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Biomedical subjects

F Gonzalez-Scarano

Publications and source records attributed to F Gonzalez-Scarano.

At least 37 records · Page 2Linked to original sources
PubMed1993

Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp120 receptor.

The gastrointestinal tract plays a major role in the pathogenesis and pathophysiology of infection by the type 1 human immunodeficiency virus (HIV-1). It is a potential route for viral entry and it is the site of a number of complications, including both opportunistic infections and a primary HIV-induced enteropathy. Correspondingly, both in vivo and in vitro studies have demonstrated HIV infection of gastrointestinal cells of lymphoid and epithelial origin. HT-29, a human colonic epithelial cell line that is infectable with many HIV-1 strains, does not express CD4 protein or mRNA. Recent studies showed that antibodies recognizing a neutral glycolipid related to galactosylceramide (GalCer) in HT-29 cells inhibited HIV-1 infection of this cell line, extending previous findings in neural cells. In the current studies, we further analyzed the neutral glycolipids of HT-29 cells and showed that they contained authentic GalCer and that recombinant gp120 bound to this glycolipid. Moreover, by analyzing GalCer expression in clones derived from HT-29 and Caco-2 (another human colonic cell line), we observed that the level of expression of this glycolipid was associated with the sensitivity to HIV-1 infection. Subclones of Caco-2 did not express GalCer and were not infectable with any of three HIV-1 strains. These results strengthen the possibility that GalCer is an alternative receptor in CD4- cell lines. Furthermore, since GalCer is a major glycolipid in epithelial cells of the small intestine and colon, these results provide a structural basis for the binding of HIV-1 by gastrointestinal epithelial cells and the entry of the virus into those cells.

Adenocarcinoma↗
PubMed1993

Expression of the La Crosse M segment proteins in a recombinant vaccinia expression system mediates pH-dependent cellular fusion.

To study the expression of La Crosse virus (LAC) glycoproteins, G1 and G2, we constructed a cDNA copy of the open reading frame (ORF) of the middle RNA segment and expressed it in a recombinant vaccinia virus (VV.ORF). Cells infected with VV.ORF expressed G1 and G2 at the cell surface and formed syncytia with a pH profile similar to that of LAC. These experiments provide a system of studying the biological functions of the LAC glycoproteins, including processing, targeting, fusion, receptor binding, and antigenicity.

Animals↗
PubMed1993

Sodium butyrate treatment of cells latently infected with HIV-1 results in the expression of unspliced viral RNA.

To investigate potential mechanisms for HIV-1 proviral latency, we generated a set of chronically HIV-1 infected and stably long terminal repeat-chloramphenicol acetyl transferase (LTR-CAT)-transfected TE671/RD cells, and studied both their virus production and LTR-driven reporter gene expression. Established tissue culture models of retroviral latency in lymphoid and monocytoid cell lines have demonstrated that the induction of virus production is associated with a shift in HIV-1-specific mRNA from a predominance of singly and multiply spliced mRNA's to the production of full-length HIV-1 RNA. We found a similar pattern in TE671/RD cells, but in contrast to U1 and ACH2 cells, could not induce viral replication by exposure to phorbol myristate acetate (PMA) alone. We demonstrated instead that production of full-length viral RNA, viral replication, and LTR-driven CAT expression could be induced by exposure to sodium butyrate. The most proximate effect of sodium butyrate is inhibition of cellular histone deacetylase(s) which results in disruption of nucleosomes relieving one level of restriction to gene expression. Consistent with this mechanism of action, we further found that sodium butyrate's effects: (i) act synergistically with PMA and TNF-alpha; (ii) are independent of protein synthesis; (iii) do not affect the constitutively expressed creatine phosphokinase gene; (iv) do not map to a discrete sequence motif in the viral LTR; and (v) are not blocked by N-acetyl cysteine but (vi) are blocked by novobiocin, an inhibitor of cellular topoisomerase II. These data show that a similar pattern of restricted viral RNA expression exists in this nonlymphoid cellular model of HIV-1 latency. In contrast however, these results suggest that in these cells there is an additional block to viral gene expression, which is overcome with sodium butyrate. These results are discussed in the context of histone-mediated repression of HIV-1 gene expression.

