Incorporation of podoplanin into HIV released from HEK-293T cells, but not PBMC, is required for efficient binding to the attachment factor CLEC-2

Abstract Background Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. Results Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. Conclusions Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that podoplanin expression is connected to apoptosis, a finding that deserves further investigation.</p

Proteolytic Activation of the 1918 Influenza Virus Hemagglutinin▿

Proteolytic activation of the hemagglutinin (HA) protein is indispensable for influenza virus infectivity, and the tissue expression of the responsible cellular proteases impacts viral tropism and pathogenicity. The HA protein critically contributes to the exceptionally high pathogenicity of the 1918 influenza virus, but the mechanisms underlying cleavage activation of the 1918 HA have not been characterized. The neuraminidase (NA) protein of the 1918 influenza virus allows trypsin-independent growth in canine kidney cells (MDCK). However, it is at present unknown if the 1918 NA, like the NA of the closely related strain A/WSN/33, facilitates HA cleavage activation by recruiting the proprotease plasminogen. Moreover, it is not known which pulmonary proteases activate the 1918 HA. We provide evidence that NA-dependent, trypsin-independent cleavage activation of the 1918 HA is cell line dependent and most likely plasminogen independent since the 1918 NA failed to recruit plasminogen and neither exogenous plasminogen nor the presence of the A/WSN/33 NA promoted efficient cleavage of the 1918 HA. The transmembrane serine protease TMPRSS4 was found to be expressed in lung tissue and was shown to cleave the 1918 HA. Accordingly, coexpression of the 1918 HA with TMPRSS4 or the previously identified HA-processing protease TMPRSS2 allowed trypsin-independent infection by pseuodotypes bearing the 1918 HA, indicating that these proteases might support 1918 influenza virus spread in the lung. In summary, we show that the previously reported 1918 NA-dependent spread of the 1918 influenza virus is a cell line-dependent phenomenon and is not due to plasminogen recruitment by the 1918 NA. Moreover, we provide evidence that TMPRSS2 and TMPRSS4 activate the 1918 HA by cleavage and therefore may promote viral spread in lung tissue

Rectal Application of a Highly Osmolar Personal Lubricant in a Macaque Model Induces Acute Cytotoxicity but Does Not Increase Risk of SHIV Infection

<div><p>Background</p><p>Personal lubricant use is common during anal intercourse. Some water-based products with high osmolality and low pH can damage genital and rectal tissues, and the polymer polyquaternium 15 (PQ15) can enhance HIV replication <i>in vitro</i>. This has raised concerns that lubricants with such properties may increase STD/HIV infection risk, although <i>in vivo</i> evidence is scarce. We use a macaque model to evaluate rectal cytotoxicity and SHIV infection risk after use of a highly osmolar (>8,000 mOsm/kg) water-based lubricant with pH of 4.4, and containing PQ15.</p><p>Methods</p><p>Cytotoxicity was documented by measuring inflammatory cytokines and epithelial tissue sloughing during six weeks of repeated, non-traumatic lubricant or control buffer applications to rectum and anus. We measured susceptibility to SHIV_SF162P3 infection by comparing virus doses needed for rectal infection in twenty-one macaques treated with lubricant or control buffer 30 minutes prior to virus exposure.</p><p>Results</p><p>Lubricant increased pro-inflammatory cytokines and tissue sloughing while control buffer (phosphate buffered saline; PBS) did not. However, the estimated AID₅₀ (50% animal infectious dose) was not different in lubricant- and control buffer-treated macaques (p = 0.4467; logistic regression models).</p><p>Conclusions</p><p>Although the test lubricant caused acute cytotoxicity in rectal tissues, it did not increase susceptibility to infection in this macaque model. Thus neither the lubricant-induced type/extent of inflammation nor the presence of PQ15 affected infection risk. This study constitutes a first step in the <i>in vivo</i> evaluation of lubricants with regards to HIV transmission.</p></div

Epithelial sloughing and blood.

<p>A. Lubricant induces rectal shedding of epithelial cells. Examples of epithelial sloughing in a control- (top left) and lubricant-treated animal (top right). The lower panel shows a representative H&E stain of sloughed rectal epithelial cells (20x); B. Blood associated with rectal washes; photographs of microfuges containing rectal lavages; C,D. Epithelial sloughing measured at acute time points collected after the 2nd weekly lubricant application (C), and those measured over the entire study (D). The panel D in this figure shows three collections per week (day 1, pre-lubricant; day 2 pre-lubricant; day 2 post-lubricant), as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120021#pone.0120021.g001" target="_blank">Fig. 1</a>.</p

Hematoxylin and eosin stain (20x) of rectal biopsies.

<p>Showing biopsies from one animal (ID: 604962) collected before lubricant application (A) and 30 minutes after (B) product application; B shows focal infiltrates of inflammatory cells (square box), predominately mononuclear, seen in the lamina propria; there is no disruption of architecture. C is a magnified section (30x) of the square box with the green arrows showing mononuclear cells.</p

Cytokine concentration in rectal lavages at acute time points post lubricant/control buffer application.

<p>The p-values were calculated using unpaired t-tests (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120021#sec006" target="_blank">Materials & Methods</a> for details); the statistically significant ones are indicated in bold; CI = confidence interval.</p><p>¹We calculated the geometric means (GMs) of cytokines concentrations as shown, combining the measurements at 15m, 30m, 2- and 4-h acute time points. Levels of eight other cytokines were determined (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120021#sec006" target="_blank">Methods</a>) but many of the data points were below the assay limit of detection, and not suitable for accurate statistical analyses</p><p>Cytokine concentration in rectal lavages at acute time points post lubricant/control buffer application.</p

SHIV162p3 challenge doses and infection.

<p>‘+’ = animal infected at the indicated dose;</p><p>‘0’ = animal not infected;</p><p>PBS = phosphate buffered saline; TCID₅₀ = tissue culture 50% infectious dose. The challenges were performed in six sets of macaques; sets1 and 2 were phosphate buffered saline (PBS)-treated controls; sets 3, 4, 5 and 6 were lubricant-treated.</p><p>¹Historical data from 5 uninfected and 1 infected animals were included at 250 TCID₅₀, and 4 uninfected and 1 infected animals at 50 TCID₅₀; these animals were non-PBS-treated</p><p>SHIV162p3 challenge doses and infection.</p

Cytotoxicity study design (Phase I) and induction of pro-inflammatory cytokines.

<p>Study design showing the cytotoxicity phase of the study (A); black rectangles = lubricant application; grey triangles = sample collections immediately prior to each product application (longitudinal time points); black triangles = samples taken 15 minutes to 48 hours after product application (acute time points); open hexagons = rectal biopsies, taken from one animal at 30 minutes post lubricant-application, and from one animal a week after last lubricant application; m = minutes; h = hours; B. Induction of pro-inflammatory cytokine TNF-α at acute time points (15 or 30 m, and 2, 4, 24, or 48 h post-product application); circles represent individual macaques; C. Induction of pro-inflammatory cytokine TNF-α at all time points during 6 weeks of product application; medians and ranges are graphed.</p