Variation of HIV-1 Mutation Spectra among Cell Types

ABSTRACT Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.

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Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR

Viruses ◽

10.3390/v13071235 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1235

Author(s):

Leah D. Brandt ◽

Shuang Guo ◽

Kevin W. Joseph ◽

Jana L. Jacobs ◽

Asma Naqvi ◽

...

Keyword(s):

Integration Site ◽

Cell Types ◽

Sequencing Data ◽

Site Specific ◽

Cell Clones ◽

Droplet Pcr ◽

Relative Rarity ◽

Deep Integration ◽

Multiple Samples ◽

Hiv 1

Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

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The Differential Anti-HIV Effect of a New Humic Substance-Derived Preparation in Diverse Cells of the Immune System

Acta Naturae ◽

10.32607/20758251-2019-11-2-68-76 ◽

2019 ◽

Vol 11 (2) ◽

pp. 68-76

Author(s):

G. V. Kornilaeva ◽

A. E. Siniavin ◽

A. Schultz ◽

A. Germann ◽

C. Moog ◽

...

Keyword(s):

Dendritic Cells ◽

T Lymphocytes ◽

Humic Substance ◽

Immune Cells ◽

Cell Types ◽

Inhibitory Effect ◽

Infection Inhibition ◽

Toxic Concentrations ◽

Anti Hiv ◽

Hiv 1

The anti-HIV activity of a new humic substance-derived preparation has been studied in individual pools of immune cells (CD4+ T lymphocytes, macrophages, dendritic cells). Near-complete inhibition of the HIV infection (by more than 90%) was achieved by treating each of the abovementioned cell types with non-toxic concentrations of the preparation. The inhibitory effect demonstrates the possibility of preventing the depletion of a significant portion of functionally important immune cells. A comparative study of infection inhibition in individual cell pools has allowed us to reveal the differences in the preparations effectiveness in each of the cell populations. A R5-tropic HIV-1 infection in macrophages exhibited maximum sensitivity to the preparation: 90% and 50% inhibition of the infection were observed in the presence of concentrations as low as 1.4 and 0.35 g/ml, respectively. A 15- and 19-fold higher concentration was required to achieve the same extent of inhibition in dendritic cells infected with the same strain. The effectiveness of the drug in CD4 + T lymphocytes is quite comparable to its effectiveness in macrophages. The drug is universally effective for both the T- and M-tropic variants of HIV-1.

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Selection following isolation of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and herpesvirus saimiri-transformed T cells is comparable

Journal of General Virology ◽

10.1099/0022-1317-83-6-1343 ◽

2002 ◽

Vol 83 (6) ◽

pp. 1343-1352 ◽

Cited By ~ 7

Author(s):

Natalie N. Zheng ◽

Cherelyn Vella ◽

Philippa J. Easterbrook ◽

Rod S. Daniels

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Peripheral Blood Mononuclear Cells ◽

Mononuclear Cells ◽

Cell Types ◽

Herpesvirus Saimiri ◽

Peripheral Blood Mononuclear ◽

Immunodeficiency Virus ◽

Blood Mononuclear Cells ◽

Hiv 1

In attempts to improve isolation rates and virus yields for human immunodeficiency virus (HIV), the use of herpesvirus saimiri-immortalized T cells (HVS T cells) has been investigated as an alternative to/improvement over peripheral blood mononuclear cells (PBMCs). Here we characterize isolates rescued, in the two cell types, from two asymptomatic, long-term non-progressing HIV-1-infected individuals. All rescued viruses replicated in PBMCs and HVS T cells only, displaying a non-syncytium inducing (NSI) phenotype, and using CCR5 as co-receptor. Furthemore, PBMC/HVS T cell virus pairs displayed similar neutralization profiles. Full-length, expression-competent env genes were rescued from all virus isolates and directly from the patient samples using proviral DNA and viral RNA as templates. Compared with the sequences retrieved directly from the patient samples, both cell types showed similar selection characteristics. Whilst the selections were distinct for individual patient samples, they shared a common characteristic in selecting for viruses with increased negative charge across the V2 domain of the viral glycoproteins. The latter was observed at the env gene sequencing level for three other patients whose HIV strains were isolated in PBMCs only. This further supports a common selection for viral sequences that display a macrophage-tropic/NSI phenotype and shows that HVS T cells are a viable alternative to PBMCs for HIV-1 isolation.

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Intron-containing RNA from the HIV-1 provirus activates type I interferon and inflammatory cytokines

10.1101/128447 ◽

2017 ◽

Author(s):

Sean Matthew McCauley ◽

Kyusik Kim ◽

Anetta Nowosielska ◽

Ann Dauphin ◽

Leonid Yurkovetskiy ◽

...

