2OHpβCD treatment impairs XMRV release from LNCaP cells.

<p>LNCaP cells chronically infected with XMRV were treated with increasing concentrations of 2OHpβCD for 1 hr at 37°C, after which cholesterol free medium was replaced and the released virus was collected 5 hrs later. (<b>A</b>) The cell viability with different concentration of 2OHpβCD treatment was determined by 7AAD staining followed by flow cytometry, the quantified data are normalized to untreated cells, (arbitrarily set as 100%). (<b>B</b>) The released virus and (<b>C</b>) cell lysates were subject to the Western blot with anti-p30 Gag antibody. (<b>D</b>) After treatment of with 2OHpβCD, the LNCaP-XMRV cells were washed and resuspended in RPMI containing 1 mM 2OHpβCD pre-complexed with cholesterol (48 µg/ml) and incubated for 2 hrs, after which cholesterol free medium was replaced. After a 5 hrs incubation the released virus was collected and subjected to the Western blot analysis with anti-p30 Gag antibody. Data are representative of five independent experiments. The band intensities of (B), (C) and (D) were shown in the bottom of each blot (quantified and normalized to untreated infected cells, arbitrarily set as 1). <b>E</b>, The virus released from 2OHpβCD treated LNCaP-XMRV was normalized by RT and used to infect naïve LNCaP cells; the viral infectivity was determined by viral protein expression in the infected cells with goat antiserum to MLV p30 Gag.</p

Cholesterol Depletion Inactivates XMRV and Leads to Viral Envelope Protein Release from Virions: Evidence for Role of Cholesterol in XMRV Infection

<div><p>Membrane cholesterol plays an important role in replication of HIV-1 and other retroviruses. Here, we report that the gammaretrovirus XMRV requires cholesterol and lipid rafts for infection and replication. We demonstrate that treatment of XMRV with a low concentration (10 mM) of 2-hydroxypropyl-β-cyclodextrin (2OHpβCD) partially depleted virion-associated cholesterol resulting in complete inactivation of the virus. This effect could not be reversed by adding cholesterol back to treated virions. Further analysis revealed that following cholesterol depletion, virus-associated Env protein was significantly reduced while the virions remained intact and retained core proteins. Increasing concentrations of 2OHpβCD (≥20 mM) resulted in loss of the majority of virion-associated cholesterol, causing disruption of membrane integrity and loss of internal Gag proteins and viral RNA. Depletion of cholesterol from XMRV-infected cells significantly reduced virus release, suggesting that cholesterol and intact lipid rafts are required for the budding process of XMRV. These results suggest that unlike glycoproteins of other retroviruses, the association of XMRV glycoprotein with virions is highly dependent on cholesterol and lipid rafts.</p> </div

High concentrations of 2OHpβCD treatment results in loss of viral RNA and other structural proteins.

<p>(<b>A</b>) The same 2OHpβCD-treated XMRV as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048013#pone-0048013-g004" target="_blank">Figure 4</a> was probed with goat antiserum to MLV. Data is representative of five independent experiments. (<b>B</b>) 2OHpβCD-treated XMRV was analyzed for particle-associated viral RNA by real-time RT-PCR. Error bars reflect standard derivation of three independent experiments. *, <i>P</i><0.05, **, <i>P</i><0.001, compared to the virions without 2OHpβCD treatment, #, below the threshold of detection.</p

Depletion of cholesterol abolishes XMRV infectivity.

<p>Purified virus was incubated with indicated concentrations of 2OHpβCD in RPMI 1640 medium for 1 hr at 37°C and the treated virus was pelleted. (<b>A</b>) The residual cholesterol in 2OHpβCD-treated virions was measured by Amplex Red assay. Error bars reflect standard derivation of three independent experiments. **, <i>P</i><0.001, compared to the virions without 2OHpβCD treatment. (<b>B</b>) The infectivity of 2OHpβCD-treated XMRV was tested in LNCaP cells. Comparable amounts of treated virus used to infect LNCaP cells were confirmed by immunoblot with an anti-MLV goat serum. (<b>C</b>) Cell lysates and virus-containing supernatants of infected LNCaP cells were harvested at 48 hrs of culture after exposing cells to virus and subjected to western blot analysis. Top panel: cell lysate from infected cells was analyzed with goat antiserum to MLV p30 Gag or β tubulin; bottom panel: released virus in the supernatant was analyzed with goat antiserum to MLV p30 Gag. Mock: cells exposed to medium alone without virus. Data are representative of five independent experiments (<b>D</b>) LNCaP cells were infected with 12, 6, 3 and 1 µg (total protein) of 2OHpβCD-treated XMRV as indicated. The released virus in the cell supernatants was harvested at 48 hrs post-infection and quantified by qRT-PCR. (<b>E</b>) LNCaP cells were infected with 12 µg (total protein) of 2OHpβCD-treated XMRV. After 48 hrs, proviral DNA from the infected cells was quantified by Q-PCR. Quantified data are normalized to cells infected untreated control virus (arbitrarily set as 1). Error bars reflect standard derivation of three independent experiments (D, E).</p

