Fred C. Krebs

Inactivation of human immunodeficiency virus type 1 by nonoxynol-9, C31G, or an alkyl sulfate, sodium dodecyl sulfate

A highly desirable approach to prevention of human immunodeficiency virus type 1 (HIV-1) transmission during sexual intercourse is the development of nontoxic, topical, broad spectrum microbicides effective against transmission of cell-associated and cell-free virus. Toward this end, the HIV-1 inactivation potential of surface active agents C31G and an alkyl sulfate, sodium dodecyl sulfate (SDS) was assessed. Because of its extensive use as a microbicidal agent, nonoxynol-9 (N-9) was used as a reference against which C31G and SDS were compared. Viral inactivation was measured using HIV-1 LTR-beta-galactosidase indicator cells (expressing CD4 or CD4/CCR5) derived from HeLa cells, a cell line of human cervical adenocarcinoma origin. In experiments which examined inactivation of cell-free HIV-1, C31G was generally more effective than N-9. Viral inactivation by SDS occurred at twice the concentration necessary to achieve similar levels of inactivation using either N-9 or C31G. Using HeLa and HeLa-derived cells in cytotoxicity studies, it was demonstrated that SDS is as much as 11 and five times less cytotoxic than N-9 or C31G, respectively, during 48 h of continuous exposure. SDS (unlike C31G and N-9) can inactivate non-enveloped viruses such as human papillomavirus (HPV) [Howett, M.K., Neely, E.B., Christensen, N.D., Wigdahl, B., Krebs, F.C., Malamud, D., Patrick, S.D., Pickel, M.D., Welsh, P.A., Reed, C.A., Ward, M.G., Budgeon, L.R., Kreider, J.W., 1999. A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses. Antimicrob. Agents Chemother. 43(2), 314-321]. Since addition of SDS to C31G or N-9 may make the resulting microbicidal mixtures broadly effective against both enveloped and non-enveloped viruses, several surface active agent combinations were evaluated for their abilities to inactivate HIV-1. Addition of SDS to either C31G or N-9 resulted in mixtures that were only slightly less effective than equivalent concentrations of C31G or N-9 alone. To investigate inactivation of cell-associated infectivity, HIV-1 IIIB-infected SupT1 cells were treated with N-9, C31G, or SDS. Inactivation of cell-associated infectivity required higher microbicide concentrations than were needed for inactivation of cell-free virus. However, the relative activities of N-9, C31G, or SDS were similar to those seen in assays of inactivation using cell-free virus. These studies suggest that C31G and SDS may be attractive candidates for human trials as topical microbicides effective against HIV-1 transmission since both function at concentrations that provide effective viral inactivation with low levels of cytotoxicity. SDS microbicides (used alone or with other microbicides) may provide the added advantage of protection from HPV infection.

Sodium Dodecyl Sulfate and C31G as Microbicidal Alternatives to Nonoxynol 9: Comparative Sensitivity of Primary Human Vaginal Keratinocytes

ABSTRACT A broad-spectrum vaginal microbicide must be effective against a variety of sexually transmitted disease pathogens and be minimally toxic to the cell types found within the vaginal epithelium, including vaginal keratinocytes. We assessed the sensitivity of primary human vaginal keratinocytes to potential topical vaginal microbicides nonoxynol-9 (N-9), C31G, and sodium dodecyl sulfate (SDS). Direct immunofluorescence and fluorescence-activated cell sorting analyses demonstrated that primary vaginal keratinocytes expressed epithelial cell-specific keratin proteins. Experiments that compared vaginal keratinocyte sensitivity to each agent during a continuous, 48-h exposure demonstrated that primary vaginal keratinocytes were almost five times more sensitive to N-9 than to either C31G or SDS. To evaluate the effect of multiple microbicide exposures on cell viability, primary vaginal keratinocytes were exposed to N-9, C31G, or SDS three times during a 78-h period. In these experiments, cells were considerably more sensitive to C31G than to N-9 or SDS at lower concentrations within the range tested. When agent concentrations were chosen to result in an endpoint of 25% viability after three daily exposures, each exposure decreased cell viability at the same constant rate. When time-dependent sensitivity during a continuous 48-h exposure was examined, exposure to C31G for 18 h resulted in losses in cell viability not caused by either N-9 or SDS until at least 24 to 48 h. Cumulatively, these results reveal important variations in time- and concentration-dependent sensitivity to N-9, C31G, or SDS within populations of primary human vaginal keratinocytes cultured in vitro. These investigations represent initial steps toward both in vitro modeling of the vaginal microenvironment and studies of factors that impact the in vivo efficacy of vaginal topical microbicides.

