Detection of p24 in HIV-1 infected cells embedded in LR White and Lowicryl K4M

1991 ◽  
Vol 23 (8) ◽  
pp. 381-384 ◽  
Author(s):  
Gregor Stransky ◽  
Robert F. Garry ◽  
Steffen Gay
Keyword(s):  
Lr White ◽  
Infected Cells ◽  
Lowicryl K4m ◽  
Hiv 1

Localization of constituent proteins of HIV Type 1 (HIV-1)

1990 ◽  
Vol 48 (3) ◽  
pp. 340-341
Author(s):  
Toshiyuki Goto ◽  
Chizuko Morita ◽  
Taisuke Ashina ◽  
Kazuyoshi Ikuta ◽  
Shiro Kato ◽  
...  
Keyword(s):  
Cell Lines ◽  
Uranyl Acetate ◽  
Mouse Serum ◽  
Infected Cells ◽  
Hiv Type 1 ◽  
Lowicryl K4m ◽  
Specific Reactions ◽  
Phosphate Buffered Saline ◽  
Hiv 1

The localization of the constituent proteins of HIV-1 in the virions and HIV-1-infected cells was examined by indirect immuno-gold labeling with monoclonal antibodies (MoAb) to gag proteins (p18 and p24/p53), to elucidate viral formation of HIV.Persistently HIV-1 (HTLV—IIIB,LAV-1)-infected MT-4 and MOLT-4 cell lines and their cloned cell lines were used as infected cells. Mouse MoAb against HIV-1 gag p18 (V17) and p24/p53 (V107) were used. The cells were fixed with 0.5-1% glutar-aldehyde in phosphate-buffered saline (pH 7.2), dehydrated in ethanol and embedded in epoxy (at 45° Cor 60°C) or Lowicryl K4M resin (at −30°C). The sections were incubated in MoAb at room temperature for 1 h and then incubated in anti-mouse goat IgG conjugated with gold (IgG-gold, 5 or 15 nm; Janssen) for 40 min. After being washed, the sections were stained with uranyl acetate and lead citrate, and were observed in an electron microscope with a tilting apparatus.The spećific reactions with V17 and V107 were detected on HIV-1 particles and in the infected cells. No reactivity was noted between uninfected control cells and the MoAb, or between the infected cells and normal mouse serum. More than 97% of the HIV-1 particles embedded in epoxy resinat 60°C were labeled with gold after exclusion of the HIV particles that were attached to supporting film or completely embedded in the section (Fig. 1). Increased labeling was observed with Lowicryl (Fig. 2)and epoxy resin embedding at 45°C.

scholarly journals Ultrastructural localization of HTLV-I gag proteins p19 and p24 by single and double immunogold labeling.

1991 ◽  
Vol 39 (2) ◽  
pp. 185-191 ◽  
Author(s):  
G Stransky ◽  
S Gay
Keyword(s):  
Osmium Tetroxide ◽  
Decisive Role ◽  
Ultrastructural Localization ◽  
Immunogold Labeling ◽  
Gag Proteins ◽  
Lr White ◽  
Positive Immunostaining ◽  
Infected Cells ◽  
Lowicryl K4m

We developed a post-embedding immunogold labeling procedure for the ultrastructural localization of the HTLV-I gag proteins p19 and p24 by the use of monoclonal antibodies (MAb). Both antigens were shown to withstand fixation with 1% glutaraldehyde. In addition, p19 antigenicity was found not to be affected by post-fixation with 1% osmium tetroxide. The choice of resin played a decisive role in the retention of antigenicity. P19 was preserved in Lowicryl K4M as well as in LR White, whereas p24 was preserved only in Lowicryl. Both p19 and p24 were found to be localized on the HTLV-I virions themselves, whereas no positive immunostaining could be observed on the infected cells. In Lowicryl-embedded samples, in which both antigens had been preserved, a double immunogold labeling procedure was performed that allowed the co-localization of p19 and p24 on the same section. In osmicated LR White-embedded samples the quality of ultrastructural preservation of HTLV-I virions was found to be comparable to results obtained with the traditional glutaraldehyde-osmium tetroxide-epoxy resin processing.

scholarly journals Induction of Autophagy to Achieve a Human Immunodeficiency Virus Type 1 Cure

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1798
Author(s):  
Grant R. Campbell ◽  
Stephen A. Spector
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiretroviral Therapy ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Functional Cure ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Infected Cells ◽  
Hiv 1