Pseudotyped HIV-1 infects primary endocervical epithelial cells.

<p>(<b>A, B</b>) Visualization of HIV-1 infection in primary endocervical epithelial cells by dual immunofluorescence staining with FITC-anti-HIV-1 Gag and anti-CK19 MAbs. Primary endocervical epithelial cells were exposed to progeny viruses from infected CEMX174 cells and immunofluorescence staining was performed 5 days post-infection. Epithelial cells exposed to virus in presence of AZT or anti-MLV polyclonal sera diluted 1∶300 are shown as indicated (<b>B</b>, left two panels). HIV-1 Gag fluorescence is shown as green and CK19 as red. The corresponding bright field images are shown on the right (<b>A</b>) or at the bottom (<b>B</b>). HIV-1 Gag staining merged with CK19 staining and enlarged views of HIV-1 infected cells are shown in (A). <b>C and D:</b> XMRV RNA (<b>C</b>) and HIV-1 RNA (<b>D</b>) in supernatants from the same infected primary endocervical epithelial cells from (<b>A</b>) and (B) above were quantified by qRT-PCR. The input virus used to infect producer CEMX174 cells and the treatments are indicated on the X-axis of the graphs (pAb 1∶300 = anti-MLV polyclonal sera at dilution 1∶300; Control serum = normal goat serum diluted 1∶300; No serum = culture medium control). **, p<0.001, control serum vs indicated treatments. The data shown represent the mean ± standard deviation from three independent experiments. <b>^a</b>, HIV-1 and XMRV from CEMX174 cells infected with each virus alone were quantified and mixed at a ratio equal to the ratio of the two viruses in the progeny virus from HIV-1 and XMRV co-infected CEMX174 cells. The virus mixture was then inoculated onto primary endocervical epithelial cells and immunostaining and qPCR was performed exactly as described for progeny virus from co-infected CEMx174 cells.</p

Pseudotyped HIV-1 infection of primary endocervical cells via cell-associated virus.

<p>Primary endocervical epithelial cells were co-cultured for two days with an equal number of mitomycin C-treated CEMX174 cells infected with HIV-1 alone, XMRV alone or co-infected with both viruses. After washing to remove non-adherent cells, dual immunofluorescence staining with FITC-anti-Gag and anti-CK19 MAbs was performed on the adherent primary endocervical epithelial cells on day 5. Merged images confirmed that HIV-1 infected cells were epithelial cells. Primary endocervical epithelial cells were co-cultured with (A) HIV-1/XMRV co-infected CEMX174; (B) same as (A) in presence of AZT; (C) HIV-1 infected CEMx174; (D) XMRV infected CEMx174; (E) mock infected CEMx174.</p

Pseudotyped HIV-1 infects epithelial cells derived from vagina.

<p>Immunofluorescence analysis of HIV-1 infection in VK-2. VK-2 cells were infected with progeny virus from primary CD4+ T cells or CEMX174 and were stained for HIV-1 Gag expression 5 days post-infection. The input virus used to infect producer T cells are as indicated. An enlarged view of epithelial cells exposed to progeny virus from HIV-1/XMRV co-infected CEMX174 cells is shown (second panel). HIV-1 Gag expression is indicated by green fluorescence (FITC); green fluorescence merged to the corresponding bright field image is shown in the bottom panels. B. Target epithelial cells were exposed to progeny viruses in the presence or absence of AZT as indicated on the panels. Data shown are representative of six independent experiments. Bar = 10 µm. <b>C</b> and <b>D</b>, HIV-1 and XMRV viral RNA in the supernatants of the same infected VK-2 cells as in (A) and (B) were quantified by qRT-PCR. The viruses used to infect producer cells are shown on the X axis. The data shown represent the mean ± standard deviation from three independent experiments.</p

LNcap epithelial cell line is susceptible to infection by HIV-1 co-produced with XMRV.

