VIROLOGY
190,
124-l
33 (1992)
Distinctive
Pattern of Infection and Replication in Blood-Derived Macrophages
HELENA SCHMIDTMAYEROVA,*+ IVAN HIRSCH,* *Unit& de Recherches Sciences, Bratislava,
INSERM U322 Czechoslovakia,
sur /es RBtrovirus and +Laboratoire Received
of HIV1 Strains
CHRISTINE BOLMONT,” STEPHEN BAGHDIGUIAN,+ JEAN-CLAUDE CHERMANN””
AND
et Maladies de Biologic February
AssociBes, Cellulaire
17, 1992;
Marseille, et Histologie,
accepted
May
France; tlnstitute of virology Facult6 de Mgdecine Nerd,
Slovak Academy Marseille, France
of
8, 1992
The macrophage-tropic virus HIVl-PAR, isolated from cerebrospinal fluid of HlVl-seropositive man, induced cytopathic effect accompanied by different magnitude of the virus production in blood-derived macrophages (BDM) obtained from different donors. HIVl-PAR-specific RNA was detected by in situ hybridization in 15 and 66% of BDM producing low and high levels of virus, respectively. In contrast with HIVl-PAR, infection of BDM with two laboratory strains adapted to T-cell lines, HIVl-LAV prototype and HIVl-NDK, a Zairian virus that is highly cytopathic for T-lymphocytes, resulted in a low production of HIV1 ~24~“~ in culture fluid. Expression of HIV1 -LAV and HIV1 -NDK RNA was detected by in situ hybridization in a maximum of 1% of macrophages. Only HIVl-NDK, and not HIVl-LAV, induced ultrastructural alterations in BDM. In contrast with a striking difference in the production of macrophage-tropic and T-lymphotropic viruses, no significant differences were found in the proportion of macrophages containing retrotranscribed genomes of HIV1 . HIV1 DNA was detected by in situ hybridization in 93,100, and 80% of macrophages infected with HIV1 -PAR, HIV1 -LAV, and HIV1 -NDK, respectively. A higher level of HIV1 DNA was detected by polymerase chain reaction in the BDM infected with HlVl-PAR than in that infected with HIVl-LAV and HIVl-NDK. The results indicate that both macrophage-tropic as well as T-lymphotropic viruses can enter and retrotranscribe their genomes in a vast majority of macrophages. 0 1992 Academic Press, 1~.
INTRODUCTION
1990) hidden from the host immune surveillance. These cells, harboring the virus, could function as a long-time reservoir of HIV infection. Nonproductively infected cells may spread infection to other susceptible cells over a long period of time by different mechanisms (Ho et a/., 1986; Mann et a/., 1990; Valentin et a/., 1990). HIV1 infection of macrophages in vitro shows a high variation in the replicative capacity for different viral strains. Most laboratory strains of HIV1 after long-term passage in T-cell lines replicate poorly or not at all in macrophages (Nicholson et a/., 1986; Gendelman et al., 1989; Collman eta/., 1989). A specific region of the envelope gpl20 gene was found to determine the macrophage tropism (O’Brien et al., 1990; Cheng-Mayer et al., 1991; Hwang et al., 1991). It was suggested in these studies that the major block of HIV1 replication in macrophages might be an inefficient virus/cell entry. The aim of the present study was the characterization and the comparison of infectibility of blood-derived macrophages (BDM) with the macrophage-tropic strain HIVl-PAR, isolated in this laboratory and two other laboratory strains adapted to the T-cell lines, HIV1 -LAV prototype and HIV1 -NDK, a Zairian virus that is highlycytopathic for T-lymphocytes. The results indi-
Cells of monocyte/macrophage lineage are an important target for human immunodeficiencyvirus (HIV). Infected macrophages were detected in the brains (Gartner eta/., 1986; Koenig eta/., 1986), lungs (Ziza et a/., 1985; Chayt et a/., 1986; Gartner et al., 1986), and other tissues (Tschachler et al., 1987; Pomerantz et al., 1988) of patients with acquired immune deficiency syndrome (AIDS). Monocyte/macrophages have important immunoregulatory functions, which could be compromised by HIV infection. A variety of pathological changes have been reported in monocytes from HIV-infected patients (Bender et a/., 1988), which could contribute to the impairment of the immune system in AIDS patients. Monocyte/macrophages are supposed to be among the first targets of HIV infection in a human organism (Ho et al., 1986). Virus could persist in the macrophages for a prolonged period of time within intracellular vacuoles (Orenstein et a/., 1988; Meltzer et al.,
’ To whom reprint requests should be addressed at INSERM Unit6 322, Unit6 de Recherches sur les Rktrovirus et Maladies Associires, Campus Universitaire de Luminy, BP 33, 13273 Marseille, c6dex 9. 0042-6822192
$5.00
CopyrIght 0 1992 by Academic Press, Inc. All rights of reproduction I” any form reserved
124
HIV1
OF
MACROPHAGES
125
A
60 -
INFECTION
50
“0 -I x 40 E
A
-
IOTCIU
-*-
100 TCIU
E, 30 u 2.P :I 20 2 & ‘Y 10 0
01 7
11
14
16
21
24
27
7
11
14
Days after infection
of HIV1 -PAR in PBMC FIG. 1. (A) Repkation both PBMC and CBL cultures. (B) Replication determined by virus titration on CBL