HIV-I-associated central nervous system dysfunction

Despite more than 15 years of extensive investigative efforts, a complete understanding of the neurological consequences of HIV-1 CNS infection remains elusive. Although the resources of numerous investigators have been focused on studies of HIV-1-associated CNS disease, the complex nature of the disease processes that underlie the clinical, pathological, and cellular manifestations of HIV-1 CNS infection have required a larger volume of studies than was initially envisioned. Several major areas remain as the focus of current research efforts. One of the more pressing issues facing researchers and clinicians alike is the search for correlates to the development of HIV-1-associated CNS neuropathology and the onset of HIVD. Although numerous parameters have been studied, none have been shown to be absolute predictors or markers of HIV-1-related CNS dysfunction. The identification of solid correlates of HIVD is an important goal that would permit clinical identification of individuals at risk for developing potentially crippling, life-threatening CNS abnormalities and would facilitate early treatment of nascent neurological problems. A more complete comprehension of the cellular foundations of CNS dysfunction and HIVD is also a fundamental part of strategies designed to treat or prevent HIV-1-associated CNS disease. Future investigations will strive to expand the body of knowledge concerning the complex interactions between infected and uninfected neuroglial cells and the roles of numerous cytokines, chemokines, and other soluble agents that are deregulated during HIV-1 CNS infection. In particular, a thorough understanding of the mechanisms of neurotoxicity may facilitate the development of new therapies that alleviate or eliminate the clinical consequences of CNS infection. Finally, investigators will continue to study HIVD within the context of single and combination drug therapies used in the treatment of HIV-1 infection and AIDS. As newer and more effective systemic treatments for HIV-1 infection and AIDS are introduced, the effects of these treatments on the onset, incidence, and severity of HIVD will also require intensive study. The impact of drug therapies on the ability of the CNS to act as an HIV-1 reservoir will also need to be addressed. Introduction of each new drug or drug combination will necessitate studies of drug penetration into the CNS and efficacy against the development of CNS abnormalities. Furthermore, as more effective treatments prolong the lifespan of individuals infected with HIV-1, the impact of extended survival on the occurrence and severity of HIVD will also require further investigations. The quest for answers to these and other questions will be complicated by the diversity of experimental systems used to study different aspects of HIV-1 CNS infection and HIVD. Each system has its own unique strengths and weaknesses. Clinical observations provide a continuous spectrum of symptomatic findings but reveal little about the underlying mechanisms of disease. In vivo imaging techniques, such as CT and MRI, also provide a continuum of observations, but the images are limited in their resolution. Neuropathological examinations of postmortem HIV-1-infected brains offer gross, cellular, and molecular views (including phenotypic and genotypic analyses of CNS viral isolates) of the diseased brain, but only provide a snapshot of the end-stage neurologic dysfunction. Studies that rely on animal surrogates for HIV-1, including SIV, simian-HIV (SHIV), feline immunodeficiency virus (FIV), visna virus, and HIV-1 SCID-hu models, permit experimental protocols that cannot be carried out in humans, but are limited by the fidelity with which each virus and animal model emulates the conditions and events observed in the human host. Finally, in vitro techniques, which include the use of primary cells and cell lines, adult or fetal human cell cultures, and BBB barrier model systems, are also convenient means by which aspe

