scholarly journals Comparison of G-to-A Mutation Frequencies Induced by APOBEC3 Proteins in H9 Cells and Peripheral Blood Mononuclear Cells in the Context of Impaired Processivities of Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Variants

2008 ◽  
Vol 82 (13) ◽  
pp. 6536-6545 ◽  
Author(s):  
Stefanie Andrea Knoepfel ◽  
Nadine Christina Salisch ◽  
Peter Michael Huelsmann ◽  
Pia Rauch ◽  
Hauke Walter ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Virus Variant ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Apobec3 Proteins ◽  
Hiv 1

ABSTRACT APOBEC3 proteins can inhibit human immunodeficiency virus type 1 (HIV-1) replication by inducing G-to-A mutations in newly synthesized viral DNA. However, HIV-1 is able to overcome the antiretroviral activity of some of those enzymes by the viral protein Vif. We investigated the impact of different processivities of HIV-1 reverse transcriptases (RT) on the frequencies of G-to-A mutations introduced by APOBEC3 proteins. Wild-type RT or the M184V, M184I, and K65R+M184V RT variants, which are increasingly impaired in their processivities, were used in the context of a vif-deficient molecular HIV-1 clone to infect H9 cells and peripheral blood mononuclear cells (PBMCs). After two rounds of infection, a part of the HIV-1 env gene was amplified, cloned, and sequenced. The M184V mutation led to G-to-A mutation frequencies that were similar to those of the wild-type RT in H9 cells and PBMCs. The frequencies of G-to-A mutations were increased after infection with the M184I virus variant. This effect was augmented when using the K65R+M184V virus variant (P < 0.001). Overall, the G-to-A mutation frequencies were lower in PBMCs than in H9 cells. Remarkably, 38% ± 18% (mean ± standard deviation) of the env clones derived from PBMCs did not harbor any G-to-A mutation. This was rarely observed in H9 cells (3% ± 3%). Our data imply that the frequency of G-to-A mutations induced by APOBEC3 proteins can be influenced by the processivities of HIV-1 RT variants. The high number of nonmutated clones derived from PBMCs leads to several hypotheses, including that additional antiretroviral mechanisms of APOBEC3 proteins other than their deamination activity might be involved in the inhibition of vif-deficient viruses.

scholarly journals The Dimer Initiation Sequence Stem-Loop of Human Immunodeficiency Virus Type 1 Is Dispensable for Viral Replication in Peripheral Blood Mononuclear Cells

2003 ◽  
Vol 77 (15) ◽  
pp. 8329-8335 ◽  
Author(s):  
M. K. Hill ◽  
M. Shehu-Xhilaga ◽  
S. M. Campbell ◽  
P. Poumbourios ◽  
S. M. Crowe ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Stem Loop ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) contains two copies of genomic RNA that are noncovalently linked via a palindrome sequence within the dimer initiation site (DIS) stem-loop. In contrast to the current paradigm that the DIS stem or stem-loop is critical for HIV-1 infectivity, which arose from studies using T-cell lines, we demonstrate here that HIV-1 mutants with deletions in the DIS stem-loop are replication competent in peripheral blood mononuclear cells (PBMCs). The DIS mutants contained either the wild-type (5′GCGCGC3′) or an arbitrary (5′ACGCGT3′) palindrome sequence in place of the 39-nucleotide DIS stem-loop (NLCGCGCG and NLACGCGT). These DIS mutants were replication defective in SupT1 cells, concurring with the current model in which DIS mutants are replication defective in T-cell lines. All of the HIV-1 DIS mutants were replication competent in PBMCs over a 40-day infection period and had retained their respective DIS mutations at 40 days postinfection. Although the stability of the virion RNA dimer was not affected by our DIS mutations, the RNA dimers exhibited a diffuse migration profile when compared to the wild type. No defect in protein processing of the Gag and GagProPol precursor proteins was found in the DIS mutants. Our data provide direct evidence that the DIS stem-loop is dispensable for viral replication in PBMCs and that the requirement of the DIS stem-loop in HIV-1 replication is cell type dependent.