and CBL. The viral inoculum containing of HIV1 -PAR in BDM. Cultures of BDM
cate that both T-lymphotropic viruses were able to enter and retrotranscribe their genome in a vaste majority of macrophages. MATERIALS isolation
and culture
AND
METHODS
of BDM
Peripheral blood mononuclear cells (PBMC) of healthy donors undergoing leukopheresis were separated on Ficoll-Hypaque gradients. Suspensions of 8 X 1 O6cells/ml, prepared in RPMI 1640 medium supplemented with 10% fetal calf serum, 5% normal human serum, 10 mM HEPES buffer and antibiotics (PSN), were allowed to adhere for 2 hr at 37”. After removal of nonadherent cells by extensive washing, the cells were cultured in the presence of 50 U/ml of recombinant
TABLE
1
INFECTION OF BLOOD-DERIVED MACROPHAGES WITH HlVlmLAV AND HIV&NDK
BDM RT activity
Infectious virusa
+
-
+
-
24
27
~24 bgiml)
of as
human granulocyte-macrophage colony stimulating factor (GMCSF, Genzyme). Resting nonadherent cells were removed by additional washing 2 and 5 days after isolation. On Day 5 more than 90% of the cells consisted of macrophages, as determined by reactivity with anti-CD1 4 monoclonal antibody (IOM2, Immunotech S.A.) and by cytochemical staining for nonspecific esterase (Alfa-Naphtyl Acetate Esterase Kit, Sigma Diagnostics). Viruses and infection
of BDM
Three different strains of HIV1 were used in the study: HIV1 -PAR, a strain isolated from the cerebrospinal fluid of an HIVl-seropositive man suffering from acute encephalopathy (Gout et al., 1988; Chermann, 1990), the HIVl-LAV prototype strain (Barre-Sinoussi et a/., 1983), and the Zairian strain HIV1 -NDK (Ellrodt, 1984). The stocks of HIV1 -LAV and HIV1 -NDK were prepared and titrated on CEM cells and stocks of HIV1 PAR on cord blood lymphocytes (CBL) (Rey et al., 1991). BDM cultures were infected 5 days after isolation. Cells were washed and incubated with 1000 tis-
Cocultivation with CEMb E
-
21
1.5 X 1 O4 cpm/ml of RT activity was used for infection were infected with 100, 10, and 1 TCIU of HIVl-PAR,
2 Donor 1 HIV1 -LAV HIVl-NDK Donor 2 HIV1 -LAV HIVl-NDK
18
Days after infection
31 330
+ +
11 30
+ +
a Culture fluid from 10 days infected BDM was filtered through 0.45 pm Millipore filter and 2-ml aliquots were used for infection of 2 x 10” CEM cells. b The CEM cells at the final concentration of 5 X 1 O5 cells/ml were added to the BDM cultures 10 days after infection.
HIVI-LAV -
HIVl-NDK ~
-HIM-PAR
of HIV1 specific DNA by amplification of 172-bp FIG. 2. Detection fragment from HIV1 ral gene. Cell lysates prepared 10 days after BDM infection with HIV+LAV, HIVl-NDK, and HIVl-PAR and containing about 100 ng of DNA were treated with 50 U/ml of RNase (lane 2) or RNase free DNase (lane 3), amplified and analyzed by Southern blot hybridization. Lane 1 represents nontreated control.
126
SCHMIDTMAYEROVA TABLE
2
HIV-1 -SPECIFIC RNA AND DNA DETECTED s~hvSm IN BDM, 3 AND 10 DAYSAFTER~NFECTION
HYBRIDIZATION
% Frequency RNA-positive
HIV1 -PARb HIV1 -PARC HIV&LAV HIVl-NDK
cells
DNA-positive
cells
3 days
10 days
3 days
10 days
8 6 ND ND
15 66 0.3 1
96 ND 97 ND
93 ND 100 80
aThe frequency was calculated from a counted cells for cultures with high and low tively. b HIV1 -PAR-infected BDM producing low ’ HIV+PAR-infected BDM producing high
total of 500 and 6000 positivity rate, respecRT levels. RT levels.
sue culture infectious units (TCIU) of HIVl-LAV or HlVl-NDK (corresponding to 5 X 1O4 cpm/ml of reverse transcriptase (RT) activity) or with 100 TCIU of HIV1 -PAR (corresponding to 3 X 1 O4 cpm/ml of RT activity) for 2 hr at 37”. After adsorption and another washing, a fresh medium containing 50 U/ml of GMCSF was added. Reverse transcriptase
and p24 antigen
assay
The activity of RT was measured by standard RT assay (Rey et a/., 1984) in the 0.1% Triton X-l 00 disrupted viral pellet prepared from 1 ml of culture fluid. The p24 antigen capture assay was done according to the manufacturer’s directions (DuPont). In situ hybridization The 9-kb Sacl fragment of plasmid pBT1 containing the HIV1 -LAV prototype nucleotide sequence (Alizon et al., 1984) was used as a hybridization probe. Detection of viral RNA. BDM cultured on chamber slides (Nunc) were fixed with 4% paraformaldehyde and treated with 1 pglml of Proteinase K. Hybridization was carried out overnight at 37” in a solution containing 50% formamide; 4 X SSC (600 mM NaCI, 60 mM sodium citrate); 0.02% each of Ficoll, polyvinylpyrrolidone, and bovine serum albumine; and 400 pg/ml each of herring sperm DNA, salmon testis DNA, and yeast RNA. [35S]DNA probe labeled by nick translation was denatured at 100” for 3 min and used at a final conceniration of 0.3 pg/ml. After autoradiography performed at 4” for 2 to 7 days, the cells were stained with eosinhematoxylin. Detection of viral DNA. To detect specific HIV1 DNA, the fixed infected BDM were treated by Proteinase K as
ET AL.