Acetylcysteine↗
PubMed1993

HIV-1 pseudotype virus containing a Cocal virus genome and an HIV envelope: construction, assay and use.

A method is described for the production and assay of pseudotype viruses between human immunodeficiency virus type 1 (HIV-1) and Cocal virus (COV), containing an HIV-1 envelope and a COV genome (COV(HIV)). COV(HIV) pseudotype virus is a useful tool for the investigation of a variety of questions regarding HIV entry into susceptible cells, including steps in virus binding, fusion, and internalization, and the role of molecules which inhibit entry. COV, a rhabdovirus closely related to vesicular stomatitis virus (VSV), replicated and caused cytopathic effect in primary cultures of human peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDM), and in human cell lines of lymphocytoid or monocytoid origin, making it an ideal candidate for pseudotype production. 174XCEM cells, which were permissive for selected macrophage-tropic strains as well as most lymphocyte-tropic strains of HIV-1, were used to produce stocks of putative pseudotype virus. To neutralize parental COV in these stocks, a rabbit antiserum was produced which had a neutralization index of > 10(7) at a dilution of 1:100. Using these methods, pseudotype viruses were produced with a titer of about 10(4) PFU per ml; these same stocks contained HIV-1 at a titer of about 10(5) TCD50 per ml and COV at a titer of about 10(8) PFU per ml. CD4-expressing HeLa cells were used to assay pseudotype stocks made with lymphocyte-tropic strains of HIV-1. The authenticity of the pseudotype stocks was validated by several controls, including their failure to register on congenic CD4-negative HeLa cells and their inhibition by monoclonal anti-CD4 antibodies such as Leu 3a.

Animals↗
PubMed1993

HIV-1 Tat alters normal organization of neurons and astrocytes in primary rodent brain cell cultures: RGD sequence dependence.

The HIV-1 trans-activator protein Tat has been implicated as a mediator of neuronal dysfunction in several model systems. To explore the possibility that Tat can affect primary brain cells, we examined the effect of recombinant Tat protein on rat cortical brain cell cultures. Tat induced marked aggregation of neurons and astrocytes in developing cultures and caused the neuritic processes to coalesce into fascicles. Cell death was not seen and brain macrophages were not affected. These effects mapped to a different region from the trans-activation domain of Tat, as mutating the RGD (arginine-glycine-aspartic acid) sequence within the second exon abrogated aggregation and fascicle formation without affecting trans-activation capacity. Such effects on primary neurons and astrocytes may reflect specific interactions of Tat with uninfected cells within the CNS in vivo.

Amino Acid Sequence↗
PubMed1993

Monoclonal anti-idiotypic antibodies that mimic the epitope on gp120 defined by anti-HIV-1 monoclonal antibody 0.5 beta.

OBJECTIVE: To develop effective, specific and safe anti-idiotypic antibody (Ab2) vaccines against HIV-1. DESIGN: Murine monoclonal Ab2 were generated against anti-HIV-1 antibody 0.5 beta (Ab1), which binds to gp120, neutralizes HIV-1 and inhibits virus-induced syncytia formation. METHODS: Mice were immunized with Ab1, and Ab2 were produced from immunized mice by the hybridoma technique. The Ab2 were characterized in vitro, injected into rabbits, and the anti-anti-idiotypes (Ab3) induced in the rabbits were analyzed for binding and antiviral reactivities by enzyme-linked immunosorbent assay, p24gag release and syncytia formation assays. RESULTS: Seven Ab2 bound to the antigen-combining site of Ab1, one of which (UD7) induced Ab3 in rabbits that were Ab1-like in their binding reactivities to PB1 (recombinant gp120 fragment) or peptides of gp120, and shared idiotypes with the Ab1. Crude Ab3-containing sera specifically and effectively neutralized the virus. CONCLUSION: Monoclonal Ab2 UD7 has potential as a vaccine against HIV-1.

AIDS Vaccines↗
PubMed1993

Replication of a macrophage-tropic strain of human immunodeficiency virus type 1 (HIV-1) in a hybrid cell line, CEMx174, suggests that cellular accessory molecules are required for HIV-1 entry.

