R and some of U3 from the LTR of another human retrovirus, HIV-2/LAV-2. The STLV-III LTR contains an imperfect 28 bp direct repeat in the R region which is not present in HIV. There are no obvious direct repeats in U3 homologous to the 10 bp repeat in the U3 of HTLV-III.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Spectrum of natural antibodies against five HTLV-III antigens in infected individuals: correlation of antibody prevalence with clinical status.

The genome of the HTLV-III/LAV retrovirus, the etiologic agent of the acquired immunodeficiency syndrome (AIDS), encodes the viral structural proteins (envelope and core proteins), the reverse transcriptase, a transactivation protein (tat-III), as well as two other proteins (3'orf, sor) of unknown function. We studied the prevalence of natural antibodies against envelope, gag, 3'orf, sor, and tat-III in the sera of HTLV-III infected individuals in an attempt to correlate clinical status with seropositivity to specific HTLV-III antigens. We selected 101 sera; 16 were obtained from normal donors with no known risk factors, and 85 were from patients with full-fledged AIDS (28 cases), AIDS-related complex (ARC, 22 cases), and healthy people at risk (homosexuals, intravenous [IV] drug users, relatives of AIDS patients; 35 cases). Seropositivity for antibodies against the envelope (gp41) and gag antigens (p15, p24) was determined by Western blot using disrupted HTLV-III virions. Of the 101 sera, all 16 from nonrisk donors and 3/35 from healthy at-risk donors were negative for antibodies against either the gp41 or p15 and p24. The remaining 82 sera were seropositive for either the gp41 and/or the p15 and p24. All sera were then tested against the three known HTLV-III antigens (3'orf, sor, and tat-III) that have been synthesized in bacteria. Our data indicate that all the HTLV-III antigens tested are immunogenic in vivo. No significant difference in antibody prevalence to gp41 (close to 100%) and to the 3'orf, sor, and tat-III proteins (approximately 50%) was observed with regard to stage of the disease. In contrast, the prevalence of antibodies against the core antigens decreased from approximately 100% in infected people with no clinical signs of disease to 50% in ARC and AIDS patients. The percentage of patients seropositive for all five antigens tested was increased in the AIDS group. These results indicate that the greatest antibody prevalence was obtained using viral envelope antigen and further suggest that screening with the newly identified 3'orf, sor, and tat-III proteins as antigens would confer no further diagnostic advantage. The pattern of natural antibodies observed during disease progression did not suggest any pathogenetic mechanism.

AIDS-Related Complex↗

PubMed1986

Identification of HTLV-III/LAV sor gene product and detection of antibodies in human sera.

The nucleotide sequence of the genome of HTLV-III, the infectious agent etiologically associated with the acquired immune deficiency syndrome, predicts a small open reading frame, termed sor, located between the pol and env genes. A DNA segment containing 82 percent of the sor region was inserted into a prokaryotic expression vector, pJL6, to determine whether sor encodes a viral protein and to gain some insight into its possible function. The bacterially synthesized sor protein reacted with sera from individuals infected with HTLV-III, indicating that sor is expressed as a protein product or products that are immunogenic in vivo. Antibodies to the purified, bacterially synthesized sor protein were found to react specifically with the same protein and also with a protein of molecular weight 23,000 (23K) in HTLV-III-infected H9 cell extracts. The 23K protein comigrated with a protein immunoprecipitated by the serum of a hemophiliac patient with antibodies to HTLV-III, suggesting that this protein is probably the sor gene product.

Acquired Immunodeficiency Syndrome↗

PubMed1986

Cytoplasmic localization of the HTLV-III 3' orf protein in cultured T cells.

HTLV-III, the etiological agent of the acquired immunodeficiency syndrome, contains in its genome coding regions for several novel proteins. One of these, the 3' open reading frame (3'orf) encodes proteins of 26-27 kDa which are expressed in infected cells both in vivo and in vitro. A specific antiserum has been raised against the recombinant 3'orf protein synthesized in bacteria and used to localize the viral proteins by subcellular fractionation and immunofluorescence on HTLV-III infected cells. The antiserum specifically immunoprecipitated the 26- to 27-kDa proteins from both the cytoplasmic (S100) and the membrane fractions, with an enrichment in the latter. The proteins were not detected in the nucleus or organelle (S100 pellet) fractions. These proteins were also recognized in the same subcellular fractions by human sera from patients with AIDS. Indirect immunofluorescence on fixed infected cells confirmed the presence of the proteins in the cytoplasm. Immunoprecipitation and Western blot analysis of total proteins from disrupted HTLV-III virions with the specific antiserum failed to detect the 3'orf protein products, suggesting that they are not a major component of mature virions and may be involved in the intracellular regulation of viral replication.