described above. A total intracellular RNA was then digested with 200 pg/ml of RNAse type A. The intracellular DNA on chamber slides was subsequently denatured at 100” for 3 min together with hybridization mixture. Detection of HI//l-specific chain reaction (PCR)
DNA by polymerase
Approximately 7 X 1 O5 BDM were lysed with 1 ml of PCR buffer (Higuchi, 1989). After protein digestion, 25 ~1of cell lysate were subjected to 30 cycles of polymerase chain reaction in a total volume of 50 ~1 containing 10 pmol of oligonucleotide primers, nt 5419-5446 and 5563-5590 of HIV1 -LAV tat sequence (CisBio International), 200 PLM of each deoxynucleotide, 50 mM KCI, 10 mMTris, pH 8.3, 1.5 mM MgCI,, and 1.2 U of Taq polymerase (Cetus Corp.). Each cycle comprised 1 min denaturation step (94”), 1.5 min annealing step (SS’C), and 2 min extension (72°C). After agarose gel electrophoresis, amplified DNA was analyzed by Southern blot hybridization with 3*P-labeled probe corresponding to the HIV1 -LAV tat sequence between nucleotides 5447 and 5474 (CisBio International). Electron
microscopy
examination
BDM grown in 25-cm* plastic flasks were fixed with 2.5% glutaraldehyde and processed for electron microscopy as described by Gelderblom et al. (1987). RESULTS Replication of HIVl-PAR in peripheral blood mononuclear cells, cord blood lymphocytes, and blood derived macrophages Production of HIV1 -PAR in PBMC and CBL cells was examined in cell-free supernatants at 3-4 day intervals (Fig. 1A). The CBL, which are less differentiated than PBMC, allowed a rapid and high replication of HIVlPAR. The same virus had a property of slow/low virus in PBMC (Asjo et a/., 1986; Von Briesen et al., 1987; Tersmette et al., 1988). The transient cytopathic effect, characterized by marked vacuolization of cytoplasm, was observed in CBL 3-4 days before virus production (not shown). No cytopathic changes were seen in PBMC infected with HIV1 -PAR. These results illustrate variability in the biological properties of viral isolate that depends on the cell culture system used. With respect to the high production of HIV-PAR in CBL, we used these cells for preparation and standardization of viral stock for infection of BDM. Five-day-old cultures of BDM grown in 24-well plates were infected with 100, 10, and 1 TCIU determined by the virus titration on CBL. Virus production was dependent on the
HIV1
FIG. 3. in sifu hybridization analysis of viral RNA in BDM HIVl-LAV (A), HIVl-NDK (B), and HIVl-PAR (C); uninfected
viral inoculum dose (Fig. 1 B) and accompanied mation of multinucleated giant cells. Infection of blood-derived LAV and HIV1 -NDK
macrophages
INFECTION
of HIV1 -specific
MACROPHAGES
10 days after infection BDM (D). Magnification,
by for-
with HIV1 -
The BDM grown in 24-well plates were infected with 1000 TCIU of HIVl-LAV and HIVl-NDK. RT production or cytopathic changes were never observed in BDM cultures obtained from different donors. However, infection of BDM with both T-lymphotropic viruses could be demonstrated by p24 antigen production and after cocultivation with CEM (Table 1). Production of infectious virus into the filtered culture fluid was demonstrated only after infection with HIV1 -NDK. Detection
OF
proviral DNA in BDM
Cell lysates prepared from BDM 10 days after infection were treated with 50 U/ml of RNAse or DNAse
with
three X205.
127
different
HIV1
strains.
BDM
cultures
infected
with
before amplification. The specific viral DNA was detected in both HIVl-LAV- and HIVl-NDK-infected BDM, although in a lower quantity than that found in the BDM infected with HIVl-PAR (Fig. 2). The results indicate that all three viruses were able to enter BDM and retrotranscribe their genomes. Higher levels of HIV1 provirus copy number were present in HIVl-PAR infected BDM than in BDM infected with T-lymphotropit viruses. In situ hybridization
analysis of viral RNA and DNA
Ten days after infection, the frequency of BDM productively infected with HIVl-PAR detected by in situ hybridization reached 15 and 66% in the cultures producing low and high RT levels, respectively(Table 2). In situ hybridization data obtained in BDM from different blood donors correlated well with results of ultrastruc-
128
FIG. 4. In situ hybridization analysis of viral infection of BDM with HIVl-LAV (A), HIVl-NDK
SCHMIDTMAYEROVA
ET AL.