Human serum alpha 1 acid glycoprotein reduces uptake, intracellular concentration, and antiviral activity of A-80987, an inhibitor of the human immunodeficiency virus type 1 protease.

1996 ◽  
Vol 40 (6) ◽  
pp. 1491-1497 ◽  
Author(s):  
J A Bilello ◽  
P A Bilello ◽  
K Stellrecht ◽  
J Leonard ◽  
D W Norbeck ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Peripheral Blood Mononuclear Cells ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Acid Glycoprotein ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Hiv 1

The therapeutic utility of a human immunodeficiency virus type 1 (HIV-1) protease inhibitor may depend on its intracellular concentration, which is a property of its uptake, metabolism, and/or efflux. Previous studies in our laboratory indicated that the addition of alpha 1 acid glycoprotein (alpha 1 AGP) to the medium markedly increased the amount of the drug required to limit infection in vitro. In this study, physiologically relevant concentrations of alpha 1 AGP and a radiolabeled inhibitor, A-80987, were used to determine both the uptake and activity of the agent in HIV-1-infected human peripheral blood mononuclear cells and cell lines. Both the uptake and efflux of 14C-labeled A-80987 were rapid (t1/2, < 5 min). Uptake of the drug was linearly dependent on the concentration but insensitive to the metabolic inhibitors KF, sodium cyanide, or CCCP (carbonyl cyanide m-chlorophenyl hydrazone). The amount of A-80987 which entered the cells was inversely proportional to the concentration of alpha 1 AGP (r2, 0.99) and directly proportional to the amount of extracellular non-protein-bound drug (r2, 0.99). Most importantly, the antiviral activity of the drug in HIV-1-infected peripheral blood mononuclear cells and MT-2 cells was directly related to the amount of intracellular A-80987. This study demonstrates that A-80987 binds to alpha 1 AGP, resulting in a free fraction below 10%. Cellular uptake of A-80987 is proportionally decreased in the presence of alpha 1 AGP, which results in less-than-expected antiviral activity. Importantly, we demonstrate for the first time that the inhibition of HIV protease is highly correlated with the amount of intracellular inhibitor.

scholarly journals Human Immunodeficiency Virus Type 1 DNA Sequences Genetically Damaged by Hypermutation Are Often Abundant in Patient Peripheral Blood Mononuclear Cells and May Be Generated during Near-Simultaneous Infection and Activation of CD4+ T Cells

2001 ◽  
Vol 75 (17) ◽  
pp. 7973-7986 ◽  
Author(s):  
Mario Janini ◽  
Melissa Rogers ◽  
Deborah R. Birx ◽  
Francine E. McCutchan
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Hiv 1

ABSTRACT G-to-A hypermutation has been sporadically observed in human immunodeficiency virus type 1 (HIV-1) proviral sequences from patient peripheral blood mononuclear cells (PBMC) and virus cultures but has not been systematically evaluated. PCR primers matched to normal and hypermutated sequences were used in conjunction with an agarose gel electrophoresis system incorporating an AT-binding dye to visualize, separate, clone, and sequence hypermutated and normal sequences in the 297-bp HIV-1 protease gene amplified from patient PBMC. Among 53 patients, including individuals infected with subtypes A through D and at different clinical stages, at least 43% of patients harbored abundant hypermutated, along with normal, protease genes. In 70 hypermutated sequences, saturation of G residues in the GA or GG dinucleotide context ranged from 20 to 94%. Levels of other mutants were not elevated, and G-to-A replacement was entirely restricted to GA or GG, and not GC or GT, dinucleotides. Sixty-nine of 70 hypermutated and 3 of 149 normal sequences had in-frame stop codons. To investigate the conditions under which hypermutation occurs in cell cultures, purified CD4+ T cells from normal donors were infected with cloned NL4-3 virus stocks at various times before and after phytohemagglutinin (PHA) activation. Hypermutation was pronounced when HIV-1 infection occurred simultaneously with, or a few hours after, PHA activation, but after 12 h or more after PHA activation, most HIV-1 sequences were normal. Hypermutated sequences generated in culture corresponded exactly in all parameters to those obtained from patient PBMC. Near-simultaneous activation and infection of CD4+ T cells may represent a window of susceptibility where the informational content of HIV-1 sequences is lost due to hypermutation.