Cell Compartmentation↗

PubMed1986

Molecular analysis of a deletion mutant provirus of type I human T-cell lymphotropic virus: evidence for a doubly spliced x-lor mRNA.

The genome of the human T-cell leukemia/lymphotropic virus type I (HTLV-I) contains a functional gene denominated x-lor that may be important in HTLV-I transformation of human T cells. To study the role of x-lor and other HTLV-I genes in cellular transformation, we obtained a transformed nonproducer human T-cell line containing a single defective HTLV-I provirus (HTLV-I 55/PL). This 7-kilobase provirus had undergone a deletion involving the entire envelope gene and the nonconserved region. The point of the deletion corresponded to the junction of a donor splice site, located between the polymerase gene and the envelope gene (nucleotide 5183), and the acceptor site for the mRNA of the x-lor gene (nucleotide 7302). The juxtaposition of nucleotides 5182 and 7302 brings the initiating methionine codon of the envelope gene immediately 5' to the x-lor region, leaving the DNA sequence in frame for expression of a protein product. This finding suggests that a double splicing mechanism is used to express the x-lor gene, and that the defective provirus 55/PL was generated through the reverse transcription of a partially spliced mRNA. Analysis of the x-lor mRNA of other HTLV-I-transformed cell lines revealed that a double splicing process is commonly used. Furthermore, since 55/PL can be faithfully transmitted and is able to immortalize recipient T cells, we can conclude that the envelope gene is not necessary for in vitro transformation by HTLV-I.

Base Sequence↗

PubMed1986

Differential response to the cytopathic effects of human T-cell lymphotropic virus type III (HTLV-III) superinfection in T4+ (helper) and T8+ (suppressor) T-cell clones transformed by HTLV-I.

We isolated six human T-cell lymphotropic virus type I (HTLV-I)-transformed T-cell clones carrying the phenotypic markers of helper and suppressor T cells. Five of the transformed T-cell clones produced infectious HTLV-I, but one (clone 55) contained a defective provirus and was therefore not competent for viral replication. To test whether there is interference between HTLV-I and the cytopathic virus HTLV-III in infection and/or their biological effects, we superinfected these T-cell clones with HTLV-III. The recipient cells that we used displayed either the OKT4 or the OKT8 membrane antigens (helper or suppressor phenotype, respectively). The superinfection was successful in all cases, regardless of phenotype of the recipient cells and status of viral production. Both HTLV-III and HTLV-I were expressed by the infected cell lines containing complete HTLV-I proviruses, as demonstrated by electron microscopy and immunofluorescence. However, only HTLV-III in the virus mixture obtained from the culture supernatants was transmitted to the human neoplastic T-cell line H9. The nonproducer clone 55 did not express HTLV-I upon superinfection with HTLV-III. HTLV-III exerted its cytopathic effect on all but one of the superinfected T-cell clones 15-20 days after infection. The exception, clone 67, is also the only cell clone that expresses the phenotypic marker of suppressor T lymphocytes (OKT8); the other clones carry the OKT4 antigen, correlated with helper functions. The virus released from the superinfected clone 67 is cytopathic for fresh peripheral and umbilical-cord blood lymphocytes, suggesting that cellular factors, rather than a genetic change in the virus, may be responsible for the lack of cytopathic effect of HTLV-III on the suppressor T-cell clone 67.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1986

Expression of the protein encoded by the 3' open reading frame of human T-cell lymphotropic virus type III in bacteria: demonstration of its immunoreactivity with human sera.

The genome of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), the infectious agent etiologically associated with the acquired immunodeficiency syndrome, contains, in addition to the genes for the polymerase, core, and envelope proteins, several open reading frames. To investigate whether the 3' open reading frame (3' orf) located between the envelope gene and the 3' long terminal repeat is a gene expressed in vivo in infected individuals, we inserted a fragment of 3' orf in a prokaryotic expression vector. The protein product synthesized in bacteria was purified and allowed to react with sera from individuals infected with human T-cell lymphotropic virus type III as indicated by seropositivity for other viral proteins. Two-thirds of the sera, regardless of the clinical status of the individuals, reacted with the purified protein indicating that 3' orf is a viral gene the product of which is immunogenic in vivo. A polyclonal rabbit antiserum reacting against the 3' orf gene product was obtained by serial injection of rabbits with the purified bacterial protein. The antiserum recognized a 27-kDa protein in the human T-cell lymphotropic virus type III-infected lymphocytes.

Acquired Immunodeficiency Syndrome↗

PubMed1985

Clonal selection of T lymphocytes infected by cell-free human T-cell leukemia/lymphoma virus type I: parameters of virus integration and expression.