DNA in BDM cultures after RNAse (B), and HIVl-PAR (C). Uninfected
tural analysis and with RT production. Variation of infectibility of macrophages from different donors was in agreement with the recent data by Olafsson e[ al, (1991). The infection of BDM with HIVl-LAVand HIVlNDK resulted in a low frequency of HIV1 -RNA-positive cells (0.3 and l%, respectively) and a much lower density of silver grains associated with BDM than that found in HIVl-PAR-infected cells (Fig. 3). Completely different distribution was obtained by in situ hybridization analysis of viral DNA (Fig. 4). In this case no significant differences between BDM infected with HIVlPAR, HIVl-LAV, or HIVl-NDK were found. A high frequency of HIV1 -DNA-containing cells was associated with all three HIV1 strains (Table 2). In contrast to significant difference in intensity of PCR signal between macrophage-tropic and T-lymphotropic viruses (Fig. 2) the results of in situ hybridization of DNA did not show a great difference in intensity.
treatment. The hybridization was BDM (D). Magnification, X205.
performed
10 days
after
This could be caused by a lower sensitivity of in situ hybridization, by differences in accessibility of HIV1 DNA target sequences in BDM, or by nonspecific hybridization. ln situ hybridization of viral RNA provided a control of specificity for DNA experiments: The same labeled viral probe was used and the same infected BDM target cells were analyzed by in situ hybridization of viral RNA and DNA. Therefore, the negative results of in situ hybridization of viral RNA in a great majority (>98%) of BDM infected with HIV1 -LAV and HIV1 -NDK (Fig. 3A and 3B) indicate that the signal obtained by in situ hybridization of viral DNA in these cells was specific. Electron
microscopy
examination
The BDM cultures were examined by electron microscopy 17 days after infection with the three HIV1
HIV1
INFECTION
OF
MACROPHAGES
g low RT FIG 5. Ultrastructure of HIV-PAR-infected BDM examined 17 days after infection. (A, B) HIVl-PAR-infected BDM cultures producrn level: ; show large number of secretory products (sp) processing from the Golgi apparatus. The higher magnification of the insert fr om A (B) show s the viral particles in the saccules of the cis and trans.Golgi complex (arrows) and the accumulation of viral particles (*) in the pros ;ecretoty (C) overall morphology of HIV1 -PAR-infected BDM producing high RT levels, with arrows indicating cytc oplasmic grant rle (pg) of the trans.Golgi. proce SSUS. Higher magnifications (D, E, F) of the insert from C shows the accumulation of viral particles (VP) in the hypertrophic tubular network (htn). Arrows indicate typical invagination of produced virus and the close association of some viral panrcles with secretory product: 3 (*). The relatic Inship of Golgi complex to the hypertrophic tubular network containing viral particles is shown (G). Peripheral portron of HIV1 -PAR -infected BDM (H, I) shows intracisternal viral partrcles and their presence also in the intercellular space (is; arrow). Magnification: A, Xl 1 ,200; B, D, X22,800; E, F, X58,400; G, I, X76,800; H, X9200. X28,1 100; C, X31,200;
130
SCHMIDTMAYEROVA
ET AL
FIG. 6. Ultrastructural alterations induced by HIV1 -NDK in BDM 17 days after infection. Cytoplasm of uninfected (A) and HIV1 -LAV-infected (B) BDM shows numerous mitochondria (m), endoplasmic reticulum (er) and lipidic droplets (Id). Ultrastructural changes induced by HIVl-NDK in the BDM are manifested by the accumulation of lysosomes (ly) and tuboreticular inclusions (tri) (C), which are exocyted (arrows) from BDM (D) as well as by cylindrical lamellar structures (E, arrows). In some lysosomes the presence of one or several internal dense “crystals” in the middle of a gray matrix (arrows) is shown (F, G). Intensive production of secretory vesicles (vs) and secretory granules (sg) by Golgi apparatus (g) (H) with the accumulation of secretory granules in the cytoplasm (I) is shown. Nucleus, n. Magnification: A, B, H, X31,200; C, D, Xl 6,800; E, I, Xl 2,400; F, G, X58,400.
HIV1 TABLE
INFECTION
3
ULTRASTRUCTURAL ANALYSIS OF HIV1 -INFECTED BLOOD DERIVED MACROPHAGES Detected
lntratubular viral particles ilV1 -PARb ilV1 -PAR” IIV-LAV Wl-NDK
cytopiasmic
structures
Viral particles associated with hypersecretion
-
10
80
-
-
-
(Yo)
TRI
CLS
HS
L
LCDC
-
-
100
+
t
-
-
+
10
-
10
5
20
Note. TRI, tuboreticular inclusions; CLS, cytoplasmic lamellar structures; HS, hypersecretion; L, lysosomes; LCDC, lysosomes containing dense “crystals.” a Over 100 cell sections from each infected culture were examined by electron microscopy. The numbers indicate the percentage of positive cell sections. f, few indicated structures were observed; -, no structures. b HIV1 -PAR-infected BDM producing low RT levels. c HIV1 -PAR-infected BDM producing high RT levels.
strains studied. Over 100 cell sections from each culture were analyzed. Different ultrastructural changes were observed in BDM prepared from the blood of two donors after infection with HIV1 -PAR in vitro. Surprisingly, hypersecretion was seen in 100% of cell sections of low RT level producing BDM, in which were found a relatively low number of viral particles in the saccules and prosecretory granules of Golgi complex. No hypersecretion was seen in BDM producing high amounts of RT, where high accumulation of viral particles in the hypertrophic tubular network (HTN) was observed in 80% of the cell sections (Fig. 5). HIV1 not only accumulated within HTN, but also assembled and budded from the vacuolar membranes producing typical invaginations. Only rare viral particles associated with the plasma membrane were observed in the infected macrophages (Orenstein et a/., 1988). In contrast to the lack of cytopathic changes in BDM infected with HIVl-LAV and HlVl-NDK as observed in optical microscopy, the ultrastructural analysis of BDM infected with HIVl-NDK revealed alterations represented by tuboreticular inclusions in more than 10% of cell sections analyzed and the accumulation of lysosomes and hypersecretion in 20% of cell sections. These structures were not observed in the noninfected controls or in the BDM infected with HIV1 -LAV (Fig. 6b; Table 3).