scholarly journals CNI-H0294, a Nuclear Importation Inhibitor of the Human Immunodeficiency Virus Type 1 Genome, Abrogates Virus Replication in Infected Activated Peripheral Blood Mononuclear Cells

1998 ◽  
Vol 42 (5) ◽  
pp. 1133-1138 ◽  
Author(s):  
Omar K. Haffar ◽  
Molly D. Smithgall ◽  
Serguei Popov ◽  
Peter Ulrich ◽  
A. Gregory Bruce ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Virus Replication ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Hiv 1

ABSTRACT Active nuclear importation of the human immunodeficiency virus (HIV) type 1 (HIV-1) preintegration complex (PIC) is required for the productive infection of nondividing cells, but it is believed to be dispensable for the infection of proliferating cells, such as activated T lymphocytes. To investigate this question, we exploited the properties of the small arylene bis (methyl ketone) compound CNI-H0294. We have previously shown that this compound associated with the HIV-1 matrix protein nuclear localization sequence and blocked binding of the HIV-1 PIC to yeast karyopherin α. CNI-H0294 abrogated nuclear importation of the HIV-1 genome in macrophages and effectively inhibited infection of nondividing cells. In this study we demonstrate that CNI-H0294 inhibits binding of the HIV-1 PIC to human karyopherin α and reduces nuclear importation of the viral genome in primary peripheral blood mononuclear cells (PBMCs). We also demonstrate that CNI-H0294 inhibits acute infection of PBMC cultures in vitro with a primary isolate of HIV-1 and reduces virus replication and virus load in cultures of endogenously infected PBMCs from seropositive individuals. Thus, as for infection of nondividing, terminally differentiated macrophages, HIV-1 uses active nuclear importation of the virus genome to infect activated CD4+ T cells. These results support nuclear importation as a novel target and CNI-H0294 and its derivatives as novel compounds for therapeutic intervention in HIV infection and AIDS.

scholarly journals The Replicative Fitness of Primary Human Immunodeficiency Virus Type 1 (HIV-1) Group M, HIV-1 Group O, and HIV-2 Isolates

2005 ◽  
Vol 79 (14) ◽  
pp. 8979-8990 ◽  
Author(s):  
Kevin K. Ariën ◽  
Awet Abraha ◽  
Miguel E. Quiñones-Mateu ◽  
Luc Kestens ◽  
Guido Vanham ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Hiv 1

ABSTRACT The main (M) group of human immunodeficiency virus type 1 (HIV-1) is responsible for the global AIDS epidemic while HIV-1 group O (outlier) and HIV type 2 are endemic only in west and central Africa. The failure of HIV-2 and especially HIV-1 group O to spread following the initial zoonotic jumps is not well understood. This study was designed to examine the relative replicative capacities between these human lentiviruses. A pairwise competition experiment was performed with peripheral blood mononuclear cells with eight HIV-2 isolates, 6 group O viruses, and 15 group M viruses of subtype A (2 viruses), B (5 viruses), C (4 viruses), D (2 viruses) and CRF01_AE (2 viruses). HIV-1 group M isolates of any subtype were typically 100-fold-more fit than group O or HIV-2 strains when competed in peripheral blood mononuclear cells from various humans. This order in replicative fitness was also observed when virus pairs were added to human dendritic cells and then cocultured with primary, quiescent T cells, which is the model for HIV-1 transmission. These results suggest that reduced replicative and transmission fitness may be contributing to the low prevalence and limited geographical spread of HIV-2 and group O HIV-1 in the human population.

