Limits...
Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment.

Suyama M, Daikoku E, Goto T, Sano K, Morikawa Y - Retrovirology (2009)

Bottom Line: Such studies would provide a better understanding of a reservoir for HIV.When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation.Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan. maris@lisci.kitasato-u.ac.jp

ABSTRACT

Background: It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV.

Results: We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-alpha stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1) and U1 cells (latently infected U937 cells with HIV-1) displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-alpha, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane.

Conclusion: Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute infection. Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.

Show MeSH

Related in: MedlinePlus

HIV particle production is accompanied by mRNA upregulation of CD44 and endocytic molecules. (A) Differential gene expression upon reactivation. J1.1 and U1 cells were either unstimulated or stimulated with 50 ng/ml TNF-α (gray columns). For comparison, uninfected Jurkat and U937 cells were similarly stimulated (white columns). Expression of each gene was quantified by qRT-PCR and normalized to the level of GAPDH. The fold increase of expression of each gene upon stimulation was shown. *, p < 0.01; **, p < 0.05. Expression levels of HIV-1 gag and tat mRNAs were quantified using specific primers (HIV-1 nucleotide positions 701–720 and 787–806 for gag and 5965–5987 and 8389–8411 for tat, respectively) (black columns). (B) Differential gene expression upon acute infection. Jurkat and U937 cells were infected with HIV-1 and subjected to qRT-PCR. The fold increase of each gene expression upon infection was shown (gray columns). *, p < 0.01; **, p < 0.05. (C) Protein expression of J1.1, U1, Jurkat, and U937 cells. TNF-α stimulation and infection were similarly performed. Cells were subjected to Western blotting using anti-CD44, anti-CD63, anti-HRS, anti-actin, anti-HIV-1 p17MA, and anti-p24CA antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2714482&req=5

Figure 2: HIV particle production is accompanied by mRNA upregulation of CD44 and endocytic molecules. (A) Differential gene expression upon reactivation. J1.1 and U1 cells were either unstimulated or stimulated with 50 ng/ml TNF-α (gray columns). For comparison, uninfected Jurkat and U937 cells were similarly stimulated (white columns). Expression of each gene was quantified by qRT-PCR and normalized to the level of GAPDH. The fold increase of expression of each gene upon stimulation was shown. *, p < 0.01; **, p < 0.05. Expression levels of HIV-1 gag and tat mRNAs were quantified using specific primers (HIV-1 nucleotide positions 701–720 and 787–806 for gag and 5965–5987 and 8389–8411 for tat, respectively) (black columns). (B) Differential gene expression upon acute infection. Jurkat and U937 cells were infected with HIV-1 and subjected to qRT-PCR. The fold increase of each gene expression upon infection was shown (gray columns). *, p < 0.01; **, p < 0.05. (C) Protein expression of J1.1, U1, Jurkat, and U937 cells. TNF-α stimulation and infection were similarly performed. Cells were subjected to Western blotting using anti-CD44, anti-CD63, anti-HRS, anti-actin, anti-HIV-1 p17MA, and anti-p24CA antibodies.

