Involvement of Integrin <Alpha>

Virology 297, 31–38 (2002) doi:10.1006/viro.2002.1399

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provided by Elsevier - Publisher Connector Involvement of Integrin ␣v␤3 in the Pathogenesis of Human Immunodeficiency Virus Type 1 Infection in Monocytes

Robert M. Lafrenie,*,1 Sherwin F. Lee,† Indira K. Hewlett,† Kenneth M. Yamada,‡ and Subhash Dhawan†

*Northeastern Ontario Regional Cancer Centre, Sudbury, Ontario, Canada P3E 5J1; †Immunopathogenesis Section, Laboratory of Molecular Virology, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20852; and ‡Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892 Received October 26, 2001; returned to author for revision November 15, 2001; accepted January 28, 2002

Attachment of HIV to macrophages is a critical early event in the establishment of infection. In the present study, we demonstrate the involvement of integrin ␣v␤3 (vitronectin receptor) in HIV infection of peripheral blood monocyte-derived macrophages. Culturing monocytes in the presence of M-CSF for 3 days upregulated expression of the ␣v-containing integrins, ␣v␤3 and ␣v␤5. The increase in ␣v␤3 expression was accompanied by increased HIV-1 replication by monocytes. Immunoblot analysis showed that purified HIV-gp120 protein interacted with CD4 and ␣v␤3 in immunoprecipitation experiments. Neutralizing antibodies against the ␣v␤3 integrin interfered with the coprecipitation of ␣v␤3 with an anti-gp120 antibody and substantially inhibited HIV infection of monocytes. Neutralizing antibodies against ␣v␤5or␤1 integrins did not significantly affect HIV infection. These results indicate that HIV infection of primary monocytes requires differentiation of these cells and may involve ␣v␤3 interaction with the HIV-1 envelope protein gp120 for productive infection. © 2002 Elsevier Science (USA) Key Words: human immunodeficiency virus type 1; integrin; vitronectin receptor; monocyte/macrophage; pathogenesis.

INTRODUCTION macrophages are susceptible to in vitro infection by monocytotrophic HIV (5–8). However, several studies Binding of HIV to the plasma membrane of cells is the have demonstrated that freshly isolated monocytes are first step in a series of events by which the viral genome difficult to infect with HIV and that successful viral infec- is introduced into the cell during infection. The interaction occurs only after the cells are allowed to differenti- tion of HIV with its cellular receptor is thought to result in ate by culture for several days in the presence of human alterations in the conformation of the receptor that are serum, growth factors, or cytokines (9–11). required for viral entry. Identification of cellular receptors In the present study, we have examined the possible for HIV has been of particular interest, since this is an involvement of integrin adhesion receptors in HIV infec- important target for clinical therapy and could be used to tion. Integrins are utilized during infection of host cells by prevent viral infection and spreading. CD4 has been a variety of intracellular parasites or viruses (12–15). HIV identified as a primary receptor for HIV and binds tightly infection of primary monocytes requires that the mono- to the HIV envelope glycoprotein, gp120 (1, 2). This bind- cytes be prepared by culturing in suspension or under ing alters the conformation of gp120, but viral binding adherent conditions in the presence of M-CSF for at least and these conformational changes are not sufficient for 3 days (9–11). These conditions also result in the upregu- HIV infection of cells (3, 4). Therefore, other factors are lated expression of monocyte ␣v-containing integrins likely involved in HIV infection. (16–18), and these changes in integrin expression have Monocyte/macrophages play a major role in HIV trans- been previously shown to be required for monocyte in- mission and throughout all stages of HIV infection and fection with adenovirus-derived vectors (19). Therefore, disease (5, 6). For example, HIV transmission may re- we have analyzed the role of the ␣v-containing integrins, quire transfer of infected monocytes, and long-lived tis- ␣v␤3 and ␣v␤5, in HIV-1 infection of primary monocyte- sue macrophages may provide a source of viral particles derived macrophages. and act as a reservoir of HIV. Human monocyte-derived RESULTS

1 To whom correspondence and reprint requests should be ad- Expression of various integrins on monocyte surface dressed at Northeastern Ontario Regional Cancer Centre, 41 Ramsey Lake Road, Sudbury, Ontario, Canada P3E 5J1. Fax: (705) 523-7326. Primary monocytes were cultured on tissue culture E-mail: [email protected]. substrates in the presence of M-CSF for various dura-

expression of certain monocyte integrins was upregulated by culture on adherent substrates in the presence of M-CSF. In particular, the expression of the ␤3 and ␣v integrin subunits were strongly upregulated following 3–5 days of culture under conditions typically required for HIV infectivity.