Effective antiretroviral therapy has led to significant human immunodeficiency virus type 1 (HIV-1) suppression and improvement in immune function. However, the persistence of integrated proviral DNA in latently infected reservoir cells, which drive viral rebound post-interruption of antiretroviral therapy, remains the major roadblock to a cure. Therefore, the targeted elimination or permanent silencing of this latently infected reservoir is a major focus of HIV-1 research. The most studied approach in the development of a cure is the activation of HIV-1 expression to expose latently infected cells for immune clearance while inducing HIV-1 cytotoxicity—the “kick and kill” approach. However, the complex and highly heterogeneous nature of the latent reservoir, combined with the failure of clinical trials to reduce the reservoir size casts doubt on the feasibility of this approach. This concern that total elimination of HIV-1 from the body may not be possible has led to increased emphasis on a “functional cure” where the virus remains but is unable to reactivate which presents the challenge of permanently silencing transcription of HIV-1 for prolonged drug-free remission—a “block and lock” approach. In this review, we discuss the interaction of HIV-1 and autophagy, and the exploitation of autophagy to kill selectively HIV-1 latently infected cells as part of a cure strategy. The cure strategy proposed has the advantage of significantly decreasing the size of the HIV-1 reservoir that can contribute to a functional cure and when optimised has the potential to eradicate completely HIV-1.

scholarly journals PCIP-seq: simultaneous sequencing of integrated viral genomes and their insertion sites with long reads

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Maria Artesi ◽  
Vincent Hahaut ◽  
Basiel Cole ◽  
Laurens Lambrechts ◽  
Fereshteh Ashrafi ◽  
...  
Keyword(s):  
Viral Genome ◽  
Clonal Expansion ◽  
Endogenous Retroviruses ◽  
Viral Genomes ◽  
Short Read Sequencing ◽  
Long Reads ◽  
Infected Cells ◽  
Insertion Sites ◽  
Viral Insertion ◽  
Hiv 1

AbstractThe integration of a viral genome into the host genome has a major impact on the trajectory of the infected cell. Integration location and variation within the associated viral genome can influence both clonal expansion and persistence of infected cells. Methods based on short-read sequencing can identify viral insertion sites, but the sequence of the viral genomes within remains unobserved. We develop PCIP-seq, a method that leverages long reads to identify insertion sites and sequence their associated viral genome. We apply the technique to exogenous retroviruses HTLV-1, BLV, and HIV-1, endogenous retroviruses, and human papillomavirus.

scholarly journals Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

scholarly journals Highly Productive Infection with Pseudotyped Human Immunodeficiency Virus Type 1 (HIV-1) Indicates No Intracellular Restrictions to HIV-1 Replication in Primary Human Astrocytes

2001 ◽  
Vol 75 (17) ◽  
pp. 7925-7933 ◽  
Author(s):  
Mario Canki ◽  
Janice Ngee Foong Thai ◽  
Wei Chao ◽  
Anuja Ghorpade ◽  
Mary Jane Potash ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Productive Infection ◽  
Human Astrocytes ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Virus Expression ◽  
Hiv 1

ABSTRACT Human astrocytes can be infected with human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo, but, in contrast to T lymphocytes and macrophages, virus expression is inefficient. To investigate the HIV-1 life cycle in human fetal astrocytes, we infected cells with HIV-1 pseudotyped with envelope glycoproteins of either amphotropic murine leukemia virus or vesicular stomatitis virus. Infection by both pseudotypes was productive and long lasting and reached a peak of 68% infected cells and 1.7 μg of viral p24 per ml of culture supernatant 7 days after virus inoculation and then continued with gradually declining levels of virus expression through 7 weeks of follow-up. This contrasted with less than 0.1% HIV-1 antigen-positive cells and 400 pg of extracellular p24 per ml at the peak of astrocyte infection with native HIV-1. Cell viability and growth kinetics were similar in infected and control cells. Northern blot analysis revealed the presence of major HIV-1 RNA species of 9, 4, and 2 kb in astrocytes exposed to pseudotyped (but not wild-type) HIV-1 at 2, 14, and 28 days after infection. Consistent with productive infection, the 9- and 4-kb viral transcripts in astrocytes infected by pseudotyped HIV-1 were as abundant as the 2-kb mRNA during 4 weeks of follow-up, and both structural and regulatory viral proteins were detected in infected cells by immunoblotting or cell staining. The progeny virus released by these cells was infectious. These results indicate that the major barrier to HIV-1 infection of primary astrocytes is at virus entry and that astrocytes have no intrinsic intracellular restriction to efficient HIV-1 replication.

scholarly journals Exosomes from HIV-1-infected Cells Stimulate Production of Pro-inflammatory Cytokines through Trans-activating Response (TAR) RNA

2015 ◽  
Vol 291 (3) ◽  
pp. 1251-1266 ◽  
Author(s):  
Gavin C. Sampey ◽  
Mohammed Saifuddin ◽  
Angela Schwab ◽  
Robert Barclay ◽  
Shreya Punya ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Infected Cells ◽  
Tar Rna ◽  
Hiv 1 ◽  
Pro Inflammatory Cytokines
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