Keyword(s):

T Cells ◽

Antiviral Therapy ◽

Antiviral Drug ◽

Cell Types ◽

Innate Immune ◽

Type I ◽

Infected People ◽

Innate Immune Signaling ◽

Post Transcriptional Regulation ◽

Hiv 1

ABSTRACTHIV-1-infected people who take drugs that suppress viremia to undetectable levels are protected from developing AIDS. Nonetheless, these individuals have chronic inflammation associated with heightened risk of cardiovascular pathology. HIV-1 establishes proviruses in long-lived CD4+memory T cells, and perhaps other cell types, that preclude elimination of the virus even after years of continuous antiviral therapy. Though the majority of proviruses that persist during antiviral therapy are defective for production of infectious virions, many are expressed, raising the possibility that the HIV-1provirus or its transcripts contribute to ongoing inflammation. Here we found that the HIV-1 provirus activated innate immune signaling in isolated dendritic cells, macrophages, and CD4+T cells. Immune activation required transcription from the HIV-1 provirus and expression of CRM1-dependent, Rev-dependent, RRE-containing, unspliced HIV-1 RNA. Ifrevwas providedin trans, all HIV-1 coding sequences were dispensable for activation except thosecis-acting sequences required for replication or splicing. These results indicate that the complex, post-transcriptional regulation intrinsic to HIV-1 RNA is detected by the innate immune system as a danger signal, and that drugs which disrupt HIV-1 transcription or HIV-1 RNA metabolism would add qualitative benefit to current antiviral drug regimens.

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The HIV-1 Transgenic Rat: Relevance for HIV Noninfectious Comorbidity Research

Microorganisms ◽

10.3390/microorganisms8111643 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1643

Author(s):

Frank Denaro ◽

Francesca Benedetti ◽

Myla D. Worthington ◽

Giovanni Scapagnini ◽

Christopher C. Krauss ◽

...

Keyword(s):

Neuronal Degeneration ◽

Cell Types ◽

Transgenic Rat ◽

Specific Cell ◽

Rat Tissues ◽

Local Production ◽

Specific Tissue ◽

Effective Models ◽

The Brain ◽

Hiv 1

HIV noninfectious comorbidities (NICMs) are a current healthcare challenge. The situation is further complicated as there are very few effective models that can be used for NICM research. Previous research has supported the use of the HIV-1 transgenic rat (HIV-1TGR) as a model for the study of HIV/AIDS. However, additional studies are needed to confirm whether this model has features that would support NICM research. A demonstration of the utility of the HIV-1TGR model would be to show that the HIV-1TGR has cellular receptors able to bind HIV proteins, as this would be relevant for the study of cell-specific tissue pathology. In fact, an increased frequency of HIV receptors on a specific cell type may increase tissue vulnerability since binding to HIV proteins would eventually result in cell dysfunction and death. Evidence suggests that observations of selective cellular vulnerability in this model are consistent with some specific tissue vulnerabilities seen in NICMs. We identified CXCR4-expressing cells in the brain, while specific markers for neuronal degeneration demonstrated that the same neural types were dying. We also confirm the presence of gp120 and Tat by immunocytochemistry in the spleen, as previously reported. However, we observed very rare positive cells in the brain. This underscores the point that gp120, which has been reported as detected in the sera and CSF, is a likely source to which these CXCR4-positive cells are exposed. This alternative appears more probable than the local production of gp120. Further studies may indicate some level of local production, but that will not eliminate the role of receptor-mediated pathology. The binding of gp120 to the CXCR4 receptor on neurons and other neural cell types in the HIV-1TGR can thus explain the phenomena of selective cell death. Selective cellular vulnerability may be a contributing factor to the development of NICMs. Our data indicate that the HIV-1TGR can be an effective model for the studies of HIV NICMs because of the difference in the regional expression of CXCR4 in rat tissues, thus leading to specific organ pathology. This also suggests that the model can be used in the development of therapeutic options.

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CA Mutation N57A Has Distinct Strain-Specific HIV-1 Capsid Uncoating and Infectivity Phenotypes

Journal of Virology ◽

10.1128/jvi.00214-19 ◽

2019 ◽

Vol 93 (9) ◽

Cited By ~ 2

Author(s):

Douglas K. Fischer ◽

Akatsuki Saito ◽

Christopher Kline ◽

Romy Cohen ◽

Simon C. Watkins ◽

...

Keyword(s):

