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HIV-1 Tropism Determines Different Mutation Profiles in Proviral DNA.

Nascimento-Brito S, Paulo Zukurov J, Maricato JT, Volpini AC, Salim AC, Araújo FM, Coimbra RS, Oliveira GC, Antoneli F, Janini LM - PLoS ONE (2015)

Bottom Line: Here we investigated the effect of viral binding to either CCR5 or CXCR4 co-receptors on viral diversity after a single round of reverse transcription.In addition, we present pioneering results of a recently developed method for the analysis of SOLiD generated sequencing data applicable to the study of viral quasi-species.Additional investigations are needed to further clarify this question, which is relevant to viral diversification process and consequent disease progression.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia e Imunologia Veterinária, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Rio de Janeiro, Brazil; Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil.

ABSTRACT
In order to establish new infections HIV-1 particles need to attach to receptors expressed on the cellular surface. HIV-1 particles interact with a cell membrane receptor known as CD4 and subsequently with another cell membrane molecule known as a co-receptor. Two major different co-receptors have been identified: C-C chemokine Receptor type 5 (CCR5) and C-X-C chemokine Receptor type 4 (CXCR4) Previous reports have demonstrated cellular modifications upon HIV-1 binding to its co-receptors including gene expression modulations. Here we investigated the effect of viral binding to either CCR5 or CXCR4 co-receptors on viral diversity after a single round of reverse transcription. CCR5 and CXCR4 pseudotyped viruses were used to infect non-stimulated and stimulated PBMCs and purified CD4 positive cells. We adopted the SOLiD methodology to sequence virtually the entire proviral DNA from all experimental infections. Infections with CCR5 and CXCR4 pseudotyped virus resulted in different patterns of genetic diversification. CCR5 virus infections produced extensive proviral diversity while in CXCR4 infections a more localized substitution process was observed. In addition, we present pioneering results of a recently developed method for the analysis of SOLiD generated sequencing data applicable to the study of viral quasi-species. Our findings demonstrate the feasibility of viral quasi-species evaluation by NGS methodologies. We presented for the first time strong evidence for a host cell driving mechanism acting on the HIV-1 genetic variability under the control of co-receptor stimulation. Additional investigations are needed to further clarify this question, which is relevant to viral diversification process and consequent disease progression.

No MeSH data available.


Related in: MedlinePlus

Expression of Activation Markers using flow cytometry assays.Histograms showing the expression of CD25, CD69, CD38 and HLA-DR from gated CD3+CD4+ cell populations from PBMCs are shown in the upper row. Histograms indicating the expression of CD25, CD69 from gated CD3+CD4+ cell populations from purified T CD4+ cells appear in the lower row. X axis indicates the fluoresce intensity of a specific marker and Y axis indicates the percentage of cells in the population expressing this specific marker. Graphics under each histogram represent the MFI (Median of Fluorescence Intensity) of each activation marker in the gated CD3+CD4+ cell populations. Results are shown as mean values ± SD and are representative from three independent experiments. Statistical significance was assessed by the Two-tailed Student’s t-test yielding * when p < 0.05; and **, when p < 0.005. Gray area: unstained control cells, green/blue line: non-stimulated cells, red line: stimulated cells.
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pone.0139037.g002: Expression of Activation Markers using flow cytometry assays.Histograms showing the expression of CD25, CD69, CD38 and HLA-DR from gated CD3+CD4+ cell populations from PBMCs are shown in the upper row. Histograms indicating the expression of CD25, CD69 from gated CD3+CD4+ cell populations from purified T CD4+ cells appear in the lower row. X axis indicates the fluoresce intensity of a specific marker and Y axis indicates the percentage of cells in the population expressing this specific marker. Graphics under each histogram represent the MFI (Median of Fluorescence Intensity) of each activation marker in the gated CD3+CD4+ cell populations. Results are shown as mean values ± SD and are representative from three independent experiments. Statistical significance was assessed by the Two-tailed Student’s t-test yielding * when p < 0.05; and **, when p < 0.005. Gray area: unstained control cells, green/blue line: non-stimulated cells, red line: stimulated cells.

Mentions: A total of 1.2 x 108 viable PBMCs/mL were obtained from a single seronegative donor. Part of those cells was further used for selection of CD4 positive cells. In order to access the Δ32CCR5 deletion in those cells, an aliquot of PBMCs was used for PCR amplification of the CCR5 coding gene. Fig 1 shows the wild type homozygous state assuring those cells could be infected by R5 HIV-1 variants. Part of each group of cells was stimulated as described in the Methods section, and the stimulation of cells was indirectly assessed by flow cytometry assay (Figs 1 and 2). Resting state was suggested by the low density or absence of phenotypic lymphocyte activation markers at the surface of non-stimulated cells, allowing us to conclude that these cells were at the G0 phase (quiescent state) of the cell cycle [24]. On the other hand, stimulated cells showed an increase in lymphocyte activation markers. Differences in the expression of activation markers before and after stimulation were assessed by MFI (Median of Fluoresce Intensity). Values of MFI for all tested markers were significantly different (p values bellow 0.05) between unstimulated and stimulated cells. Pseudotyped HIV-1 viruses were harvested from the supernatant of transfected HeLa cells. A mean value of 1.3 x 107 viral particles/mL for pseudotyped viruses (X4 and R5) were obtained and infections of blood derived cells performed. After 72 hours of infection no evidence of cytopathic effects was observed for neither pseudotyped viruses. Also no virus was detected in the supernatant of all primary cultures after pelleting down infected cells (see Methods section).