DISCUSSION Our results characterized further the biological properties of macrophage-tropic virus HIV1 -PAR (Cher-
OF
MACROPHAGES
131
mann, 1990). A striking difference was demonstrated in BDM between production of HIVl-PAR and that of two laboratory strains adapted to T-cell lines, HIVlLAV and HIV1 -NDK. In contrast to the virus production, no significant difference in the proportion of infected macrophages containing retrotranscribed HIV1 genomes was found between the macrophage-tropic and T-lymphotropic viruses. A vast majority of macrophages infected with any of the three HIV1 viruses tested contained HIV1 DNA, as detected by in situ hybridization. These results indicate that the strains that are poorly tropic for macrophages can enter these cells and retrotranscribe their genomes. The quantitative difference in amount of HIV1 DNA detected by PCR could be explained by a higher amount of macrophage-tropic virus particles entering individual cells. Alternatively, the difference between both groups of viruses occurs after their cell entry, perhaps at the level of retrotranscription or integration. These mechanisms are still poorly understood in macrophages and are the center of present investigation in this laboratory. A difference in the quantity of HIV1 DNA in macrophages infected with macrophage-tropic strain HIV1 -JR-FL and T-lymphotropic strain HIVl-NL4-3, as determined by PCR amplification, was recently demonstrated by O’Brien et a/. (1990). Comparison of a greater number of HIV1 strains by in situ hybridization studies is necessary to determine more generally the efficacy of HIV1 entry into macrophage cells. Both viruses adapted to T-cell lines, HIVl-LAV and HIVl-NDK, replicated poorly in BDM cultures. Although both of them produced comparably low amounts of p24gag into culture fluid, only HIVl-NDK was detected in the form infectious for T-cell lines after multiplication in BDM. Further experiments are necessay to decide whether HIV1 -LAV lost its T lymphotropism after passage in BDM or whether this difference resulted from a lower infectivity of HIV1 -LAV for T lymphocytes (Spire et al,, 1989). HIVl-NDK also induced ultrastructural alterations in BDM. This indicates that significant difference between the growth and cytopathic properties of HIV-LAV and HIVl-NDK demonstrated in T-lymphocytes (Spire et a/., 1989; Hirsch er al., 1992) have been reflected also in BDM cultures. Pathological changes very similar to those found in BDM infected with HIVl-NDK, and characterized by apparent tuboreticular inclusions, were detected in PBMC isolated from patients with AIDS and AIDS-related complex (Yoffe et al., 1989). The chimeric viruses between HlVl-LAV and HIV1 -NDK (Hirsch et a/., 1992) could be used to elucidate the genetic nature of these phenotypic differences. The correlation between development of disease and emergence of HIV1 variants with highercytopathic-
132
SCHMIDTMAYEROVA
ity in vitro was clearly documented in several laboratories (Cheng-Mayer eta/., 1988; Tersmette eta/., 1988). Very few data are available on evolution of macrophage-tropic viruses in infected humans and their evolutionary relationship to T-lymphotropic HIV1 viruses. The partial permissivity of macrophages for the Zairian virus HIV1 -NDK, highly cytopathic for T lymphocytes, might be of special interest from this point of view. Both HIVl-LAV and HlVl-NDK retained their infectivity for T-cells when rescued from BDM by direct cocultivation. This indicates that macrophages could efficiently spread HIV1 infection to secondary susceptible targets cells. ACKNOWLEDGMENTS We are grateful to Dr. C. Cotte (Regional Center of Blood Transfusion, Marseille) for kindly providing leukopheresis. We thank J. BailIon for his critical comments. I. Hirsch is a recipient of ANRS senior research fellowship. This work was financially supported by INSERM.
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HIV1
INFECTION
immunodeficiency virus(HIV) infection of the uterine cervix. Ann. Intern. Med. 108, 32 l-327. REY, M. A., SPIRE, B., DORMONT, D., BARR~~INOUSSI, F., MONTAGNIER, L., and CHERMANN, J. C. (1984). Characterization of the RNA dependent DNA polymerase of a new human T-lymphotropic retrovirus (lymphadenopathy associated virus). Biochem. Biophys. Res. Commun. 121, 126-133. REY, F., DONKER, G., HIRSCH, I., and CHERMANN, J. C. (1991). Productive infection of CD4+ cells by selected HIV strains is not inhibited by anti-CD4 monoclonal antibodies. Virology 181, 165-l 71. SPIRE, B.. SIRE, I., ZACHAR, V., REY, F., BARR&~INOUSSI, F., GALIBERT, F., HAMPE, A., and CHERMANN, 1. C. (1989). Nucleotide sequence of HIV1 -NDK: A highly cytopathic strain of the human immunodeficiency virus. Gene 81, 275-284. TERSMETTE. M., DE GOEDE, R. E. Y., AL, B. J. M., WINKEL, I. N., GRUTERS, R. A., CUYPERS, H. T., HUISMAN, H. G., and MIEDEMA, F. (1988). Differential syncytium-inducing capacity of human immunodeficiency virus isolates: Frequent detection of syncytiuminducing isolates in patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex. J. Viral. 62, 2026-2032. TSCHACHLER, E., GROH, V., POPOVIC, M., MANN, D. L.. KONRAD, K., SAFI. B., ERON, L., DIMARZO VERONESE, F., WOLFF, K., and STINGL, G.