Time course of HIV-1 infection is coincident with ␣v expression Primary human monocytes cultured on tissue culture plastic substrates in the presence of M-CSF for various

durations were infected with HIVBa-L. Infection was determined by measuring the amount of HIV-gag-pol mRNA present by RT-PCR followed by Southern blot analysis or by detection of HIV-p24 in culture supernatants by ELISA assay. HIV mRNA was not detected in monocytes cultured for only 1 day prior to inoculation with virus, suggesting that these cells could not be infected by HIV (Fig. 2A). HIV gag-pol mRNA was detected in monocytes that had been precultured for 3 days prior to HIV infection and became more prominent in cells precultured for longer durations. The quality of the amplified cDNA was tested and normalized by measuring the amount GAPDH determined by PCR of the cDNA (Fig. 2B). HIV infection was FIG. 1. (A) Expression of integrin mRNA in cultured monocytes. Primary monocytes cultured on culture substrates in the presence of also examined by measuring HIV-p24 in the culture me- M-CSF for 1, 2, 3, or 5 days were harvested and the RNA was purified. dia of monocytes cultured on plastic substrates in the RNA (10 ␮g) was subjected to Northern blot analysis with probes that absence or presence of M-CSF (Fig. 3). Monocytes recognize ␣v, ␤1, ␤3, and ␤5 integrin subunits. (B) Cell surface expres- treated with M-CSF were infected to a much greater sion of monocyte integrin adhesion molecules. Primary monocytes or extent than untreated monocytes, although culturing the monocytes cultured on culture substrates for 1, 3, or 5 days were harvested and fixed in 1% paraformaldehyde and then incubated with cells on plastic substrates for more than 3 days did the indicated anti-integrin antibodies for 30 min at 4°C. The cells were enhance HIV infection compared to fresh monocytes. washed and incubated with an FITC-conjugated secondary antibody for 30 min. The cells were then fixed in 1% paraformaldehyde and the fluorescence profiles were determined by FACS analysis. The fluorescence profiles of nonimmune IgG (open curve) and antibody-stained cells are shown (closed curve). tions and examined for the expression of multiple integrin subunit mRNAs by Northern blot analysis. Freshly isolated monocytes did not express the ␣v, ␤3, and ␤5 integrin subunits. However, expression of these integrin subunits rose markedly after 3 days of culture and became more prominent in cells cultured for longer durations (Fig. 1A). In contrast, the ␤1 integrin subunit was constitutively expressed through 5 days in culture. The expression of integrin adhesion molecules on the surface of the monocytes was measured by fluorescence flow cytometry using specific anti-integrin antibodies. The ␣v and ␣v␤3 integrins were not expressed on the surface of primary monocytes, but were expressed on FIG. 2. HIV-1 gag-pol amplimer of RNA from the monocytes infected the surface of monocytes that were cultured on tissue at various times in culture. (A) RNA (1 ␮g) purified from monocytes culture substrates in the presence of M-CSF for 3 and 5 cultured for various durations and then infected with HIV was subjected ␤ to RT-PCR with oligonucleotides that amplify HIV-gag-pol or GAPDH days (Fig. 1B). The 1 integrins were expressed on and then detected by Southern blot analysis with HIV-gag-pol or primary monocytes to a similar extent as on monocytes GAPDH-specific oligonucleotide probes. (B) Densitometric analysis of precultured on tissue culture substrates. Therefore, the the HIV-gag-pol-hybridizing signal normalized for GAPDH. ␣v␤3 EXPRESSION ANDHIV-1 INFECTION IN MONOCYTES 33