To investigate the mechanism underlying one aspect of the cellular tropism of human immunodeficiency virus type 1 (HIV-1), we used a macrophage-tropic isolate, 89.6, and screened its ability to infect a number of continuous cell lines. HIV-1 (89.6) was able to replicate robustly in a T-cell/B-cell hybrid line, CEMx174, while it replicated modestly or not at all in either of its parents, one of which is the CD4-positive line CEM.3. Analysis by transfection of a molecular clone, a virus uptake assay, and polymerase chain reaction all provided strong evidence that the block to HIV-1(89.6) replication in the CEM.3 line lies at the level of cellular entry. These results were complemented by preparing a CD4-expressing derivative of the B-cell parent, 721.174, and demonstrating that it is permissive for productive HIV-1(89.6) replication. Given these experimental findings, we speculate that there exist cellular accessory factors which facilitate virus entry and infection in CD4-positive cells. Furthermore, these cellular accessory factors may be quite virus strain specific, since not all macrophage-tropic strains of HIV-1 were able to replicate in the CEMx174 hybrid cell line. This experimental model provides a system for the identification of one or more of these putative cellular accessory factors.

B-Lymphocytes↗
PubMed1993

Replication of type 1 human immunodeficiency viruses containing linker substitution mutations in the -201 to -130 region of the long terminal repeat.

In previous transfection analyses using the chloramphenicol acetyltransferase reporter gene system, we determined that linker substitution (LS) mutations between -201 and -130 (relative to the transcription start site) of the human immunodeficiency virus type 1 long terminal repeat (LTR) caused moderate decreases in LTR transcriptional activity in a T-cell line (S. L. Zeichner, J. Y. H. Kim, and J. C. Alwine, J. Virol. 65:2436-2444, 1991). In order to confirm the significance of this region in the context of viral replication, we constructed several of these LS mutations (-201 to -184, -183 to -166, -165 to -148, and -148 to -130) in proviruses and prepared viral stocks by cocultivation of transfected RD cells with CEMx174 cells. In addition, two mutations between -93 and -76 and between -75 and -58 were utilized, since they affect the nuclear factor kappa B (NF-kappa B)- and Sp1-binding sites and were expected to diminish viral replication. Our results suggest that while transfection analyses offer an adequate approximation of the effects of the LS mutations, the analysis of viral replication using a mutant viral stock presents a more accurate picture, which is sometimes at variance with the transfection results. Three mutants (-201/-184 NXS, -165/-148 NXS, and -147/-130 NXS) had effects on viral replication that were much more severe than the effects predicted from their performance in transfection analyses, and the effects of two LS mutations (-201/-184 NXS and -183/-166 NXS) were not predicted by their effects in transfection. In addition, we observed cell type-specific permissiveness to replication of some mutant viruses. In the cell types tested, the LS mutations indicated an apparent requirement not only for the intact NF-kappa B and SP1-binding sites but also for several regions between -201 and -130 not previously associated with viral infectivity.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Polygenic control of neuroinvasiveness in California serogroup bunyaviruses.

The pathogenesis of the California serogroup bunyaviruses includes both extraneural and intraneural replicative phases that can be separated experimentally. The present study dissects the viral genetic determinants of extraneural replication. We have previously described two attenuated reassortant clones of California serogroup bunyaviruses which exhibit reduced neuroinvasiveness after subcutaneous inoculation into suckling mice. Clone B1-1a bears an attenuated middle RNA segment (neuroinvasiveness phenotype v alpha v), and clone B.5 bears an attenuated large RNA segment (neuroinvasiveness phenotype alpha vv). We prepared reassortant viruses between these two strains and found that the two attenuated gene segments acted independently and additively, since reassortants bearing two attenuated RNA segments were more attenuated than the parental clones. Reassortants bearing no attenuated RNA segments were much more neuroinvasive than either parental clone, indicating that a neuroinvasive strain can be derived from two attenuated clones. Pathogenesis studies demonstrated that after injection of 10(3) PFU, the attenuated reassortant clones did not replicate in peripheral tissue, failed to reach the brain, and did not cause disease. At a dose of 10(6) PFU, attenuated clones failed to replicate to a significant level in peripheral tissue and produced only a minimal passive plasma viremia during the first 24 h but nevertheless reached high titers in the brain and killed mice. Because of this result, we investigated the possibility that neuroinvasion occurs via retrograde axonal transport, by determining whether sciatic nerve sectioning could protect against virus infection after hind leg footpad inoculation. We found that nerve sectioning had no effect on lethality, ruling out this mode of entry and suggesting that passive viremia is likely to be sufficient for invasion of the central nervous system.

