Retroviral DNA integration directed by HIV integration protein in vitro

Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a lambda DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.     

Deletion in chromosome 11p associated with a hepatitis B integration site in hepatocellular carcinoma

Hepatitis B virus (HBV), a virus with known carcinogenic potential, integrates into cellular DNA during long-term persistent infection in man. Hepatocellular carcinomas isolated from viral carriers often contain clonally propagated viral DNA integrations. As small chromosomal deletions are associated with several types of carcinomas, the occurrence of chromosomal deletions in association with HBV integration in hepatocellular carcinoma was studied. HBV integration was accompanied by a deletion of at least 13.5 kilobases of cellular sequences in a human hepatocellular carcinoma. The viral DNA integration and deletion of cellular sequences occurred on the short arm of chromosome 11 at location 11p13-11p14. The cellular sequences that were deleted at the site of HBV integration were lost from the tumor cells, leaving only a single copy of the remaining cellular allele.     

Human CD4 binds immunoglobulins

T cell glycoprotein CD4 binds to class II major histocompatibility molecules and to the human immunodeficiency virus (HIV) envelope protein gp120. Recombinant CD4 (rCD4) bound to polyclonal immunoglobulin (Ig) and 39 of 50 (78%) human myeloma proteins. This binding depended on the Fab and not the Fc portion of Ig and was independent of the light chain. Soluble rCD4, HIV gp120, and sulfated dextrans inhibited the CD4-Ig interaction. With the use of a panel of synthetic peptides, the region critical for binding to Ig was localized to amino acids 21 to 38 of the first extracellular domain of CD4. CD4-bound antibody (Ab) complexed with antigen approximately 100 times better than Ab alone. This activity may contribute to the Ab-mediated enhancement of cellular HIV interaction that appears to depend on a trimolecular complex of HIV, antibodies to gp120, and CD4.     

Structure of nerve growth factor complexed with the shared neurotrophin receptor p75

Neurotrophins are secreted growth factors critical for the development and maintenance of the vertebrate nervous system. Neurotrophins activate two types of cell surface receptors, the Trk receptor tyrosine kinases and the shared p75 neurotrophin receptor. We have determined the 2.4 A crystal structure of the prototypic neurotrophin, nerve growth factor (NGF), complexed with the extracellular domain of p75. Surprisingly, the complex is composed of an NGF homodimer asymmetrically bound to a single p75. p75 binds along the homodimeric interface of NGF, which disables NGF's symmetry-related second p75 binding site through an allosteric conformational change. Thus, neurotrophin signaling through p75 may occur by disassembly of p75 dimers and assembly of asymmetric 2:1 neurotrophin/p75 complexes, which could potentially engage a Trk receptor to form a trimolecular signaling complex.     

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.     

Regulation of hepatic stellate cell differentiation by the neurotrophin receptor p75NTR

Differentiation of hepatic stellate cells (HSCs) to extracellular matrix- and growth factor-producing cells supports liver regeneration through promotion of hepatocyte proliferation. We show that the neurotrophin receptor p75NTR, a tumor necrosis factor receptor superfamily member expressed in HSCs after fibrotic and cirrhotic liver injury in humans, is a regulator of liver repair. In mice, depletion of p75NTR exacerbated liver pathology and inhibited hepatocyte proliferation in vivo. p75NTR-/- HSCs failed to differentiate to myofibroblasts and did not support hepatocyte proliferation. Moreover, inhibition of p75NTR signaling to the small guanosine triphosphatase Rho resulted in impaired HSC differentiation. Our results identify signaling from p75NTR to Rho as a mechanism for the regulation of HSC differentiation to regeneration-promoting cells that support hepatocyte proliferation in the diseased liver.     

Alterations in T4 (CD4) protein and mRNA synthesis in cells infected with HIV

Cells infected with the human immunodeficiency virus (HIV) show decreased expression of the 58-kilodalton T4 (CD4) antigen on their surface. In this study, the effect of HIV infection on the synthesis of T4 messenger RNA (mRNA) and protein products was evaluated in T-cell lines. Metabolically labeled lysates from the T4+ cell line Sup-T1 were immunoprecipitated with monoclonal antibodies to T4. Compared with uninfected cells, HIV-infected Sup-T1 cells showed decreased amounts of T4 that coprecipitated with both the 120-kilodalton viral envelope and the 150-kilodalton envelope precursor molecules. In four of five HIV-producing T-cell lines studied, the steady-state levels of T4 mRNA were also reduced. Thus, the decreased T4 antigen on HIV-infected cells is due to at least three factors: reduced steady-state levels of T4-specific mRNA, reduced amounts of immunoprecipitable T4 antigen, and the complexing of available T4 antigen with viral envelope gene products. The data suggested that the T4 protein produced after infection may be complexed with viral envelope gene products within infected cells. Retroviral envelope-receptor complexes may thus participate in a general mechanism by which receptors for retroviruses are down-modulated and alterations in cellular function develop after infection.     