Mentions: Gene expression analysis based on cDNA microarrays has extensively been employed and has provided evidence for the modulation of host cellular gene expression upon HIV infection (replication and latency) [15-20]. Although numerous host genes are modulated upon HIV infection, it is conceivable that expression levels of host membrane components may change by feedback regulation upon HIV reactivation, as HIV requires host cell membrane for particle budding. A membrane contains a number of microdomains, enriched in cholesterol (i.e., rafts) and in tetraspanins (e.g., CD63 and CD81), which accumulate at sites of HIV budding [7,21-26]. It has been shown that TSG101, a component of endosomal sorting complex required for transport (ESCRT) is recruited to the sites of particle assembly and is responsible for HIV particle budding [27,28]. Thus we chose endosomal (EEA1, CD63, HRS, TSG101, and Syntaxin12) and PM (CD44 and SNAP23) markers and quantified their mRNA levels by qRT-PCR (Fig. 2A and 2B) using the primer sets shown in Additional File 1. Their properties and functions are as follows: EEA1 is a marker molecule for early endosome; HRS is an initial molecule for the ESCRT pathway; Syntaxin12 is a SNARE molecule for endosomal membrane fusion; CD44 is an adhesion molecule implicated in cell migration; SNAP23 is a SNARE molecule for PM fusion in the exocytic pathway. When the mRNA levels in J1.1 cells stimulated with TNF-α were compared with those in unstimulated J1.1 cells, CD44 gene expression was increased, but the other genes tested were largely unaltered. No significant upregulation of CD44 was observed when cells of its uninfected parental line, Jurkat, were similarly stimulated with TNF-α, indicating that the CD44 upregulation was not simply due to cell stimulation (Fig. 2A, upper). CD44 has been reported hardly expressed even at mRNA level in unstimulated Jurkat cells [29]. A similar analysis was carried out for U1 cells. Downregulation of CD44 has been reported for chronically infected monocytic cells [30]. Upon reactivation, CD44 upregulation was apparent but the endocytic molecules (CD63 and HRS) and SNAP23 were also upregulated in U1 cells. The modulation of others such as TSG101 was not statistically significant. These upregulations were not observed when uninfected U937 cells were stimulated (Fig. 2A, lower). The gene expression profiles upon reactivation were consistent with protein expression levels of the molecules when analyzed by Western blotting (Fig. 2C). We cannot simply compare the data of J1.1 and U1 cells, since expression levels of individual genes differ between the cell lines, but these significant upregulations were not observed in their parental but uninfected cells, suggesting that the upregulations might be linked with HIV expression. To test this possibility, we quantified expression levels of the same genes in acutely infected Jurkat and U937 cells and compared them with the levels in uninfected Jurkat and U937 cells. Upregulation of CD44 was observed in acutely infected Jurkat cells (Fig. 2B, upper), and this magnitude fold of upregulation was likely due to a very low level of CD44 expression in uninfected Jurkat cells [29]. In acutely infected U937 cells, besides CD44 upregulation, upregulation of other genes (CD63, HRS, and SNAP23) was observed (Fig. 2B, lower). Together, the results indicate that the upregulation of host molecules observed here was likely to be linked with HIV production. Higher levels of gag mRNA than tat mRNA observed in this study were possibly because we analyzed at a late stage of HIV replication. Western blotting confirmed HIV antigens, p55Gag precursor and its processing products, p24CA and p17MA, appeared upon reactivation or infection and showed that unlike anti-p24CA antibody, anti-p17MA antibody used in this study (against the C-terminal region of p17MA) recognized the mature p17MA domain but not the unprocessed p55Gag (Fig. 2C).


Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment.

Suyama M, Daikoku E, Goto T, Sano K, Morikawa Y - Retrovirology (2009)

HIV particle production is accompanied by mRNA upregulation of CD44 and endocytic molecules. (A) Differential gene expression upon reactivation. J1.1 and U1 cells were either unstimulated or stimulated with 50 ng/ml TNF-α (gray columns). For comparison, uninfected Jurkat and U937 cells were similarly stimulated (white columns). Expression of each gene was quantified by qRT-PCR and normalized to the level of GAPDH. The fold increase of expression of each gene upon stimulation was shown. *, p < 0.01; **, p < 0.05. Expression levels of HIV-1 gag and tat mRNAs were quantified using specific primers (HIV-1 nucleotide positions 701–720 and 787–806 for gag and 5965–5987 and 8389–8411 for tat, respectively) (black columns). (B) Differential gene expression upon acute infection. Jurkat and U937 cells were infected with HIV-1 and subjected to qRT-PCR. The fold increase of each gene expression upon infection was shown (gray columns). *, p < 0.01; **, p < 0.05. (C) Protein expression of J1.1, U1, Jurkat, and U937 cells. TNF-α stimulation and infection were similarly performed. Cells were subjected to Western blotting using anti-CD44, anti-CD63, anti-HRS, anti-actin, anti-HIV-1 p17MA, and anti-p24CA antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2714482&req=5