Animals↗
PubMed1993

Molecular determinants of the virulence and infectivity of California serogroup bunyaviruses.

California bunyaviruses cause encephalitis in mammalian hosts after peripheral infection. The virulence of these viruses is determined by their ability to replicate sequentially in striated muscle, cause viremia, and invade and replicate in the central nervous system. These viruses are also able to infect vector mosquitoes following ingestion of a blood meal containing virus. Bunyaviruses are negative stranded RNA viruses with a trisegmented genome, and the large, medium, and small RNA segments encode the polymerase, the glycoproteins, and the nucleoprotein, respectively. Reassortants between virulent and avirulent virus clones have been used to map virulence determinants in mice as well as determinants of infectivity in mosquitoes. Attenuation in mice and infectivity in mosquitoes of some virus clones maps to the medium RNA segment, implying that the virus glycoproteins, which are involved in virus entry, play a role in virulence. Attenuation in mice and mosquito infectivity of other clones maps to the large RNA segment, suggesting that cell-specific differences in the function of the viral polymerase can also determine virulence and host range.

Animals↗
PubMed1992

Infection of cultured human adrenal cells by different strains of HIV.

OBJECTIVE: To determine whether human adrenal cells can be infected by HIV. METHODS: Cultured human fetal adrenal cells and the SW13 human adrenocortical carcinoma cell line were inoculated with several HIV-1 and HIV-2 strains. Virus replication was detected by viral core antigen enzyme-linked immunosorbent and reverse transcriptase assays. CD4 expression was measured by Northern blot and polymerase chain reaction procedures. RESULTS: HIV infection of these adrenal cells was detected and was most evident after cocultivation of the inoculated cells with peripheral blood mononuclear cells. Infection does not involve the CD4 molecule, which is not expressed by these adrenal cells. The relative level of HIV replication depended on the viral strain used. Virus production occurred best in cells that maintained evidence of adrenal cell function. Infection did not appear to disturb steroidogenesis measured in the cells. CONCLUSIONS: These observations indicate that human adrenal cells are susceptible to HIV infection, and provide further evidence of the polytropic nature of the virus.

Adrenal Cortex↗
PubMed1992

Experimental allergic encephalomyelitis and multiple sclerosis: lesion characterization with magnetization transfer imaging.

Magnetization transfer imaging (MTI) was initially performed in normal guinea pigs and human volunteers. A magnetization transfer ratio (MTR) was calculated in the normal white matter and was found to be 42%-44%, with less than 2.5% variation, which indicates the high reproducibility of the measurement. MTI was then applied to an animal model of white matter disease, acute experimental allergic encephalomyelitis (EAE). In this model of EAE, pathologically proved lesions were edematous with essentially no demyelination. MTRs decreased slightly but significantly (5%-8%) compared with the MTRs of the same tissue region measured before the onset of the lesion [corrected]. Fifteen patients with multiple sclerosis (MS) also underwent MTI. In the 15 patients with MS, all lesions (209 plaques) had a significantly decreased MTR (average, 26%). The authors believe that demyelination produced the lower MTR, and, thus, lesions varied in transfer ratio on the basis of the extent of myelin loss. In patients with MS, particularly those with chronic and/or progressive MS, the MTR of the normal-appearing white matter was significantly decreased. The data suggest that calculated MTR obtained with in vivo MTI may enable differentiation of edema from demyelination, and that MTI can demonstrate white matter abnormalities that cannot be seen with standard spin-echo or gradient-echo magnetic resonance imaging.