was determined by blot analysis with streptavidin-HRP. The monocytes were cultured for 1, 3, and 5 days in the presence or absence of M-CSF. Biotin-labeled gp120 was bound to and chemically crosslinked to the monocytes cell surface and the amount of bound gp120 determined by blotting with streptavidin-HRP. Monocytes cultured on tissue culture substrates in the presence or absence of M-CSF for at least 3 days bound gp120 (Fig. 5A). Monocytes treated with M-CSF bound greater amounts of gp120 than untreated cells, and binding was enhanced by increased durations of treatment. For de- ␣ ␤ ␤ FIG. 3. HIV-p24 levels in the media of monocytes infected with HIV. tection of cell surface v 3or 1 expression, monocytes Monocytes were cultured on culture substrates in the absence or cultured in the presence or absence of M-CSF were presence of M-CSF for 1, 2, 3, 5, or 7 days and infected with HIVBa-L surface-labeled by incubation with NHS-biotin, and cell (m.o.i. ϭ 0.05). The cells were then incubated in culture media. Culture lysates were normalized for biotin-labeled proteins, im- supernatants were harvested on Day 7 postinoculation and then as- munoprecipitated with anti-␣v␤3 or anti-␤1 antibodies, sayed for cell-free HIV-p24 antigen by ELISA using NEN/DuPont p24 ELISA test kits. and then subjected to blot analysis with streptavidin- HRP. Monocytes cultured in the absence or presence of ␣ ␤ Therefore, monocytes cultured on adherent tissue cul- M-CSF showed an enhanced expression of v 3 that ture plastic substrates for more than 3 days in the ab- directly correlated with the increase in gp120 binding to sence or presence of M-CSF could be infected with HIV. the monocyte surface. The cell surface expression of the ␤1 integrin did not change significantly during this time Inhibition of HIV-1 infection by anti-␣v␤3 integrin in culture. antibody The correlation between the time course of monocyte culture required for both HIV infectivity and ␣v␤3 expression suggests that ␣v␤3 may serve as a cofactor for HIV infection. To test this hypothesis, we infected monocytes cultured on adherent substrates with HIV in the presence of blocking anti-integrin antibodies and then measured HIV-gag-pol mRNA levels 24 h postinfection or HIV-p24 antigen in culture supernatants 6 days postinfection. The amount of GAPDH cDNA was also determined by PCR of the same samples to normalize RNA amounts. These studies show that the inclusion of anti-␣v␤3 polyclonal antibodies or inclusion of a neutralizing monoclonal antibody markedly inhibited the production of HIV gag-pol mRNA by approximately 70% relative to the GAPDH control (Fig. 4A). Similarly, inclusion of an anti-␣v␤3, neutralizing antibody blocked HIV-p24 production by approximately 40–45% (Table 1). Inclusion of the anti-␣v␤3 antibodies did not promote detectable cell retraction or cell loss from the monocyte monolayer and antibody-treated and untreated monolayers had indistinguishable mor- phologies (data not shown). However, in contrast to the inhibition by anti-␣v␤3 antibodies, neutralizing anti-␣v␤5 monoclonal antibody only marginally inhibited HIV infection. Inclusion of neutralizing antibodies against the FIG. 4. Effect of anti-integrin antibodies on HIV infection of cultured ␣2␤1or␣5␤1 integrins had no effect on HIV infection monocytes. Monocytes were cultured for 5 days in the presence of M-CSF. The cells were then incubated with HIVBa-L in the presence or as measured by the production of HIV gag-pol mRNA absence of the indicated anti-integrin antibodies. The monolayers were (Fig. 4B). washed and grown in culture media prior to the isolation of RNA. (A) RNA (1 ␮g) was subjected to RT-PCR with oligonucleotides that amplify HIV-1 gp120 interacts with ␣v␤3 integrin HIV gag-pol or GAPDH and then assayed by Southern blot analysis with HIV gag-pol or GAPDH-specific oligonucleotide probes. (B) Den- The correlation between cell surface ␣v␤3 expression sitometric analysis of the HIV gag-pol-hybridizing signal normalized for and the ability of HIV-gp120 to bind the monocyte surface GAPDH. The data are representative of three separate determinations. 34 LAFRENIE ET AL.

