scholarly journals Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release

Retrovirology ◽  
2020 ◽  
Vol 17 (1) ◽  
Author(s):  
James Kirui ◽  
Eric O. Freed
Keyword(s):  
Virus Release ◽  
Reporter Vector ◽  
Highly Sensitive ◽  
Hiv 1

Two-site enzyme immunoassay of CD4 and CD8 molecules on the surface of T lymphocytes from healthy subjects and HIV-1-infected patients

1994 ◽  
Vol 40 (1) ◽  
pp. 30-37 ◽  
Author(s):  
D Carriere ◽  
C Fontaine ◽  
A M Berthier ◽  
A M Rouquette ◽  
P Carayon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Lymphocytes ◽  
Enzyme Immunoassay ◽  
Cd8 T Cells ◽  
Envelope Glycoprotein ◽  
Healthy Subjects ◽  
Gp 120 ◽  
Highly Sensitive ◽  
One Step ◽  
Hiv 1

Abstract A highly sensitive two-site enzyme immunoassay (Capcellia) was developed to determine the concentration of CD4 and CD8 molecules expressed on the surface of human T lymphocytes. This assay, performed in one step (20 min), involves the specific immunocapture of T lymphocytes and reaction of the CD4 or CD8 molecules with an enzyme-labeled monoclonal antibody (mAb). The results were expressed as molar concentrations of the T-cell markers on the basis of results obtained with calibrated CD4 and CD8 standards. The assay was sensitive enough to detect 0.4 pmol/L CD4 or 0.8 pmol/L CD8, which corresponded to approximately 20 x 10(6) CD4+ or CD8+ T cells per liter of blood. Mean concentrations in healthy adults were 17.2 pmol/L for CD4 and 22.1 pmol/L for CD8. The CD4 concentration was < 8 pmol/L in 50% of HIV-1-infected patients and in 95% of AIDS patients. Given the epitopic specificity of the mAb to CD4 we used, these values correspond to the concentration of CD4 molecules free of envelope glycoprotein (gp)120.

scholarly journals A highly sensitive and dynamic immunofluorescent cytometric bead assay for the detection of HIV-1 p24

2009 ◽  
Vol 157 (1) ◽  
pp. 98-101 ◽  
Author(s):  
Angélique Biancotto ◽  
Beda Brichacek ◽  
Silvia S. Chen ◽  
Wendy Fitzgerald ◽  
Andrea Lisco ◽  
...  
Keyword(s):  
Highly Sensitive ◽  
Hiv 1

How Many Bloods will a ‘HIVCHEK’ Check? Multiple Tests for HIV Antibody for a Single Screening Kit

1992 ◽  
Vol 22 (4) ◽  
pp. 151-154 ◽  
Author(s):  
G J Wannan ◽  
M J Cranefield ◽  
W A M Cutting ◽  
P R Fischer ◽  
F D Hargreaves ◽  
...  
Keyword(s):  
Blood Donors ◽  
Screening Tests ◽  
Serum Samples ◽  
Hiv Seroprevalence ◽  
Multiple Tests ◽  
Test Kit ◽  
Highly Sensitive ◽  
Multiple Sample ◽  
Sample Method ◽  
Hiv 1

Detection of HIV infection in blood donors or populations is usually by testing sera for antibodies to HIV-1 and HIV-2. Screening tests are now highly sensitive and specific, but still expensive and scarce in Africa. We tested the commercially available kits ‘HIVCHEK 1 + 2′ in two field laboratories, on specimens from blood donors and antenatal women in rural Zaire. We describe a method of using one test kit for up to five serum samples, saving money and time. In 491 antenatal mothers in Eastern Zaire, among whom the HIV seroprevalence was 3.3%, we compared ‘HIVCHEK’ results with results obtained by ELISA and Western blot. The ‘HIVCHEK’ multiple-sample method had a sensitivity of 82% and a specificity of 99.6%. In an area with an HIV seroprevalence of <4%, using ‘HIVCHEK’ by the multiple sample method would lead to a saving of about £2400 for every 1000 individuals tested.

scholarly journals Molecular Characterization of Feline Immunodeficiency Virus Budding