HIV-1 Tropism Determines Different Mutation Profiles in Proviral DNA.

Nascimento-Brito S, Paulo Zukurov J, Maricato JT, Volpini AC, Salim AC, Araújo FM, Coimbra RS, Oliveira GC, Antoneli F, Janini LM - PLoS ONE (2015)

Expression of Activation Markers using flow cytometry assays.Histograms showing the expression of CD25, CD69, CD38 and HLA-DR from gated CD3+CD4+ cell populations from PBMCs are shown in the upper row. Histograms indicating the expression of CD25, CD69 from gated CD3+CD4+ cell populations from purified T CD4+ cells appear in the lower row. X axis indicates the fluoresce intensity of a specific marker and Y axis indicates the percentage of cells in the population expressing this specific marker. Graphics under each histogram represent the MFI (Median of Fluorescence Intensity) of each activation marker in the gated CD3+CD4+ cell populations. Results are shown as mean values ± SD and are representative from three independent experiments. Statistical significance was assessed by the Two-tailed Student’s t-test yielding * when p < 0.05; and **, when p < 0.005. Gray area: unstained control cells, green/blue line: non-stimulated cells, red line: stimulated cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4587555&req=5

pone.0139037.g002: Expression of Activation Markers using flow cytometry assays.Histograms showing the expression of CD25, CD69, CD38 and HLA-DR from gated CD3+CD4+ cell populations from PBMCs are shown in the upper row. Histograms indicating the expression of CD25, CD69 from gated CD3+CD4+ cell populations from purified T CD4+ cells appear in the lower row. X axis indicates the fluoresce intensity of a specific marker and Y axis indicates the percentage of cells in the population expressing this specific marker. Graphics under each histogram represent the MFI (Median of Fluorescence Intensity) of each activation marker in the gated CD3+CD4+ cell populations. Results are shown as mean values ± SD and are representative from three independent experiments. Statistical significance was assessed by the Two-tailed Student’s t-test yielding * when p < 0.05; and **, when p < 0.005. Gray area: unstained control cells, green/blue line: non-stimulated cells, red line: stimulated cells.
Mentions: A total of 1.2 x 108 viable PBMCs/mL were obtained from a single seronegative donor. Part of those cells was further used for selection of CD4 positive cells. In order to access the Δ32CCR5 deletion in those cells, an aliquot of PBMCs was used for PCR amplification of the CCR5 coding gene. Fig 1 shows the wild type homozygous state assuring those cells could be infected by R5 HIV-1 variants. Part of each group of cells was stimulated as described in the Methods section, and the stimulation of cells was indirectly assessed by flow cytometry assay (Figs 1 and 2). Resting state was suggested by the low density or absence of phenotypic lymphocyte activation markers at the surface of non-stimulated cells, allowing us to conclude that these cells were at the G0 phase (quiescent state) of the cell cycle [24]. On the other hand, stimulated cells showed an increase in lymphocyte activation markers. Differences in the expression of activation markers before and after stimulation were assessed by MFI (Median of Fluoresce Intensity). Values of MFI for all tested markers were significantly different (p values bellow 0.05) between unstimulated and stimulated cells. Pseudotyped HIV-1 viruses were harvested from the supernatant of transfected HeLa cells. A mean value of 1.3 x 107 viral particles/mL for pseudotyped viruses (X4 and R5) were obtained and infections of blood derived cells performed. After 72 hours of infection no evidence of cytopathic effects was observed for neither pseudotyped viruses. Also no virus was detected in the supernatant of all primary cultures after pelleting down infected cells (see Methods section).

Bottom Line: Here we investigated the effect of viral binding to either CCR5 or CXCR4 co-receptors on viral diversity after a single round of reverse transcription.In addition, we present pioneering results of a recently developed method for the analysis of SOLiD generated sequencing data applicable to the study of viral quasi-species.Additional investigations are needed to further clarify this question, which is relevant to viral diversification process and consequent disease progression.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia e Imunologia Veterinária, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Rio de Janeiro, Brazil; Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil.

ABSTRACT
In order to establish new infections HIV-1 particles need to attach to receptors expressed on the cellular surface. HIV-1 particles interact with a cell membrane receptor known as CD4 and subsequently with another cell membrane molecule known as a co-receptor. Two major different co-receptors have been identified: C-C chemokine Receptor type 5 (CCR5) and C-X-C chemokine Receptor type 4 (CXCR4) Previous reports have demonstrated cellular modifications upon HIV-1 binding to its co-receptors including gene expression modulations. Here we investigated the effect of viral binding to either CCR5 or CXCR4 co-receptors on viral diversity after a single round of reverse transcription. CCR5 and CXCR4 pseudotyped viruses were used to infect non-stimulated and stimulated PBMCs and purified CD4 positive cells. We adopted the SOLiD methodology to sequence virtually the entire proviral DNA from all experimental infections. Infections with CCR5 and CXCR4 pseudotyped virus resulted in different patterns of genetic diversification. CCR5 virus infections produced extensive proviral diversity while in CXCR4 infections a more localized substitution process was observed. In addition, we present pioneering results of a recently developed method for the analysis of SOLiD generated sequencing data applicable to the study of viral quasi-species. Our findings demonstrate the feasibility of viral quasi-species evaluation by NGS methodologies. We presented for the first time strong evidence for a host cell driving mechanism acting on the HIV-1 genetic variability under the control of co-receptor stimulation. Additional investigations are needed to further clarify this question, which is relevant to viral diversification process and consequent disease progression.

No MeSH data available.


Related in: MedlinePlus