Comparative In Vitro Sensitivities of Human Immune Cell Lines, Vaginal and Cervical Epithelial Cell Lines, and Primary Cells to Candidate Microbicides Nonoxynol 9, C31G, and Sodium Dodecyl Sulfate

ABSTRACT In experiments to assess the in vitro impact of the candidate microbicides nonoxynol 9 (N-9), C31G, and sodium dodecyl sulfate (SDS) on human immune and epithelial cell viability, cell lines and primary cell populations of lymphocytic and monocytic origin were generally shown to be equally sensitive to exposures ranging from 10 min to 48 h. However, U-937 cells were more sensitive to N-9 and C31G after 48 h than were primary monocyte-derived macrophages. Cytokine activation of monocytes and lymphocytes had no effect on cell viability following exposure to these microbicidal compounds. Primary and passaged vaginal epithelial cultures and cell lines differed in sensitivity to N-9 and C31G but not SDS. These studies provide a foundation for in vitro experiments in which cell lines of human immune and epithelial origin can be used as suitable surrogates for primary cells to further investigate the effects of microbicides on cell metabolism, membrane composition, and integrity and the effects of cell type, proliferation, and differentiation on microbicide sensitivity.

HIV-1 Vpr binding to HIV-1 LTR C/EBP cis-acting elements and adjacent regions is sequence-specific

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a 14 kDa virion-associated protein that transactivates the HIV-1 long terminal repeat (LTR) as well as other eukaryotic promoters. Vpr also functions in nuclear localization and import of the HIV-1 preintegration complex (PIC), cell cycle arrest at the G(2)/M interface, and virion packaging. Electrophoretic mobility shift analysis has been utilized to demonstrate a direct association between purified Vpr (strain pNL4-3) and HIV-1 LTR sequences that span the adjacent C/EBP site I, NF-kappaB site II, and ATF/CREB binding site (nt -95 to -130, relative to the start of transcription). A similar interaction has been observed between HIV-1 Vpr and LTR C/EBP site II (nt -167 to -175). A total of 94.7% of LTRs derived from peripheral blood contained C/EBP site I variants that displayed a high relative Vpr binding affinity phenotype, while only 5.3% exhibited a low relative Vpr binding affinity phenotype. All LTRs derived from peripheral blood exhibited a high relative Vpr binding phenotype at C/EBP site II. These results suggest a preference for the maintenance of two cis-acting elements with high affinity for Vpr within LTRs derived from peripheral blood. Additional studies have also demonstrated that naturally occurring sequence variation within C/EBP site I and II can dramatically alter the relative affinity of Vpr for these cis-acting elements. These studies suggest that Vpr may regulate the interaction of members of the C/EBP transcription factor family with the viral LTR.

Structural and Functional Evolution of Human Immunodeficiency Virus Type 1 Long Terminal Repeat CCAAT/Enhancer Binding Protein Sites and Their Use as Molecular Markers for Central Nervous System Disease Progression

The appearance and progression of human immunodeficiency virus type 1 (HIV-1)—associated pathogenesis in the immune and central nervous systems is dependent on the ability of the virus to replicate in these compartments, which is, in turn, controlled by numerous factors, including viral binding and entry, receptor and coreceptor usage, and regulation of viral expression by the long terminal repeat (LTR). The LTR promotes viral expression in conjunction with viral and cellular regulatory proteins, including members of the CCAAT/enhancer binding protein (C/EBP) family, which modulate LTR activity through at least two cis-acting binding sites. Previous studies have shown that these sites are necessary for HIV-1 replication in cells of the monocyte/macrophage lineage, but dispensable in T lymphocytes. To establish potential links between this important family of transcription factors and HIV-1— associated pathogenesis, C/EBP site I and II sequence variation in peripheral blood mononuclear cell (PBMC)-derived LTRs from HIV-1—infected patients with varying degrees of disease severity was examined. A high prevalence of C/EBP site variants 3T (site I) and consensus B (site II) within PBMC-derived HIV-1 LTRs was shown to correlate with late stage disease in HIV-1-infected patients. These results suggest that the increased prevalence in the PBMCs of HIV-1 LTRs containing the 3T C/EBP site I variant and the consensus B site II variant may serve as a molecular marker for disease progression within the immune system. The relative low or high binding affinity of C/EBP β to sites I and II in electrophoretic mobility shift (EMS) analyses correlated with low or high LTR activity, respectively, in transient expression analyses during both early and late disease stages. The 3T C/EBP site I was the only variant examined that was not found in LTRs derived from PBMCs of patients at early stages of HIV-1 disease, but was found at increasing frequencies in patients with late stage disease. Furthermore, the 3T C/EBP site I was not found in brain-derived LTRs of patients without HIV-1-associated dementia (HIVD), but was found in increasing numbers in brain-derived LTRs from patients diagnosed with HIVD. The C/EBP site I 3T variant appears to be exclusive to patients progressing to increasingly severe HIV-1—associated immunologic and neurologic disease.