Transfer RNA genes: landmarks for integration of mobile genetic elements in Dictyostelium discoideum

In prokaryotes and eukaryotes mobile genetic elements frequently disrupt the highly conservative structures of chromosomes, which are responsible for storage of genetic information. The factors determining the site for integration of such elements are still unknown. Transfer RNA (tRNA) genes are associated in a highly significant manner with different putative mobile genetic elements in the cellular slime mold Dictyostelium discoideum. These results suggest that tRNA genes in D. discoideum, and probably tRNA genes generally in lower eukaryotes, may function as genomic landmarks for the integration of different transposable elements in a strictly position-specific manner.     

ER cargo properties specify a requirement for COPII coat rigidity mediated by Sec13p

Eukaryotic secretory proteins exit the endoplasmic reticulum (ER) via transport vesicles generated by the essential coat protein complex II (COPII) proteins. The outer coat complex, Sec13-Sec31, forms a scaffold that is thought to enforce curvature. By exploiting yeast bypass-of-sec-thirteen (bst) mutants, where Sec13p is dispensable, we probed the relationship between a compromised COPII coat and the cellular context in which it could still function. Genetic and biochemical analyses suggested that Sec13p was required to generate vesicles from membranes that contained asymmetrically distributed cargoes that were likely to confer opposing curvature. Thus, Sec13p may rigidify the COPII cage and increase its membrane-bending capacity; this function could be bypassed when a bst mutation renders the membrane more deformable.     

Benefits and costs of HIV testing.

The benefits and costs of human immunodeficiency virus (HIV) testing in employment settings are examined from two points of view: that of private employers whose profitability may be affected by their testing policies and that of public policy-makers who may affect social welfare through their design of regulations related to HIV testing. The results reveal that HIV testing is clearly not cost-beneficial for most firms, although the benefits of HIV testing may outweigh the costs for some large firms that offer generous fringe-benefit packages and that recruit workers from populations in which the prevalence of HIV infection is high. The analysis also indicates that the testing decisions of unregulated employers are not likely to yield socially optimal economic outcomes and that existing state and federal legislation related to HIV testing in employment settings has been motivated primarily by concerns over social equity.     

Association of human papillomavirus types 16 and 18 E6 proteins with p53

Human papillomavirus type 16 (HPV-16) is a DNA tumor virus that is associated with human anogenital cancers and encodes two transforming proteins, E6 and E7. The E7 protein has been shown to bind to the retinoblastoma tumor suppressor gene product, pRB. This study shows that the E6 protein of HPV-16 is capable of binding to the cellular p53 protein. The ability of the E6 proteins from different human papillomaviruses to form complexes with p53 was assayed and found to correlate with the in vivo clinical behavior and the in vitro transforming activity of these different papillomaviruses. The wild-type p53 protein has tumor suppressor properties and has also been found in association with large T antigen and the E1B 55-kilodalton protein in cells transformed by SV40 and by adenovirus type 5, respectively, providing further evidence that the human papillomaviruses, the adenoviruses, and SV40 may effect similar cellular pathways in transformation.     

The polarity protein Par-3 directly interacts with p75NTR to regulate myelination

Cell polarity is critical in various cellular processes ranging from cell migration to asymmetric cell division and axon and dendrite specification. Similarly, myelination by Schwann cells is polarized, but the mechanisms involved remain unclear. Here, we show that the polarity protein Par-3 localizes asymmetrically in Schwann cells at the axon-glial junction and that disruption of Par-3 localization, by overexpression and knockdown, inhibits myelination. Additionally, we show that Par-3 directly associates and recruits the p75 neurotrophin receptor to the axon-glial junction, forming a complex necessary for myelination. Together, these results point to a critical role in the establishment of cell polarity for myelination.     