Figure 2: HIV particle production is accompanied by mRNA upregulation of CD44 and endocytic molecules. (A) Differential gene expression upon reactivation. J1.1 and U1 cells were either unstimulated or stimulated with 50 ng/ml TNF-α (gray columns). For comparison, uninfected Jurkat and U937 cells were similarly stimulated (white columns). Expression of each gene was quantified by qRT-PCR and normalized to the level of GAPDH. The fold increase of expression of each gene upon stimulation was shown. *, p < 0.01; **, p < 0.05. Expression levels of HIV-1 gag and tat mRNAs were quantified using specific primers (HIV-1 nucleotide positions 701–720 and 787–806 for gag and 5965–5987 and 8389–8411 for tat, respectively) (black columns). (B) Differential gene expression upon acute infection. Jurkat and U937 cells were infected with HIV-1 and subjected to qRT-PCR. The fold increase of each gene expression upon infection was shown (gray columns). *, p < 0.01; **, p < 0.05. (C) Protein expression of J1.1, U1, Jurkat, and U937 cells. TNF-α stimulation and infection were similarly performed. Cells were subjected to Western blotting using anti-CD44, anti-CD63, anti-HRS, anti-actin, anti-HIV-1 p17MA, and anti-p24CA antibodies.
Mentions: Gene expression analysis based on cDNA microarrays has extensively been employed and has provided evidence for the modulation of host cellular gene expression upon HIV infection (replication and latency) [15-20]. Although numerous host genes are modulated upon HIV infection, it is conceivable that expression levels of host membrane components may change by feedback regulation upon HIV reactivation, as HIV requires host cell membrane for particle budding. A membrane contains a number of microdomains, enriched in cholesterol (i.e., rafts) and in tetraspanins (e.g., CD63 and CD81), which accumulate at sites of HIV budding [7,21-26]. It has been shown that TSG101, a component of endosomal sorting complex required for transport (ESCRT) is recruited to the sites of particle assembly and is responsible for HIV particle budding [27,28]. Thus we chose endosomal (EEA1, CD63, HRS, TSG101, and Syntaxin12) and PM (CD44 and SNAP23) markers and quantified their mRNA levels by qRT-PCR (Fig. 2A and 2B) using the primer sets shown in Additional File 1. Their properties and functions are as follows: EEA1 is a marker molecule for early endosome; HRS is an initial molecule for the ESCRT pathway; Syntaxin12 is a SNARE molecule for endosomal membrane fusion; CD44 is an adhesion molecule implicated in cell migration; SNAP23 is a SNARE molecule for PM fusion in the exocytic pathway. When the mRNA levels in J1.1 cells stimulated with TNF-α were compared with those in unstimulated J1.1 cells, CD44 gene expression was increased, but the other genes tested were largely unaltered. No significant upregulation of CD44 was observed when cells of its uninfected parental line, Jurkat, were similarly stimulated with TNF-α, indicating that the CD44 upregulation was not simply due to cell stimulation (Fig. 2A, upper). CD44 has been reported hardly expressed even at mRNA level in unstimulated Jurkat cells [29]. A similar analysis was carried out for U1 cells. Downregulation of CD44 has been reported for chronically infected monocytic cells [30]. Upon reactivation, CD44 upregulation was apparent but the endocytic molecules (CD63 and HRS) and SNAP23 were also upregulated in U1 cells. The modulation of others such as TSG101 was not statistically significant. These upregulations were not observed when uninfected U937 cells were stimulated (Fig. 2A, lower). The gene expression profiles upon reactivation were consistent with protein expression levels of the molecules when analyzed by Western blotting (Fig. 2C). We cannot simply compare the data of J1.1 and U1 cells, since expression levels of individual genes differ between the cell lines, but these significant upregulations were not observed in their parental but uninfected cells, suggesting that the upregulations might be linked with HIV expression. To test this possibility, we quantified expression levels of the same genes in acutely infected Jurkat and U937 cells and compared them with the levels in uninfected Jurkat and U937 cells. Upregulation of CD44 was observed in acutely infected Jurkat cells (Fig. 2B, upper), and this magnitude fold of upregulation was likely due to a very low level of CD44 expression in uninfected Jurkat cells [29]. In acutely infected U937 cells, besides CD44 upregulation, upregulation of other genes (CD63, HRS, and SNAP23) was observed (Fig. 2B, lower). Together, the results indicate that the upregulation of host molecules observed here was likely to be linked with HIV production. Higher levels of gag mRNA than tat mRNA observed in this study were possibly because we analyzed at a late stage of HIV replication. Western blotting confirmed HIV antigens, p55Gag precursor and its processing products, p24CA and p17MA, appeared upon reactivation or infection and showed that unlike anti-p24CA antibody, anti-p17MA antibody used in this study (against the C-terminal region of p17MA) recognized the mature p17MA domain but not the unprocessed p55Gag (Fig. 2C).

Bottom Line: Such studies would provide a better understanding of a reservoir for HIV.When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation.Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan. maris@lisci.kitasato-u.ac.jp

ABSTRACT

Background: It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV.

Results: We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-alpha stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1) and U1 cells (latently infected U937 cells with HIV-1) displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-alpha, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane.

Conclusion: Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute infection. Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.

Show MeSH
Related in: MedlinePlus