Human Immunodeficiency Virus Type 1 Long Terminal Repeat Quasispecies Differ in Basal Transcription and Nuclear Factor Recruitment in Human Glial Cells and Lymphocytes

The generation of genomic diversity during the course of infection has the potential to affect all aspects of HIV-1 replication, including expression of the proviral genome. To gain a better understanding of the impact of long terminal repeat (LTR) sequence diversity on LTR-directed gene expression in cells of the central nervous system (CNS) and immune system, we amplified and cloned LTRs from proviral DNA in HIV-1-infected peripheral blood. Sequence analysis of nineteen LTRs cloned from 2 adult and 3 pediatric patients revealed an average of 33 nucleotide changes (with respect to the sequence of the LAI LTR) within the 455-bp U3 region. Transient expression analyses in cells of neuroglial and lymphocytic origin demonstrated that some of these LTRs had activities which varied significantly from the LAI LTR in U-373 MG cells (an astrocytoma cell line) as well as in Jurkat cells (a CD4-positive lymphocyte cell line). While LTRs which demonstrated the highest activities in U-373 MG cells also yielded high activities in Jurkat cells, the LTRs were generally more active in Jurkat cells when compared to the LAI LTR. Differences in LTR sequence also resulted in differences in transcription factor recruitment to cis-acting sites within the U3 region of the LTR, as demonstrated by electrophoretic mobility shift assays. In particular, naturally occurring sequence variation impacted transcription factor binding to an activating transcription factor/cAMP response element binding (ATF/CREB) binding site (located between the LEF-1 and distal NF-kappaB transcription factor binding sites) that we identified in previous studies of the HIV-1 LTR. These findings suggest that LTR sequence changes can significantly affect basal LTR function and transcription factor recruitment, which may, in turn, alter the course of viral replication in cells of CNS and immune system origin.

Identification of binding sites for members of the CCAAT/enhancer binding protein transcription factor family in the simian immunodeficiency virus long terminal repeat

Members of the CCAAT/enhancer binding protein (C/EBP) transcription factor family are necessary for human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) activity and viral replication in cells of monocyte/macrophage lineage. The integral roles that HIV-1-infected monocytes and macrophages play in the development and progression of HIV-1-associated disease in the immune and central nervous systems underscore the importance of the C/EBP transcription factor family within the context of regulating HIV-1 gene expression. Although there are considerable similarities between HIV-1 and simian immunodeficiency virus (SIV), including viral-induced immunopathogenesis and neurologic dysfunction, infection of CD4(+) T cells and cells of monocyte/macrophage origin, and LTR structure/function, the involvement of C/EBP factors in regulating SIV transcription has not been previously demonstrated. Analyses of the SIV(mac)239 LTR sequence indicated the presence of five putative C/EBP binding sites within the LTR. Electrophoretic mobility shift (EMS) analyses demonstrated that four of the five sites within the SIV LTR were able to bind C/EBP factors (alpha and beta) and compete for DNA-protein complexes formed by the HIV-1 C/EBP site located adjacent to the promoter-distal NF-kappaB site. DNase I protection assays indicated that purified C/EBPbeta specifically was able to occupy each of the four binding sites. These studies suggest that C/EBP factors may also have important roles in the regulation of SIV gene expression and replication, and that these factors and signal transduction pathways that regulate their activity may impact SIV-associated pathogenesis.