Rational design of peptide-based HIV proteinase inhibitors

A series of peptide derivatives based on the transition-state mimetic concept has been designed that inhibit the proteinase from the human immunodeficiency virus (HIV). The more active compounds inhibit both HIV-1 and HIV-2 proteinases in the nanomolar range with little effect at 10 micromolar against the structurally related human aspartic proteinases. Proteolytic cleavage of the HIV-1 gag polyprotein (p55) to the viral structural protein p24 was inhibited in chronically infected CEM cells. Antiviral activity was observed in the nanomolar range (with one compound active below 10 nanomolar) in three different cell systems, as assessed by p24 antigen and syncytium formation. Cytotoxicity was not detected at 10 and 5 micromolar in C8166 and JM cells, respectively, indicating a high therapeutic index for this new class of HIV proteinase inhibitors.     

Anti-HIV and anti-anti-MHC antibodies in alloimmune and autoimmune mice

Alloimmune mice (mice that have been exposed to cells from another murine strain) were shown to make antibodies against gp120 and p24 of human immunodeficiency virus (HIV), and mice of the autoimmune strains MRL-lpr/lpr and MRL-(+)/+ made antibodies against gp120. This is surprising because the mice were not exposed to HIV. Furthermore, anti-anti-MHC antibodies (molecules that have shapes similar to those of major histocompatibility complex molecules) were detected in both alloimmune sera and MRL mice. These results are discussed in the context of a possible role for allogeneic stimuli in the pathogenesis of acquired immunodeficiency syndrome, as suggested by an idiotypic network model.     

Reconstruction and future trends of the AIDS epidemic in the United States

There has been considerable uncertainty in estimates of past and current human immunodeficiency virus (HIV) infection rates in the United States. Statistical estimates of historical infection rates can be obtained from acquired immunodeficiency syndrome (AIDS) incidence data and the incubation period. However, this approach is subject to a number of sources of uncertainty and two other approaches, epidemic models of HIV transmission and surveys of HIV prevalence, are used to corroborate and refine the statistical estimates. Analyses suggest the HIV infection rate in the United States grew rapidly in the early 1980s, peaked in the mid-1980s, and subsequently declined markedly. Due both to the decline in the underlying infection rate and to the development of effective therapies that may delay AIDS diagnosis, overall AIDS incidence may plateau during the next 5 years. However, the number of individuals with advanced HIV disease without a diagnosis of AIDS who could potentially benefit from therapy is expected to increase 40% by 1995 as infected individuals progress to more advanced stages of HIV disease. Thus, although the overall HIV infection rate has declined, the demands on the U.S. health care system for treatment and care of HIV-infected individuals remain enormous.     

A quantitative bioassay for HIV-1 based on trans-activation

A bioassay that is based on trans-activation has been developed for the detection and quantitation of the human immunodeficiency virus type 1 (HIV-1). Indicator cell lines were constructed that contain the HIV-1 long terminal repeat ligated to the chloramphenicol acetyltransferase (CAT) gene. Infection of these cells by HIV activates the expression of CAT protein. Isolates of HIV-1 with divergent nucleotide sequences activated the indicator cell lines to a similar extent, approximately 500- to 1000-fold. Human T cell lymphotropic viruses types 1 and 2, equine infectious anemia virus, and herpes simplex virus 1 did not activate the indicator cell lines. Isolates of simian immunodeficiency virus and human T cell lymphotropic virus type 4 activated these cells to a much lesser extent, which suggests that these viruses contain similar, but distinct, trans-activators. This assay can be used for the detection, quantitation, and typing of HIV and for studying the effect of drugs on the replication of HIV in different cellular backgrounds.     

Epitopes of the CD4 antigen and HIV infection

The CD4 (or T4) surface antigen of human T lymphocytes is an important part of the receptor for the human immunodeficiency virus (HIV). After binding to the receptor, the HIV may enter the T cell and induce the formation of syncytia. In an attempt to identify the receptor site more closely, monoclonal antibodies (Mab's) to CD4 were tested for their ability to block HIV infection in a syncytium formation assay, and the CD4 epitopes so identified were mapped by antibody cross-blocking. The antibodies that showed strong inhibition of HIV fell into two main families while a third group of Mab's blocked syncytia formation weakly or not at all. Several different isolates of HIV as well as the laboratory strain CBL1 grown in CEM cells were used to induce the syncytia. The data indicate that only some epitopes of CD4 are important for virus binding and imply that the virus-binding site for CD4 is conserved in different isolates of HIV with substantially divergent env gene sequences. Preliminary studies of patients suggest that polymorphism of these epitopes does not play a role in determining susceptibility to infection.