Adult↗
PubMed1991

Galactosyl ceramide or a derivative is an essential component of the neural receptor for human immunodeficiency virus type 1 envelope glycoprotein gp120.

This report demonstrates that galactosyl ceramide (GalCer) or a molecule derived from it may serve as an alternative receptor for human immunodeficiency virus in the nervous system. Recombinant gp120, an envelope glycoprotein of human immunodeficiency virus type 1, specifically binds to GalCer and its derivatives. This specificity was studied by inhibiting binding of radioiodinated gp120 to GalCer with antibodies to GalCer, antibodies to gp120, and an excess of unlabeled gp120. Binding activity was also removed by absorbing gp120 with liposomes containing GalCer. In addition, studies using natural and semisynthetic lipids indicate that the linkage between galactose and ceramide is essential for binding. The significance of an alternative receptor for human immunodeficiency virus in the nervous system is discussed.

Binding Sites↗
PubMed1991

Inhibition of entry of HIV-1 in neural cell lines by antibodies against galactosyl ceramide.

Although the CD4 molecule is the principal cellular receptor for the human immunodeficiency virus (HIV), several CD4-negative cell lines are susceptible to infection with one or more HIV strains. These findings indicate that there are alternate modes of viral entry, perhaps involving one or more receptor molecules. Antibodies against galactosyl ceramide (galactocerebroside, or GalC) inhibited viral internalization and infection in two CD4-negative cell lines derived from the nervous system: U373-MG and SK-N-MC. Furthermore, recombinant HIV surface glycoprotein gp120 bound to GalC but not to other glycolipids. These results suggest a role for GalC or a highly related molecule in HIV entry into neural cells.

Antibodies↗
PubMed1991

Entry of human immunodeficiency virus-1 into glial cells proceeds via an alternate, efficient pathway.

Although the CD4 molecule is the cellular receptor for human immunodeficiency virus-1 (HIV-1) in cells of the lymphocyte/monocyte lineage, a number of investigators have also been able to infect cells, including several of central nervous system (CNS) origin, that do not express CD4 protein or mRNA. These infections are generally nonpermissive. To ascertain whether the nonpermissive nature of infection in glial cells is due to an inefficient entry pathway, we prepared a permanently transfected U373-MG cell line expressing the CD4 molecule and demonstrated that HIV-1 still replicates at a low level. Furthermore, a virus uptake assay indicated that HIV-1 enters glial cells effectively, even in the absence of CD4. These results demonstrate that HIV-1 entry is efficient and that the restrictive nature of the infection in glial cells is due to postentry mechanisms. In addition, these findings support the existence of an alternate, efficient, entry pathway in some glial cells.

Blotting, Western↗
PubMed1991

Neuroattenuation of an avirulent bunyavirus variant maps to the L RNA segment.

The derivation and characterization of a neuroattenuated reassortant clone (RFC 25/B.5) of California serogroup bunyavirus was described previously (M. J. Endres, A. Valsamakis, F. Gonzalez-Scarano, and N. Nathanson, J. Virol. 64:1927-1933, 1990). To map the RNA segment responsible for this attenuation, a panel of reassortants was constructed between the attenuated clone B.5 (genotype TLL) and a virulent clone (B1-1a) of reciprocal genotype (LTT). Parent viruses and clones representing all of the six possible reassortants were examined for neurovirulence by intracerebral injection in adult mice. Reassortants bearing the large RNA segment from the virulent parent were almost as virulent as the virulent parent virus, while reassortants bearing the large RNA segment from the avirulent parent virus exhibited low or intermediate virulence. These results indicate that the large RNA segment is the major determinant of neuroattenuation of clone B.5. In addition to its neuroattenuation, clone B.5 was temperature sensitive and exhibited an altered plaque morphology. These phenotypes also segregated with the large RNA segment. The importance of the large RNA segment (which encodes the viral polymerase) in neurovirulence contrasts with prior studies which indicate that the ability to cause lethal encephalitis after peripheral injection of suckling mice (neuroinvasiveness) is primarily determined by the middle-sized RNA segment, which encodes the viral glycoproteins.

Animals↗