TABLE 1 Monocytes and macrophages serve a variety of immu- Effect of Anti-␣v Integrin Antibodies on p24 Antigen Production noregulatory, phagocytic, and secretory functions. HIV by HIV-Infected Monocytes infection of monocytes/macrophages can alter crucial immunoregulatory functions including monocyte/endo- p24 production thelial cell interactions, cytokine production, and acces-

Experiment 1 Experiment 2

Antibody % Production % Inhibition % Production % Inhibition

None (Control) 100 0 100 0 ␣v␤353Ϯ 19 47* 61 Ϯ 11 39* ␣v␤581Ϯ 22 19 130 —

Note. Monocytes were cultured for 5 days in the presence of M-CSF. The indicated antibodies were incubated with the cells at 4°C for 1 h, and then the cells were inoculated with HIVBa-L in the presence of antibodies. After 1 h, cells were washed with medium containing antibodies and replaced with antibody-containing medium for 6 days. Viral p24 levels in culture supernatants were determined by the NEN/ DuPont p24 ELISA test kit. Data in both experiments are presented as the mean Ϯ standard deviation relative to the control for at least three determinations and differences were determined using a Student’s t test. * P Ͻ 0.05.

To determine whether gp120 and CD4 specifically in- teract with ␣v␤3, monocytes cultured in the presence of M-CSF for 5 days were incubated with, and chemically crosslinked to, biotin-labeled gp120. Cell lysates were immunoprecipitated with specific anti-integrin antibodies and the immunoprecipitated material was subjected to blot analysis with streptavidin-HRP. The results of these experiments showed that gp120 coprecipitated with antibodies that recognize ␣v␤3 but not with the anti-␣2, ␣5, or ␤1 antibodies (Fig. 5B). Immunoprecipitation of gp120- crosslinked monocytes also showed that gp120 could be ␣ ␤ ␤ coprecipitated with anti-gp120, anti-␣v␤3, and anti-CD4 FIG. 5. Levels of monocyte-bound HIV gp120, v 3, or 1 integrins. ␤ (A) Monocytes cultured in the presence or absence of M-CSF were antibodies, (but not anti- 1 integrin antibodies), indicat- surface-biotinylated or incubated with biotin-labeled gp120 at the indi- ing involvement of ␣v␤3 in a CD4-mediated interaction cated times and then immunoprecipitated with anti-gp120, anti-␣v␤3, or with HIV-gp120 (Fig. 5C). It should be noted that CD4 anti-␤1 antibodies. The immunoprecipitates were then subjected to expression was not significantly enhanced following blot analysis by incubation in 1:2000 streptavidin-horseperoxidase treatment of the monocytes with M-CSF for 3 days (data (HRP) conjugate in 0.5% BSA in TBST. The HRP was detected by incubation in Supersignal reagent and exposed to Hyperfilm-ECL X-ray not shown). Further, competitive inhibition by preincuba- film. (B) Specificity of HIV-gp120 interactions with integrin adhesion tion of monocytes with unlabeled gp120, anti-CD4, or receptors in cultured monocytes. Monocytes cultured in the presence anti-␣v␤3 antibodies each substantially reduced the of M-CSF for 5 days were incubated with biotin-labeled gp120 and then binding of biotinylated gp120 to the monocyte cell sur- treated with the DTSSP crosslinker. The cells were lysed and the face while anti-␤1 antibodies did not. These experiments lysates immunoprecipitated with the indicated anti-integrin antibodies ␣ ␤ and goat-anti-mouse IgG Sepharose. The crosslinker was cleaved by suggest that v 3 is present in a complex that includes incubation in DTT and the immunoprecipitate subjected to blot analysis CD4 and HIV-gp120 and further suggest that ␣v␤3isa with streptavidin-HRP. HRP was detected by incubation in Supersignal cofactor that promotes interaction between CD4 and HIV. reagent and exposed to X-ray film. (C) Coprecipitation of HIV gp120 with anti-CD4 or anti-␣v␤3 antibodies. (Panel a) Monocyte surface proteins DISCUSSION were labeled by incubation with biotin-labeled gp120, crosslinked, and immunoprecipitated with anti-␤1 (control), anti-gp120, anti-CD4, or anti- ␣ ␤ In the present study, we have shown that culturing v 3 antibodies. (Panel b) Monocytes were pretreated with unlabeled ␣ ␤ gp120, anti-CD4, anti-␣v␤3, or anti-␤1 (control) antibodies, as indicated, primary monocytes in M-CSF upregulates v 3 expres- prior to incubation and crosslinking with biotin-labeled gp120. The cell sion and that ␣v␤3 can be an important component in lysates were immunoprecipitated with anti-gp120 and biotin-labeled HIV infection of monocytes in vitro. gp120 was detected as described above. ␣v␤3 EXPRESSION ANDHIV-1 INFECTION IN MONOCYTES 35 sory cell function (20–22). The complex interactions be- culture with M-CSF (16–18). We hypothesized that the tween monocyte/macrophages and HIV are of special initial absence of these receptors might account for importance, since these cells are relatively refractory to the fact that productive HIV infection of freshly isolated the cytopathic effects of HIV and probably serve as a primary monocytes is difficult; infection also requires major reservoir for the virus as well as a vehicle for that monocytes be cultured for several days especially disseminating HIV to various tissues (5, 23, 24). There- in the presence of M-CSF (9–11). However, we have fore, inhibition of monocyte/macrophage infection by HIV also showed that