scholarly journals PCR-Based Assay To Quantify Human Immunodeficiency Virus Type 1 DNA in Peripheral Blood Mononuclear Cells

2000 ◽  
Vol 38 (2) ◽  
pp. 630-634 ◽  
Author(s):  
Cindy Christopherson ◽  
Yorda Kidane ◽  
Brian Conway ◽  
John Krowka ◽  
Haynes Sheppard ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
The Mean ◽  
Long Term Survivors ◽  
Hiv 1

An assay that quantifies the amount of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells has been developed. PCR amplification of the HIV-1 DNA is performed in the presence of an internal quantitation standard, and colorimetric detection of the amplified product is performed with microwell plates. The copies of HIV-1 DNA are normalized to total genomic DNA input. The assay has an analytical sensitivity of 10 input copies per amplification reaction and a three-log detection range. In an analysis of sequential samples from patients on combination therapy, HIV-1 DNA was quantifiable for all individuals tested, including those with undetectable plasma HIV-1 RNA. In a separate study, a comparison of HIV-1 DNA levels was made with a group of long-term survivors and progressors. The mean HIV-1 DNA levels were lower in the long-term survivors than in the progressors (P, 0.04). The mean HIV-1 RNA levels were also lower, but the difference was not statistically significant (P, 0.164). A quantitative DNA assay will provide an additional tool to gain insight into the natural history of infection and the continued efficacy of potent antiretroviral therapies.

scholarly journals T Cells Expressing Activated LFA-1 Are More Susceptible to Infection with Human Immunodeficiency Virus Type 1 Particles Bearing Host-Encoded ICAM-1

1998 ◽  
Vol 72 (3) ◽  
pp. 2105-2112 ◽  
Author(s):  
Jean-François Fortin ◽  
Réjean Cantin ◽  
Michel J. Tremblay
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Hiv 1

ABSTRACT The incorporation of host-derived proteins in nascent human immunodeficiency virus type 1 (HIV-1) particles is a well-established phenomenon. We recently demonstrated that the physical presence of host-encoded ICAM-1 glycoproteins on HIV-1 leads to a significant increase in virus infectivity in an ICAM-1/LFA-1-dependent fashion (J.-F. Fortin, R. Cantin, G. Lamontagne, and M. Tremblay, J. Virol. 71:3588–3596, 1997). We show here that conversion of LFA-1 to high affinity for ICAM-1 with the use of anti-LFA-1 antibodies (clones NKI-L16 and MEM83) markedly enhances the susceptibility of different target T-lymphoid cell lines, as well as of primary peripheral blood mononuclear cells, to infection by ICAM-1-bearing HIV-1 particles (6- to 95-fold). It is known that T-cell receptor (TCR) cross-linking induces a transient increase in LFA-1 affinity for ICAM-1. Treatment of peripheral blood mononuclear cells with anti-TCR antibodies (clone OKT3) resulted in a transient increase in susceptibility to infection by ICAM-1-positive virions that parallels the previously reported kinetics of the LFA-1/ICAM-1 adhesion mechanism. Our results led us to postulate that the strong interaction taking place between virally incorporated ICAM-1 and cell surface-activated LFA-1 markedly enhances the efficiency of virus binding and entry, thus favoring greater infection by ICAM-1-bearing HIV-1 particles. In view of the knowledge that primary HIV-1 isolates harbor host-derived ICAM-1 on their surfaces, these results provide new information about the role of host-derived ICAM-1 in the life cycle of HIV-1 and how it could positively modulate the dynamics of the viral infection, mainly in cellular compartments, such as the lymphoid tissues, where the level of cellular activation is high and where the probability of encountering a T cell expressing the activated LFA-1 form is also elevated.

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