2007 ◽  
Vol 82 (5) ◽  
pp. 2106-2119 ◽  
Author(s):  
Benjamin G. Luttge ◽  
Miranda Shehu-Xhilaga ◽  
Dimiter G. Demirov ◽  
Catherine S. Adamson ◽  
Ferri Soheilian ◽  
...  
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Equine Infectious Anemia Virus ◽  
Binding Motif ◽  
Virus Release ◽  
Related Sequence ◽  
Endosomal Sorting ◽  
Peptide Motifs ◽  
C Terminus ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Infection of domestic cats with feline immunodeficiency virus (FIV) is an important model system for studying human immunodeficiency virus type 1 (HIV-1) infection due to numerous similarities in pathogenesis induced by these two lentiviruses. However, many molecular aspects of FIV replication remain poorly understood. It is well established that retroviruses use short peptide motifs in Gag, known as late domains, to usurp cellular endosomal sorting machinery and promote virus release from infected cells. For example, the Pro-Thr/Ser-Ala-Pro [P(T/S)AP] motif of HIV-1 Gag interacts directly with Tsg101, a component of the endosomal sorting complex required for transport I (ESCRT-I). A Tyr-Pro-Asp-Leu (YPDL) motif in equine infectious anemia virus (EIAV), and a related sequence in HIV-1, bind the endosomal sorting factor Alix. In this study we sought to identify and characterize FIV late domain(s) and elucidate cellular machinery involved in FIV release. We determined that mutagenesis of a PSAP motif in FIV Gag, small interfering RNA-mediated knockdown of Tsg101 expression, and overexpression of a P(T/S)AP-binding fragment of Tsg101 (TSG-5′) each inhibited FIV release. We also observed direct binding of FIV Gag peptides to Tsg101. In contrast, mutagenesis of a potential Alix-binding motif in FIV Gag did not affect FIV release. Similarly, expression of the HIV-1/EIAV Gag-binding domain of Alix (Alix-V) did not disrupt FIV budding, and FIV Gag peptides showed no affinity for Alix-V. Our data demonstrate that FIV relies predominantly on a Tsg101-binding PSAP motif in the C terminus of Gag to promote virus release in HeLa cells, and this budding mechanism is highly conserved in feline cells.

scholarly journals Reactivation Kinetics of HIV-1 and Susceptibility of Reactivated Latently Infected CD4+T Cells to HIV-1-Specific CD8+T Cells

2015 ◽  
Vol 89 (18) ◽  
pp. 9631-9638 ◽  
Author(s):  
Victoria E. K. Walker-Sperling ◽  
Valerie J. Cohen ◽  
Patrick M. Tarwater ◽  
Joel N. Blankson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antiretroviral Therapy ◽  
Time Frame ◽  
Virus Release ◽  
Latently Infected ◽  
Shock And Kill ◽  
Infected Cells ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACTThe “shock and kill” model of human immunodeficiency virus type 1 (HIV-1) eradication involves the induction of transcription of HIV-1 genes in latently infected CD4+T cells, followed by the elimination of these infected CD4+T cells by CD8+T cells or other effector cells. CD8+T cells may also be needed to control the spread of new infection if residual infected cells are present at the time combination antiretroviral therapy (cART) is discontinued. In order to determine the time frame needed for CD8+T cells to effectively prevent the spread of HIV-1 infection, we examined the kinetics of HIV transcription and virus release in latently infected cells reactivatedex vivo. Isolated resting, primary CD4+T cells from HIV-positive (HIV+) subjects on suppressive regimens were found to upregulate cell-associated HIV-1 mRNA within 1 h of stimulation and produce extracellular virus as early as 6 h poststimulation. In spite of the rapid kinetics of virus production, we show that CD8+T cells from 2 out of 4 viremic controllers were capable of effectively eliminating reactivated autologous CD4+cells that upregulate cell-associated HIV-1 mRNA. The results have implications for devising strategies to prevent rebound viremia due to reactivation of rare latently infected cells that persist after potentially curative therapy.IMPORTANCEA prominent HIV-1 cure strategy termed “shock and kill” involves the induction of HIV-1 transcription in latently infected CD4+T cells with the goal of elimination of these cells by either the cytotoxic T lymphocyte response or other immune cell subsets. However, the cytotoxic T cell response may also be required after curative treatment if residual latently infected cells remain. The kinetics of HIV-1 reactivation indicate rapid upregulation of cell-associated HIV-1 mRNA and a 5-h window between transcription and virus release. Thus, HIV-specific CD8+T cell responses likely have a very short time frame to eliminate residual latently infected CD4+T cells that become reactivated after discontinuation of antiretroviral therapy following potentially curative treatment strategies.

scholarly journals Antagonism of BST-2/Tetherin Is a Conserved Function of the Env Glycoprotein of Primary HIV-2 Isolates

2016 ◽  
Vol 90 (24) ◽  
pp. 11062-11074 ◽  
Author(s):  
Chia-Yen Chen ◽  
Masashi Shingai ◽  
Sarah Welbourn ◽  
Malcolm A. Martin ◽  
Pedro Borrego ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Change ◽  
Mixed Population ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Virus Release ◽  
Host Restriction ◽  
Single Amino Acid Change ◽  
Hiv 1 ◽  
Env Glycoprotein