culturing primary monocytes on tis- and determination of factors required for viral entry are sue culture substrates without cytokines for longer important for designing clinical strategies to inhibit HIV that 3 days is also sufficient to upregulate HIV infec- infection and progression of the disease. tion. This is likely due to the observation that adherent HIV has been shown to involve several coreceptors in monocytes are able to synthesize and secrete cyto- addition to CD4 for entry into target cells. The exact kines such as IL-1␤,TNF-␣, and M-CSF (39, 40). The mechanism by which these coreceptors contribute to time course required for HIV infection correlated with HIV entry remains unclear. In the past few years, remark- the expression of the ␣v-containing integrins in par- able progress has been made in identifying chemokine ticular the expression of the ␣v␤3 integrin. Further, receptors as critical components in HIV infection of a different culture conditions also showed a direct cor- number of cell types and cell lines. Chemokine receptors relation between HIV infectivity of monocytes and up- are large molecules belonging to the family of seven- regulation of the ␣v␤3 integrin expression. In addition, transmembrane spanning GTP-binding proteins (25). inclusion of a specific anti-␣v␤3 monoclonal antibody, Binding of HIV strains which are tropic for T-cells to that is known to block ligand binding (41), partially various target cells is mediated by the CXC chemokine inhibited HIV infection of cultured monocytes as de- receptor 4 (CXCR4), or fusion (26). Binding of macro- tected both by the presence of the HIV-gag-pol mRNA phage-tropic HIV to target cells is mediated by the CC and secretion of the HIV-p24 antigen. These results chemokine receptor 5 (CCR5) and can be competitively support the notion that HIV infection of monocytes is blocked by treatment with ␤-chemokines or chemokine mediated at least in part by the ␣v␤3 integrin. The analogs (14, 27–31). A number of studies have shown anti-␣v␤3 antibody inhibited the amount of HIV-gag- that chemokine receptors are coreceptors for HIV and pol mRNA detected the day following infection by that blocking these coreceptors on various immune cells approximately 70% while the anti-␣v␤3 antibody inhib- can have strong antiviral effects (12, 13). In addition, it ited HIV-p24 secretion detected 6 days after infection has been shown that culturing monocytes under condi- by approximately 40%. This difference is likely due to tions which promote differentiation, such as treatment the release of virus by the HIV-infected monocytes and with M-CSF, can also induce CCR-5 expression, suggesting CCR5 may be involved in mediating HIV infection in reinfection of neighboring cells during the 6 days in differentiated cultured monocytes (10, 33). One study (33) culture. In these experiments, the antibodies were has shown that a derivative of RANTES, which can block added during and immediately after the original inoc- binding via CCR5, can inhibit HIV infection of PHA- ulation and proteolysis and phagocytosis are expected treated macrophages in vitro; however, other studies to decrease their ability to effectively inhibit ligand/ have shown that RANTES requires interactions with cell monocyte binding over time. Although it is also possi- surface proteoglycans, not present on M-CSF monocyte- ble that these differences could be related to signals ␣ ␤ derived macrophages, in order to be inhibitory (34, 35). induced by anti- v 3 integrin antibodies that inhibit ␣ ␤ Further, a number of studies have shown that HIV infec- HIV replication, the anti- v 3 antibodies have been tion of primary monocytes or macrophage-derived mono- shown to directly block interactions with HIV-gp120 cytes is not inhibited by inclusion of chemokines even which correlates with their effects on HIV infection. though CCR5 is expressed on these cells (36–38). There- Finally, the coimmunoprecipitation experiments re- fore, it seems likely that HIV infection of monocytes and vealed that HIV-gp120 binds to cell surface ␣v␤3 and monocyte-derived macrophages may involve other mol- is part of a complex that includes CD4. M-CSF treat- ecules in addition to or distinct from CCR5. ment does not change CD4 expression to a large Integrin adhesion receptors, in particular ␣v- enough extent to account for the changes in HIV in- containing integrins, have been shown to mediate fection or complex formation as described previously binding and internalization of a variety of intracellular (31, 33, 42). Further, the anti-␣v␤3 antibodies block the parasites and viruses (12–15, 19). Whether these re- interaction between ␣v␤3 and gp120, suggesting that ceptors could be possible candidates for mediating these antibodies are able to inhibit interactions be- HIV binding and internalization is not known. Recent tween HIV and monocytes. These results suggest a studies have demonstrated, and we confirmed in the possible mechanism by which ␣v␤3 could support HIV present study, that ␣v␤3 and ␣v␤5 integrin receptors infection by enhancing interactions of the virus with are expressed on the monocyte surface after 3 days in the monocyte and thereby promoting internalization. 36 LAFRENIE ET AL.