<p><b>A.</b> Visualization of HIV-1 infection in epithelial cells using immunofluorescence staining with anti-HIV Gag monoclonal antibody. LNcap cells were exposed to culture supernatants from CEMX174 cells infected with indicated viruses. Cells were fixed and stained for HIV-1 Gag expression 5 days after exposure to the viral supernatants. Green fluorescence (FITC) indicates HIV-1 Gag expression. Green fluorescence merged with corresponding bright field images are shown in the bottom panels. An enlarged view of cells exposed to HIV-1 co-produced with XMRV is shown in the second panel from the left. <b>B.</b> AZT inhibits HIV-1 infection of epithelial cells. LNcap cells and progeny viruses from HIV-1/XMRV co-infected CEMX174 cells were pretreated with AZT or with medium alone. The virus preparations were then added to the cells and the input virus was removed after 24 hrs. The cells were then cultured for an additional four days in medium alone or medium containing AZT as outlined in Materials and Methods. The cells were then fixed and stained for HIV-1 Gag expression. Cells infected in the absence of AZT are shown on the left. The corresponding bright field image is shown in the bottom panels. Bar = 10 µm. Data are representative of 5 independent experiments. <b>C.</b> HIV-1 RNA in the supernatants of the same LNcap cells from (<b>B.</b>) infected in the presence or absence of AZT was quantified by qRT-PCR. <b>D.</b> XMRV RNA in the supernatants of the infected LNcap cells was quantified by qPCR. The input viruses were supernatants from CEMx174 cells infected as indicated. For (C) and (D) the data shown represent the mean ± standard deviation from five independent experiments. **p<0.001, infection in the absence vs presence of AZT.</p

Inhibition of HIV-1 infection of epithelial cells by neutralizing anti-MLV polyclonal antiserum.

<p><b>A.</b> Anti-MLV polyclonal antibody neutralizes XMRV infection in a dose-dependent manner. Progeny viruses from CEMX174 cells infected with HIV-1 and/or XMRV were pre-incubated with the indicated dilutions of anti-MLV polyclonal serum at 37°C for 1 hr before addition to LNcap epithelial cells. XMRV released into the supernatants of infected LNcap cells was quantified by qRT-PCR. pAb = Anti-MLV polyclonal antibody; Con. Serum = normal goat serum diluted 1∶100. The input viruses used to infect producer CEMx174 cells are indicated at the bottom. The data shown represent the mean ± standard deviation from three independent experiments. <b>B.</b> Immunofluorescence analysis of HIV-1 infection in LNcap cells in the presence or absence of anti-MLV neutralizing antibody. The same LNcap cells infected with progeny viruses from HIV-1/XMRV co-infected cells (shown in A) in the presence of indicated dilutions of anti-MLV neutralizing pAb or control serum were stained for HIV-1 Gag (green). The corresponding bright field image for each panel is shown on the right. Bar = 10 µm. Images are representative of three independent experiments. <b>C.</b> HIV-1 viral RNA in supernatants of from the same infected LNcap cells in (B) was quantified by qRT-PCR. The input virus used to infect producer cells and the dilutions of neutralizing or control sera are indicated. The data shown represent the mean ± standard deviation from three independent experiments. *, p<0.05; **, p<0.001, control serum vs anti-MLV pAb.</p

Pseudotyped HIV-1 infects primary vaginal squamous epithelial cells.

<p>(<b>A, B</b>) Dual immunofluorescence staining with FITC-anti-HIV-1 Gag and anti-CK19 MAbs was performed on primary vaginal squamous epithelial cells which were exposed to progeny virus from infected CEMX174 cells. HIV-1 Gag is shown as green and CK19 is shown as red. Corresponding bright field images are also shown as indicated. The input viruses used to infect producer CEMX174 cells is indicated on the panels. Epithelial cells exposed to progeny virus in the presence of AZT or anti-MLV polyclonal sera diluted 1∶300 are shown in the left two columns in <b>B</b>.</p