Cell Cycle ◽

Amino Acid ◽

Cell Types ◽

Careful Consideration ◽

Immunodeficiency Virus ◽

Cell Cycle Dependence ◽

Cycle Dependent ◽

Multiple Strains ◽

Different Cell Types ◽

Hiv 1

ABSTRACTThe ability of human immunodeficiency virus type 1 (HIV-1) to transduce nondividing cells is key to infecting terminally differentiated macrophages, which can serve as a long-term reservoir of HIV-1 infection. The mutation N57A in the viral CA protein renders HIV-1 cell cycle dependent, allowing examination of HIV-1 infection of nondividing cells. Here, we show that the N57A mutation confers a postentry infectivity defect that significantly differs in magnitude between the common lab-adapted molecular clones HIV-1NL4-3(>10-fold) and HIV-1LAI(2- to 5-fold) in multiple human cell lines and primary CD4+T cells. Capsid permeabilization and reverse transcription are altered when N57A is incorporated into HIV-1NL4-3but not HIV-1LAI. The N57A infectivity defect is significantly exacerbated in both virus strains in the presence of cyclosporine (CsA), indicating that N57A infectivity is dependent upon CA interacting with host factor cyclophilin A (CypA). Adaptation of N57A HIV-1LAIselected for a second CA mutation, G94D, which rescued the N57A infectivity defect in HIV-1LAIbut not HIV-1NL4-3. The rescue of N57A by G94D in HIV-1LAIis abrogated by CsA treatment in some cell types, demonstrating that this rescue is CypA dependent. An examination of over 40,000 HIV-1 CA sequences revealed that the four amino acids that differ between HIV-1NL4-3and HIV-1LAICA are polymorphic, and the residues at these positions in the two strains are widely prevalent in clinical isolates. Overall, a few polymorphic amino acid differences between two closely related HIV-1 molecular clones affect the phenotype of capsid mutants in different cell types.IMPORTANCEThe specific mechanisms by which HIV-1 infects nondividing cells are unclear. A mutation in the HIV-1 capsid protein abolishes the ability of the virus to infect nondividing cells, serving as a tool to examine cell cycle dependence of HIV-1 infection. We have shown that two widely used HIV-1 molecular clones exhibit significantly different N57A infectivity phenotypes due to fewer than a handful of CA amino acid differences and that these clones are both represented in HIV-infected individuals. As such minor differences in closely related HIV-1 strains may impart significant infectivity differences, careful consideration should be given to drawing conclusions from one particular HIV-1 clone. This study highlights the potential for significant variation in results with the use of multiple strains and possible unanticipated effects of natural polymorphisms.

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Persistent NF-κB activation in renal epithelial cells in a mouse model of HIV-associated nephropathy

AJP Renal Physiology ◽

10.1152/ajprenal.00208.2005 ◽

2006 ◽

Vol 290 (3) ◽

pp. F657-F665 ◽

Cited By ~ 32

Author(s):

Scott Martinka ◽

Leslie A. Bruggeman

Keyword(s):

Transcription Factor ◽

Epithelial Cells ◽

Cell Types ◽

Nuclear Accumulation ◽

Mobility Shift ◽

Glomerular Epithelial Cells ◽

Host Virus Interactions ◽

Virus Interactions ◽

Electrophoretic Mobility Shift Assays ◽

Hiv 1

Human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) is caused, in part, by direct infection of kidney epithelial cells by HIV-1. In the spectrum of pathogenic host-virus interactions, abnormal activation or suppression of host transcription factors is common. NF-κB is a necessary host transcription factor for HIV-1 gene expression, and it has been shown that NF-κB activity is dysregulated in many naturally infected cell types. We show here that renal glomerular epithelial cells (podocytes) expressing the HIV-1 genome, similar to infected immune cells, also have a dysregulated and persistent activation of NF-κB. Although podocytes produce p50, p52, RelA, RelB, and c-Rel, electrophoretic mobility shift assays and immunocytochemistry showed a predominant nuclear accumulation of p50/RelA-containing NF-κB dimers in HIV-1-expressing podocytes compared with normal. In addition, the expression level of a transfected NF-κB reporter plasmid was significantly higher in HIVAN podocytes. The mechanism of NF-κB activation involved increased phosphorylation of IκBα, resulting in an enhanced turnover of the IκBα protein. There was no evidence for regulation by IκBβ or the alternate pathway of NF-κB activation. Altered activation of this key host transcription factor likely plays a role in the well-described cellular phenotypic changes observed in HIVAN, such as proliferation. Studies with inhibitors of proliferation and NF-κB suggest that NF-κB activation may contribute to the proliferative mechanism in HIVAN. In addition, because NF-κB regulates many aspects of inflammation, this dysregulation may also contribute to disease severity and progression through regulation of proinflammatory processes in the kidney microenvironment.

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CCR5, CXCR4, and CD4 Are Clustered and Closely Apposed on Microvilli of Human Macrophages and T Cells

Journal of Virology ◽

10.1128/jvi.75.8.3779-3790.2001 ◽

2001 ◽

Vol 75 (8) ◽

pp. 3779-3790 ◽

Cited By ~ 111

Author(s):

Irwin I. Singer ◽

Solomon Scott ◽

Douglas W. Kawka ◽

Jayne Chin ◽

Bruce L. Daugherty ◽

...

Keyword(s):

T Cells ◽

Chemokine Receptors ◽

Inflammatory Responses ◽

Cell Types ◽

Cellular Membrane ◽

Major Elements ◽

Cellular Migration ◽

Directional Information ◽

Virus Adsorption ◽

Hiv 1

ABSTRACT The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.

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Altered host range of HIV-1 after passage through various human cell types

Virology ◽

10.1016/0042-6822(91)90494-v ◽

1991 ◽

Vol 181 (1) ◽

pp. 288-294 ◽

Cited By ~ 26

Author(s):

Cecilia Cheng-Mayer ◽

Deborah Seto ◽

Jay A. Levy

Keyword(s):

Host Range ◽

Human Cell ◽

Cell Types ◽

Hiv 1

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