MATERIALS AND METHODS a monocytotrophic HIV strain (Advanced Biotechnologies Inc., Columbia, MD), at a multiplicity of infection of 0.05 Monocyte isolation infectious virus particles/target cell (7). After a period of Monocytes were isolated from the peripheral blood 2 h to allow viral adsorption, cultures were washed with mononuclear cells of donors seronegative for HIV and medium and harvested after 24 h for RT-PCR analysis. hepatitis after leukapheresis and purification by counter- Alternately, infected monocytes were grown in culture for current centrifugal elutriation (43). 6 days, and then culture supernatants were assayed for HIV-p24 using the NEN/Dupont ELISA analysis kit, ac- Detection of cell surface integrins expressed on cording to the manufacturer’s instructions (7). For some monocytes experiments, HIV infection was carried out in the pres- ␮ Monocytes (106) cultured in the presence of M-CSF ence of 10 g/ml of anti-integrin monoclonal antibodies ␣ ␤ ␣ were harvested at selected times and fixed in 1% para- against 2 1 (clone P1E6, Life Technologies), 5 ␤ ␣ ␣ ␤ ␣ ␤ formaldehyde, washed twice in PBS, and resuspended in (mAb16), 1 (mAb13), v (LM142), v 3 (LM609), v 5 FACS binding buffer (PBS containing 2% FBS, 1% BSA, (P1F6), or with anti-vitronectin receptor polyclonal antibodies at a titer of 1:1000. and 0.1% NaN3) (44). Cells were incubated with monoclonal antibodies against ␣v (LM142, Chemicon Interna- tional, Temecula, CA) (41), ␣v␤3 (LM609), ␣5 (mAb16), or RT-PCR for gag-pol HIV transcripts ␤1 (mAb13) (45) at 4°C for 45 min and washed 3 times Monocytes were cultured in the presence of M-CSF for

with binding buffer. The washed cells were incubated 1, 2, 3, or 5 days, infected with 0.05 HIV-1Ba-L particles/ with a sheep anti-mouse IgG-FITC or sheep anti-rat IgG- target cell in the presence or absence of different anti- FITC-conjugated secondary antibody (Life Technology, integrin antibodies and cultured for 24 h. Total RNA was Burlington, ON) at 4°C for 30 min. Monocytes were isolated and 1 ␮g was reverse-transcribed using 10 ng of