We have successfully transmitted cell-free HTLV-I to normal cord blood and peripheral blood lymphocytes and have exploited this system to study the kinetics of infection and transformation of these cells. Transmission was successful in 4 out of 23 attempts. In all 4 cases, the infected cells progressed from an initial stage of polyclonality to predominantly monoclonal cells within 4-6 weeks. Both complete and defective proviruses were transmitted to the recipient cells initially, but cells with a complete provirus were preferentially maintained. The monoclonally infected cells have persisted in culture for more than 6 months and may be considered immortalized. Expression of core antigens as detected by immunoflourescence and the reverse transcriptase activity in the medium at least in one case was not observed until weeks after the cells had become monoclonal, suggesting that expression of virus or viral structural proteins is not necessary for selected growth of the infected cells in vitro.

Bone Marrow Cells↗

PubMed1985

HTLV-I infection of T and B cells of a patient with adult T-cell leukemia-lymphoma (ATLL) and transmission of HTLV-I from B cells to normal T cells.

We analysed the DNA of different tissues of a patient (HS) with adult T-cell leukemia/lymphoma virus (HTLV-I). We detected viral sequences in fresh specimens from spleen, thymus, liver, skin and peripheral blood neoplastic lymphocytes. The pattern of HTLV-I integration is identical in the leukemic cells and in all other tissues analysed, but the signal intensity is strongest in the leukemic cells, indicating the source of HTLV-I proviral sequences was the leukemic T-cells which had infiltrated these tissues. In fact, the cultured skin fibroblasts of the patient did not contain HTLV-I sequence. However, cultured lymphocytes of this patient was consistently an immortalized B-cell line containing HTLV-I sequences in a manner indicative of a polyclonal infection. This cell line was also infected with the Epstein-Barr virus (EBV). In order to determine whether HTLV-I alone was sufficient for B-cell immortalization, we obtained single cell clones by limiting dilution. The DNA of all the cell clones that we analysed contained both the HTLV-I and EBV genomes, suggesting that immortalization of the B-cell was more likely due to the EBV rather than HTLV-I. Infectious HTLV-I viruses produced by the B-cell line still had the propensity to infect and transform T-lymphocytes in normal human umbilical cord blood. Unlike the parental B cells, the transformed T lymphocytes were clonally selected. Our results indicate that although the predominant infected cell population of the patient was his leukemic T lymphocytes, some of his EBV-positive B-lymphocytes were also polyclonally infected. The latter had a growth advantage in culture over the T lymphocytes but the virus produced by these immortalized B cells has not been adapted and has maintained its tropism for T cells.

B-Lymphocytes↗

PubMed1985

trans-Activation of the human T-cell leukemia virus long terminal repeat correlates with expression of the x-lor protein.

Cell lines established directly from adult T-cell leukemia-lymphoma patients or immortalized by human T-cell leukemia virus type I (HTLV-I) in vitro that do not produce complete HTLV virions were characterized both for the content of viral proteins and for the presence of trans-acting factors activating gene expression under the control of the HTLV long terminal repeat. The expression of the 42-kilodalton HTLV x-lor product correlated with trans-activation of the long terminal repeat. The implications of this study for understanding the role of the HTLV x-lor product in the initiation and maintenance of T-lymphocyte transformation are discussed.

Deltaretrovirus↗

PubMed1985

Molecular biology of human T-lymphotropic retroviruses.

The generic name for a family of human T-lymphotropic retroviruses is HTLV. Two of the three members in this family have been linked etiologically to human diseases: HTLV-I with adult T-cell leukemia and HTLV-III with the acquired immunodeficiency syndrome. In addition to their T-cell tropism and a number of other common biological and biochemical properties, the most unique common features of these viruses from a molecular biological point of view are the presence of the x-lor gene towards the 3' end of the genome and the phenomenon of a virus-induced trans-acting factor in activation of transcription initiated in the viral long terminal repeat. These features may not only be key in understanding the mechanism of transformation or cell killing by these viruses, but they also provide a basis for new classification of retroviruses. In spite of these similarities among HTLV-I, -II, -III, and bovine leukemia virus, the genome of HTLV-III is only distantly related to these other viruses. Instead, it shows greater homology to members of the Lentivirus family. Therefore, all these viruses may have a common progenitor. Two other salient features arose from the analyses of HTLV-III and acquired immunodeficiency syndrome. (a) HTLV-III frequently infects the brain of acquired immunodeficiency syndrome patients who suffer from central nervous system disorders. This not only identifies HTLV-III as the direct candidate in these central nervous system disorders but also poses the problem of crossing the blood-brain barrier in therapy strategies to eradicate the virus. (b) Different HTLV-III isolates comprise a spectrum of related viruses, with the degree of divergence varying from virtual identity to 10-15% difference. The most divergent region resides in the envelope gene. Whether this finding has implications in the development of an effective vaccine for acquired immunodeficiency syndrome remains to be determined.