ABSTRACTAlthough HIV-2 does not encode avpugene, the ability to antagonize bone marrow stromal antigen 2 (BST-2) is conserved in some HIV-2 isolates, where it is controlled by the Env glycoprotein. We previously reported that a single-amino-acid difference between the laboratory-adapted ROD10 and ROD14 Envs controlled the enhancement of virus release (referred to here as Vpu-like) activity. Here, we investigated how conserved the Vpu-like activity is in primary HIV-2 isolates. We found that half of the 34 tested primary HIV-2 Env isolates obtained from 7 different patients enhanced virus release. Interestingly, most HIV-2 patients harbored a mixed population of viruses containing or lacking Vpu-like activity. Vpu-like activity and Envelope functionality varied significantly among Env isolates; however, there was no direct correlation between these two functions, suggesting they evolved independently. In comparing the Env sequences from one HIV-2 patient, we found that similar to the ROD10/ROD14 Envs, a single-amino-acid change (T568I) in the ectodomain of the TM subunit was sufficient to confer Vpu-like activity to an inactive Env variant. Surprisingly, however, absence of Vpu-like activity was not correlated with absence of BST-2 interaction. Taken together, our data suggest that maintaining the ability to antagonize BST-2 is of functional relevance not only to HIV-1 but also to HIV-2 as well. Our data show that as with Vpu, binding of HIV-2 Env to BST-2 is important but not sufficient for antagonism. Finally, as observed previously, the Vpu-like activity in HIV-2 Env can be controlled by single-residue changes in the TM subunit.IMPORTANCELentiviruses such as HIV-1 and HIV-2 encode accessory proteins whose function is to overcome host restriction mechanisms. Vpu is a well-studied HIV-1 accessory protein that enhances virus release by antagonizing the host restriction factor BST-2. HIV-2 does not encode avpugene. Instead, the HIV-2 Env glycoprotein was found to antagonize BST-2 in some isolates. Here, we cloned multiple Env sequences from 7 HIV-2-infected patients and found that about half were able to antagonize BST-2. Importantly, most HIV-2 patients harbored a mixed population of viruses containing or lacking the ability to antagonize BST-2. In fact, in comparing Env sequences from one patient combined with site-directed mutagenesis, we were able to restore BST-2 antagonism to an inactive Env protein by a single-amino-acid change. Our data suggest that targeting BST-2 by HIV-2 Env is a dynamic process that can be regulated by simple changes in the Env sequence.

scholarly journals Elucidation of the Molecular Consequences of Two Unique p6Gag Mutations Derived from HIV-1 CRF07_BC-infected Patients

2020 ◽  
Author(s):  
Zetao Cheng ◽  
Sherimay D. Ablan ◽  
Eric O. Freed ◽  
Haiying Wang ◽  
Shixing Tang
Keyword(s):  
Amino Acid ◽  
Disease Progression ◽  
Binding Domain ◽  
Host Protein ◽  
Virus Infectivity ◽  
Virus Release ◽  
Amino Acid Deletion ◽  
Gag Protein ◽  
Subtype B ◽  
Hiv 1

Abstract Background We previously observed that individuals infected with HIV-1 CRF07_BC showed slower disease progression than those infected with HIV-1 subtype B or CRF01_AE. CRF07_BC viruses carry two unique mutations in the p6 Gag protein: insertion of PTAPPE sequences downstream of the original Tsg101 binding domain, and deletion of a seven-amino-acid sequence ( 30 PIDKELY 36 ) that partially overlaps with the Alix binding domain. To further define the role of these mutations in virus release and replication, we introduced them into the HIV-1 proviral clone pNL4-3 for functional characterization. Results We found that the seven-amino-acid deletion, but not the PTAPPE insertion, significantly decreased virus release, Gag processing, and virus infectivity. The seven-amino-acid deletion also resulted in a virus replication defect in both T-cell lines and peripheral blood mononuclear cells. We found that these defects were caused by the seven-amino-acid deletion in p6 Gag , especially deletion of Tyr-36 of p6 Gag , not the deletion of the overlapping p6* sequence in the HIV-1 GagPol protein. The p6 Gag deletion mutant was resistant to a dominant-negative Alix fragment, suggesting a loss of binding between p6 Gag and Alix. Conclusions Our results indicate that the patient-derived seven-amino-acid deletion in p6 Gag of HIV-1 CRF07_BC virus affects virus release, infectivity and replication capacity by disrupting the interaction between HIV-1 p6 Gag and host protein Alix. These results may explain the slower disease progression observed in the subjects infected with HIV-1 CRF07_BC bearing this unique mutation.

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