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(1987). Epidermal Langerhans cells: A target for HTLV-IWLAV infection. /. Invest. Dermatol. 88, 233-237. VALENTIN, A., ALBERT, J., FENYB, E. M., and As~b, B. (1990). HIV-1 infection of normal human macrophage cultures: Implication for silent infection. Virology 177, 790-794. VON BRIESEN, H., BECKER, W. B., HENCO. K., HELM, E. B., GELDERBLOM, H. R., BREDE, H. D., and ROBSAMEN-WAIGMANN, H. (1987). Isolation frequency and growth properties of HIV-variants: Multiple simultaneous variants in a patient demonstrated by molecular cloning. /. Med. Vkol. 23, 51-66. YAHI, N., FANTINI, J., HIRSCH, I., and CHERMANN, J. C. (1992). Structural variability of env and gag gene products from a highly cytopathic strain of HIV1 Arch. Viol., 125, in press. YOFFE, B., PETRIE, B. L., NOONAN, C. A., and HOLLINGER, F. B. (1989). ln viva and in vitro ultrastructural alterations induced by human immunodeficiency virus in human lymphoid cells. Lab. Invest. 61, 303-309. ZIZA, J. M., BRUN-VEZINET, F., VENET, A., ROUZIOUX. C. H., TRAVERSAT, J., ISRAEL-BIET, B., BARRY-SINOUSSI, F., CHERMANN, J. C., and GODEAU, P. (1985). Lymphadenopathy-associated virus isolated from bronchoalveolar lavage fluid in AIDS-related complex with lymphoid interstitial pneumonitis. N. Engl. 1. Med. 313(3), 183.
190,
124-l
33 (1992)
Distinctive
Pattern of Infection and Replication in Blood-Derived Macrophages
HELENA SCHMIDTMAYEROVA,*+ IVAN HIRSCH,* *Unit& de Recherches Sciences, Bratislava,
INSERM U322 Czechoslovakia,
sur /es RBtrovirus and +Laboratoire Received
of HIV1 Strains
CHRISTINE BOLMONT,” STEPHEN BAGHDIGUIAN,+ JEAN-CLAUDE CHERMANN””
AND
et Maladies de Biologic February
AssociBes, Cellulaire
17, 1992;
Marseille, et Histologie,
accepted
May
France; tlnstitute of virology Facult6 de Mgdecine Nerd,
Slovak Academy Marseille, France
of
8, 1992
The macrophage-tropic virus HIVl-PAR, isolated from cerebrospinal fluid of HlVl-seropositive man, induced cytopathic effect accompanied by different magnitude of the virus production in blood-derived macrophages (BDM) obtained from different donors. HIVl-PAR-specific RNA was detected by in situ hybridization in 15 and 66% of BDM producing low and high levels of virus, respectively. In contrast with HIVl-PAR, infection of BDM with two laboratory strains adapted to T-cell lines, HIVl-LAV prototype and HIVl-NDK, a Zairian virus that is highly cytopathic for T-lymphocytes, resulted in a low production of HIV1 ~24~“~ in culture fluid. Expression of HIV1 -LAV and HIV1 -NDK RNA was detected by in situ hybridization in a maximum of 1% of macrophages. Only HIVl-NDK, and not HIVl-LAV, induced ultrastructural alterations in BDM. In contrast with a striking difference in the production of macrophage-tropic and T-lymphotropic viruses, no significant differences were found in the proportion of macrophages containing retrotranscribed genomes of HIV1 . HIV1 DNA was detected by in situ hybridization in 93,100, and 80% of macrophages infected with HIV1 -PAR, HIV1 -LAV, and HIV1 -NDK, respectively. A higher level of HIV1 DNA was detected by polymerase chain reaction in the BDM infected with HlVl-PAR than in that infected with HIVl-LAV and HIVl-NDK. The results indicate that both macrophage-tropic as well as T-lymphotropic viruses can enter and retrotranscribe their genomes in a vast majority of macrophages. 0 1992 Academic Press, 1~.