Acquired Immunodeficiency Syndrome↗

PubMed1984

Human T-cell leukemia virus (HTLV-I) transcripts in fresh and cultured cells of patients with adult T-cell leukemia.

We examined the DNA and RNA of fresh leukemic cells (five patients) and long-term cultured T lymphocytes (four patients) from patients with adult T-cell leukemia for the presence of human T-cell leukemia virus (HTLV-I) sequences. In all cases HTLV-I provirus was found and several species of viral mRNAs were observed in all cell lines. We used HTLV DNA probes representing the gag, pol, and env genes and pX regions and the U3- and U5-specific sequences to characterize the genetic content of the viral transcripts. Although similar RNA sequences were expressed in one HTLV-I provirus-positive specimen of fresh primary leukemic cells, no viral transcripts were found in four other similar specimens. These findings are consistent with the idea that the expression of one or more HTLV-I genes may be involved in initiation of transformation, but consistent expression is not needed for maintaining the neoplastic state.

Adult↗

PubMed1984

Molecular cloning of a unique human T-cell leukemia virus (HTLV-IIMo).

Human T-cell leukemia virus IIMo (HTLV-IIMo) is a human retrovirus isolated from a patient with a T-cell hairy cell leukemia. This virus has been shown to have core protein (gag) antigens similar to, but distinct from, those of all known isolates of the prototype human T-cell leukemia virus (HTLV-I). We have used a subgenomic clone of the HTLV-I env-pX region to detect and characterize HTLV-IIMo proviral sequences by performing Southern blot hybridization under conditions of low stringency. Using the HTLV-I probe, we cloned a partial integrated HTLV-IIMo provirus from a genomic library of the producer Mo cell line. These sequences could be characterized by low stringency hybridization with different subgenomic clones of HTLV-I. An HTLV-IIMo-specific subclone was made by isolating a 3.6-kilobase BamHI fragment of the partial provirus. This was used to clone two full-length integrated viral genomes. Using the HTLV-IIMo viral probe, we also showed by hybridization under stringent conditions to DNA and RNA of various infected and uninfected cell lines that these HTLV-IIMo sequences are unique.

Base Sequence↗

PubMed1984

Molecular studies of human T-cell leukemia virus and adult T-cell leukemia.

We describe previously published work as well as new data on the molecular biology of human T-cell leukemia virus (HTLV) and its associated disease, adult T-cell leukemia-lymphoma (ATLL). This specific kind of disease is endemic to certain areas of Japan and the Caribbean, and several isolated cases have been described also in the United States, Israel, South America, and Africa. The disease is probably also endemic to Africa and South America, but sufficient studies of these areas have not been performed. We have molecularly cloned the HTLV genome and used the viral DNA as a probe in a large molecular study of the DNAs of human hematopoietic malignancies. The results showed that HTLV sequences could be detected in the fresh leukemic cells of all cases of ATLL tested. The neoplastic cells are of clonal origin and contain one or few copies of integrated HTLV. Detailed comparative analyses by restriction enzyme mapping of the proviral DNA in U.S., Japanese, Caribbean, and Israeli cases revealed that the viruses are almost indistinguishable. The DNA from neoplastic cells of other cases of hematopoietic neoplasias analyzed were negative for HTLV sequences with the exception of two. DNA from cells of a patient (MO) with a T-cell variant of hairy cell leukemia did contain a provirus only distantly related to HTLV (less than 10% of DNA sequence homology). The virus isolated from the MO cells has been designated HTLV-II. The second case was a patient with chronic myeloid leukemia whose cells contained exogenous DNA sequences distantly related to HTLV. Different fragments of the clones HTLV genome have been used to hybridize to DNA from uninfected normal tissues of several vertebrate species, including humans, in a search for cell-derived sequences related to the HTLV genome. No homologous sequences were found except sequences distantly related to the pol and env genes, indicating that HTLV does not carry a cellularly derived onc gene. Surprisingly, however, infection of normal human fresh T cells by HTLV transforms them into cells with permanent growth and with several other properties similar to neoplastic T cells. We have also studied the expression of viral and cellular genes in fresh and cultured neoplastic cells from patients with ATLL. Several species of viral mRNAs are always detected in the cultured neoplastic cells, whereas in some fresh samples expression of normal mRNA was not detected.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