C/EBP- and Tat-mediated activation of the HIV-1 LTR in CD34+ hematopoietic progenitor cells.

Human immunodeficiency virus type 1 (HIV-1) infection of cells of the monocyte/macrophage lineage within the bone marrow and peripheral blood plays an important role in the pathologic events leading to the development of the acquired immune deficiency syndrome (AIDS) as well as HIV-1 dementia (HIVD). The TF-1 erythro-myeloid cell line is being utilized as a model cellular phenotype to examine HIV-1 infection of a hematopoietic progenitor cell population. Expression of TF-1 cell surface marker RNAs and proteins was characterized by RT-PCR and FACS, respectively, and compared to those of the well characterized U-937 monocytic cell line. Transcription factors in TF-1 and U-937 cells that have been shown to be important for sustaining the expression of HIV-1 LTR activity were also examined. TF-1 cells were shown to contain the transcription factors C/EBP, Sp1, and NF-kappaB. C/EBP- and Tat-mediated induction of the YU-2 LTR was examined. Relative C/EBP induction of the HIV-1 strain YU-2 LTR was greater in TF-1 cells than in U-937 cells. When the C/EBP sites I and II were mutated to sequences with a low relative affinity for C/EBP factors, there was a reduction of Tat-mediated trans-activation in TF-1 cells, but not in U-937 cells. These studies form the foundation for investigations into the relationship between HIV-1 infection of bone marrow and peripheral blood precursor cells of the monocyte/macrophage lineage and pathogenesis associated with HIV-1 infection of the immune and central nervous system (CNS).

Spl and related factors fail to interact with the NF-kB-proximal G/C box in the LTR of a replication competent, brain-derived strain of HIV-1 (YU-2)

The HIV-1 LTR promoter proximal G/C box array has been demonstrated to function by interacting with the Sp1 transcription factor family whose members can act as either activators or repressors of transcription. In this regard, we have examined the interaction of the HIV-1 Sp binding sites with nuclear factors that are present in cell types that support HIV-1 replication, including those of lymphocytic, monocytic, and astrocytic origin. As determined by electrophoretic mobility shift (EMS) competition analyses using oligonucleotides containing the sequences of each of the Sp1 sites of HIV-1 strain LAI, the NF-kappaB-proximal Sp site (site III) displayed the highest binding activity compared to sites I and II with regard to Sp1 and related factors present in lymphocytic (Jurkat) and astrocytic (U-373 MG) nuclear extracts. Sp1 and two Sp3 isoforms were detected as the primary cellular constituents of DNA-protein complexes formed with the NF-kappaB-proximal site. Only modest differences in Sp1:Sp3 binding ratios were observed when this site was reacted with either astrocytic or lymphocytic nuclear extract. However, when nuclear extracts derived from two monocytic cell types that differ in the degree of differentiation were reacted with the HIV-1 LAI Sp site III, a large difference in Sp1 and Sp3 binding was observed. To determine if naturally occurring and replication-competent strains of HIV-1 contain base pair alterations within the Sp elements that affect the ability of the site to interact with Sp1 and related factors, a series of Sp site III variants were constructed and examined by EMS analyses. One of these sites, obtained from the published sequence of the YU-2 strain (a brain-derived macrophage tropic strain of HIV-1), displayed almost no Sp1 or Sp3 binding activity as a result of a single base pair alteration in Sp site III. This base-pair alteration, when placed in the context of an HIV-1 LAI LTR-luciferase construct, resulted in a 40-50% reduction in LTR activity in transiently transfected Jurkat and U-373 MG cells. Overall, these results suggest that specific G/C box sequence alterations present in the brain-derived HIV-1 variant YU-2, or possibly other brain-derived variants, may exhibit altered replication properties as a result of the low affinity of the NF-kappaB-proximal G/C box for members of the Sp transcription factor family.