washed 3 times with PBS, fixed with 1% paraformalde- an oligo(dT)12–18 primer, 10 units of Moloney murine leu- hyde, and analyzed for fluorescence on an EpicsElite kemia virus-reverse transcriptase (MMLV-RT; Life Tech- fluorescence cytometer (Coulter Corporation, Burlington, nologies), in 1ϫ PCR buffer, and 1 mM dNTPs, in a total ON). volume of 20 ␮l at 42°C for 60 min (48, 49). The mixture was inactivated at 94°C for 10 min, and 10 ␮l of the cDNA RNA isolation and Northern blot analysis of integrin was then amplified by PCR to detect HIV gag-pol or mRNA GAPDH using 100 pmol of each of the appropriate primer Monocytes (107) cultured in the presence of rM-CSF pairs, 1 mM dNTPs, and 5 U Taq DNA polymerase (Life were harvested at 1, 3, and 5 days in a guanidium Technologies). The cycle program consisted of an initial isothiocyanate solution and were extracted with phenol- denaturation step at 94°C, followed by 30 cycles of 1 min chloroform to isolate total RNA as previously described at 94°C, 1 min at 55°C, and 2 min at 72°C. The samples (46). Total RNA (10 ␮g) was subjected to electrophoresis were subjected to electrophoresis on 2% agarose-Tris- on 1% agarose gels containing formaldehyde. The RNA buffered EDTA (TBE) gels and transferred to 0.22 ␮m was transferred to Nytran Plus membranes (Schleicher nitrocellulose filters (Schleicher and Scheull). The result- and Scheull, Mandel Scientific Co., Mississauga, ON) by ing blots were then probed with appropriate end-labeled capillary diffusion with 20ϫ SSPE. Individual cDNA oligonucleotide probes that recognize HIV gag-pol, or probes against the ␤1, ␤3, ␣5, and ␣v integrin subunits GAPDH, washed, and exposed to Hyperfilm-MP X-ray were labeled with [␣32P]dCTP (New England Nuclear, film (49). Electronic images of the resulting X-rays were Mandel Scientific Co.) using a random primer labeling kit captured using a VideoMaster VDS gel documentation (Stratagene, La Jolla, CA). Hybridizations were carried system (Pharmacia Biotechnology, Baie d’Urfe, QU) and out at 42°C in 50% formamide, 5ϫ SSPE, 5ϫ Denhardt’s the densities of the HIV gag-pol hybridizing bands deter- solution, 0.5% SDS, and 100 ␮g/ml salmon sperm DNA mined and normalized for the densities of the corre- (47). The blots were washed once with 4ϫ SSPE, 0.1% sponding GAPDH-hybridizing bands. SDS at room temperature for 30 min followed by 30 min washes in 0.5ϫ SSPE, 0.1% SDS at 42°C and at 55°C. Biotinylated-gp120 crosslinking and integrin The blots were then exposed to Hyperfilm-MP (Amer- immunoprecipitation Ϫ sham, Oakville, ON) at 80°C. Monocytes, cultured for 1, 3, or 5 days, were incubated for1hat4°Cwith 50 ␮M biotin-labeled gp120 (Advanced HIV-1 infection of monocytes Biotechnologies Inc.). The gp120 was crosslinked to Primary monocytes or monocytes cultured for 1, 2, 3, or monocytes using 3,3-dithiobis(sulfosuccinimidyl propio- 5 days in the presence or absence of M-CSF were nate) (DTSSP) as recommended by the supplier (Pierce washed, incubated in macrophage-SFM media (Life Chemical Co., Rockford, IL). For some experiments, the

Technology, Gaithersbury, MD), and exposed to HIV-1Ba-L, monocytes were surface-labeled by incubation in 100 ␣v␤3 EXPRESSION ANDHIV-1 INFECTION IN MONOCYTES 37

␮g/ml biotin-NHS (Pierce Chemical Co.) in PBS for 30 type 1 T-cell or macrophage tropism, cytopathogenicity, and min. The monocytes were harvested and then lysed in CD4 antigen modulation. J. Virol. 64, 4390–4398. 150 mM octylglucoside, 150 mM NaCl, 10 mM HEPES, 10. Di Marzio, P., Tse, J., and Landau, N. R. (1998). Chemokine receptor regulation and HIV type 1 tropism in monocyte-macrophages. pH 7.4, 1 mM CaCl2, and 1 mM MgCl2. The lysates were AIDS Res. Hum. Retroviruses 14, 129–138. immunoprecipitated with anti-integrin antibodies (1 ␮g) 11. Gruber, M. F., Weih, K. A., Boone, E. J., Smith, P. D., and Clouse, K. A. against ␣v␤3 (LM609), ␣v (LM142), ␤1 (mAb13), or ␣5 (1995). Endogenous macrophage CSF production is associated (mAb16) overnight at 4°C and then incubated with 25 ␮l with viral replication in HIV-1-infected human monocyte-derived of 10% protein A-Sepharose (Pharmacia) for 2 h (47). For macrophages. J. Immunol. 154, 5528–5535. 12. Hayashi, T. S., Rao, S. 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Involvement of Integrin &lt;Alpha&gt;

Involvement of Integrin <Alpha>