INTRODUCTION
1990) hidden from the host immune surveillance. These cells, harboring the virus, could function as a long-time reservoir of HIV infection. Nonproductively infected cells may spread infection to other susceptible cells over a long period of time by different mechanisms (Ho et a/., 1986; Mann et a/., 1990; Valentin et a/., 1990). HIV1 infection of macrophages in vitro shows a high variation in the replicative capacity for different viral strains. Most laboratory strains of HIV1 after long-term passage in T-cell lines replicate poorly or not at all in macrophages (Nicholson et a/., 1986; Gendelman et al., 1989; Collman eta/., 1989). A specific region of the envelope gpl20 gene was found to determine the macrophage tropism (O’Brien et al., 1990; Cheng-Mayer et al., 1991; Hwang et al., 1991). It was suggested in these studies that the major block of HIV1 replication in macrophages might be an inefficient virus/cell entry. The aim of the present study was the characterization and the comparison of infectibility of blood-derived macrophages (BDM) with the macrophage-tropic strain HIVl-PAR, isolated in this laboratory and two other laboratory strains adapted to the T-cell lines, HIV1 -LAV prototype and HIV1 -NDK, a Zairian virus that is highlycytopathic for T-lymphocytes. The results indi-
Cells of monocyte/macrophage lineage are an important target for human immunodeficiencyvirus (HIV). Infected macrophages were detected in the brains (Gartner eta/., 1986; Koenig eta/., 1986), lungs (Ziza et a/., 1985; Chayt et a/., 1986; Gartner et al., 1986), and other tissues (Tschachler et al., 1987; Pomerantz et al., 1988) of patients with acquired immune deficiency syndrome (AIDS). Monocyte/macrophages have important immunoregulatory functions, which could be compromised by HIV infection. A variety of pathological changes have been reported in monocytes from HIV-infected patients (Bender et a/., 1988), which could contribute to the impairment of the immune system in AIDS patients. Monocyte/macrophages are supposed to be among the first targets of HIV infection in a human organism (Ho et al., 1986). Virus could persist in the macrophages for a prolonged period of time within intracellular vacuoles (Orenstein et a/., 1988; Meltzer et al.,
’ To whom reprint requests should be addressed at INSERM Unit6 322, Unit6 de Recherches sur les Rktrovirus et Maladies Associires, Campus Universitaire de Luminy, BP 33, 13273 Marseille, c6dex 9. 0042-6822192
$5.00
CopyrIght 0 1992 by Academic Press, Inc. All rights of reproduction I” any form reserved
124
HIV1
OF
MACROPHAGES
125
A
60 -
INFECTION
50
“0 -I x 40 E
A
-
IOTCIU
-*-
100 TCIU
E, 30 u 2.P :I 20 2 & ‘Y 10 0
01 7
11
14
16
21
24
27
7
11
14
Days after infection
of HIV1 -PAR in PBMC FIG. 1. (A) Repkation both PBMC and CBL cultures. (B) Replication determined by virus titration on CBL
and CBL. The viral inoculum containing of HIV1 -PAR in BDM. Cultures of BDM
cate that both T-lymphotropic viruses were able to enter and retrotranscribe their genome in a vaste majority of macrophages. MATERIALS isolation
and culture
AND
METHODS
of BDM
Peripheral blood mononuclear cells (PBMC) of healthy donors undergoing leukopheresis were separated on Ficoll-Hypaque gradients. Suspensions of 8 X 1 O6cells/ml, prepared in RPMI 1640 medium supplemented with 10% fetal calf serum, 5% normal human serum, 10 mM HEPES buffer and antibiotics (PSN), were allowed to adhere for 2 hr at 37”. After removal of nonadherent cells by extensive washing, the cells were cultured in the presence of 50 U/ml of recombinant
TABLE
1
INFECTION OF BLOOD-DERIVED MACROPHAGES WITH HlVlmLAV AND HIV&NDK
BDM RT activity
Infectious virusa
+
-
+
-
24
27
~24 bgiml)
of as
human granulocyte-macrophage colony stimulating factor (GMCSF, Genzyme). Resting nonadherent cells were removed by additional washing 2 and 5 days after isolation. On Day 5 more than 90% of the cells consisted of macrophages, as determined by reactivity with anti-CD1 4 monoclonal antibody (IOM2, Immunotech S.A.) and by cytochemical staining for nonspecific esterase (Alfa-Naphtyl Acetate Esterase Kit, Sigma Diagnostics). Viruses and infection
of BDM
Three different strains of HIV1 were used in the study: HIV1 -PAR, a strain isolated from the cerebrospinal fluid of an HIVl-seropositive man suffering from acute encephalopathy (Gout et al., 1988; Chermann, 1990), the HIVl-LAV prototype strain (Barre-Sinoussi et a/., 1983), and the Zairian strain HIV1 -NDK (Ellrodt, 1984). The stocks of HIV1 -LAV and HIV1 -NDK were prepared and titrated on CEM cells and stocks of HIV1 PAR on cord blood lymphocytes (CBL) (Rey et al., 1991). BDM cultures were infected 5 days after isolation. Cells were washed and incubated with 1000 tis-
Cocultivation with CEMb E
-
21
1.5 X 1 O4 cpm/ml of RT activity was used for infection were infected with 100, 10, and 1 TCIU of HIVl-PAR,
2 Donor 1 HIV1 -LAV HIVl-NDK Donor 2 HIV1 -LAV HIVl-NDK
18
Days after infection
31 330
+ +
11 30
+ +
a Culture fluid from 10 days infected BDM was filtered through 0.45 pm Millipore filter and 2-ml aliquots were used for infection of 2 x 10” CEM cells. b The CEM cells at the final concentration of 5 X 1 O5 cells/ml were added to the BDM cultures 10 days after infection.
HIVI-LAV -
HIVl-NDK ~
-HIM-PAR
of HIV1 specific DNA by amplification of 172-bp FIG. 2. Detection fragment from HIV1 ral gene. Cell lysates prepared 10 days after BDM infection with HIV+LAV, HIVl-NDK, and HIVl-PAR and containing about 100 ng of DNA were treated with 50 U/ml of RNase (lane 2) or RNase free DNase (lane 3), amplified and analyzed by Southern blot hybridization. Lane 1 represents nontreated control.
126
SCHMIDTMAYEROVA TABLE
2
HIV-1 -SPECIFIC RNA AND DNA DETECTED s~hvSm IN BDM, 3 AND 10 DAYSAFTER~NFECTION
HYBRIDIZATION
% Frequency RNA-positive
HIV1 -PARb HIV1 -PARC HIV&LAV HIVl-NDK
cells
DNA-positive
cells
3 days
10 days
3 days
10 days
8 6 ND ND
15 66 0.3 1
96 ND 97 ND
93 ND 100 80
aThe frequency was calculated from a counted cells for cultures with high and low tively. b HIV1 -PAR-infected BDM producing low ’ HIV+PAR-infected BDM producing high
total of 500 and 6000 positivity rate, respecRT levels. RT levels.
sue culture infectious units (TCIU) of HIVl-LAV or HlVl-NDK (corresponding to 5 X 1O4 cpm/ml of reverse transcriptase (RT) activity) or with 100 TCIU of HIV1 -PAR (corresponding to 3 X 1 O4 cpm/ml of RT activity) for 2 hr at 37”. After adsorption and another washing, a fresh medium containing 50 U/ml of GMCSF was added. Reverse transcriptase
and p24 antigen
assay
The activity of RT was measured by standard RT assay (Rey et a/., 1984) in the 0.1% Triton X-l 00 disrupted viral pellet prepared from 1 ml of culture fluid. The p24 antigen capture assay was done according to the manufacturer’s directions (DuPont). In situ hybridization The 9-kb Sacl fragment of plasmid pBT1 containing the HIV1 -LAV prototype nucleotide sequence (Alizon et al., 1984) was used as a hybridization probe. Detection of viral RNA. BDM cultured on chamber slides (Nunc) were fixed with 4% paraformaldehyde and treated with 1 pglml of Proteinase K. Hybridization was carried out overnight at 37” in a solution containing 50% formamide; 4 X SSC (600 mM NaCI, 60 mM sodium citrate); 0.02% each of Ficoll, polyvinylpyrrolidone, and bovine serum albumine; and 400 pg/ml each of herring sperm DNA, salmon testis DNA, and yeast RNA. [35S]DNA probe labeled by nick translation was denatured at 100” for 3 min and used at a final conceniration of 0.3 pg/ml. After autoradiography performed at 4” for 2 to 7 days, the cells were stained with eosinhematoxylin. Detection of viral DNA. To detect specific HIV1 DNA, the fixed infected BDM were treated by Proteinase K as
ET AL.
described above. A total intracellular RNA was then digested with 200 pg/ml of RNAse type A. The intracellular DNA on chamber slides was subsequently denatured at 100” for 3 min together with hybridization mixture. Detection of HI//l-specific chain reaction (PCR)
DNA by polymerase
Approximately 7 X 1 O5 BDM were lysed with 1 ml of PCR buffer (Higuchi, 1989). After protein digestion, 25 ~1of cell lysate were subjected to 30 cycles of polymerase chain reaction in a total volume of 50 ~1 containing 10 pmol of oligonucleotide primers, nt 5419-5446 and 5563-5590 of HIV1 -LAV tat sequence (CisBio International), 200 PLM of each deoxynucleotide, 50 mM KCI, 10 mMTris, pH 8.3, 1.5 mM MgCI,, and 1.2 U of Taq polymerase (Cetus Corp.). Each cycle comprised 1 min denaturation step (94”), 1.5 min annealing step (SS’C), and 2 min extension (72°C). After agarose gel electrophoresis, amplified DNA was analyzed by Southern blot hybridization with 3*P-labeled probe corresponding to the HIV1 -LAV tat sequence between nucleotides 5447 and 5474 (CisBio International). Electron
microscopy
examination
BDM grown in 25-cm* plastic flasks were fixed with 2.5% glutaraldehyde and processed for electron microscopy as described by Gelderblom et al. (1987). RESULTS Replication of HIVl-PAR in peripheral blood mononuclear cells, cord blood lymphocytes, and blood derived macrophages Production of HIV1 -PAR in PBMC and CBL cells was examined in cell-free supernatants at 3-4 day intervals (Fig. 1A). The CBL, which are less differentiated than PBMC, allowed a rapid and high replication of HIVlPAR. The same virus had a property of slow/low virus in PBMC (Asjo et a/., 1986; Von Briesen et al., 1987; Tersmette et al., 1988). The transient cytopathic effect, characterized by marked vacuolization of cytoplasm, was observed in CBL 3-4 days before virus production (not shown). No cytopathic changes were seen in PBMC infected with HIV1 -PAR. These results illustrate variability in the biological properties of viral isolate that depends on the cell culture system used. With respect to the high production of HIV-PAR in CBL, we used these cells for preparation and standardization of viral stock for infection of BDM. Five-day-old cultures of BDM grown in 24-well plates were infected with 100, 10, and 1 TCIU determined by the virus titration on CBL. Virus production was dependent on the
HIV1
FIG. 3. in sifu hybridization analysis of viral RNA in BDM HIVl-LAV (A), HIVl-NDK (B), and HIVl-PAR (C); uninfected
viral inoculum dose (Fig. 1 B) and accompanied mation of multinucleated giant cells. Infection of blood-derived LAV and HIV1 -NDK
macrophages
INFECTION
of HIV1 -specific
MACROPHAGES
10 days after infection BDM (D). Magnification,
by for-
with HIV1 -
The BDM grown in 24-well plates were infected with 1000 TCIU of HIVl-LAV and HIVl-NDK. RT production or cytopathic changes were never observed in BDM cultures obtained from different donors. However, infection of BDM with both T-lymphotropic viruses could be demonstrated by p24 antigen production and after cocultivation with CEM (Table 1). Production of infectious virus into the filtered culture fluid was demonstrated only after infection with HIV1 -NDK. Detection
OF
proviral DNA in BDM
Cell lysates prepared from BDM 10 days after infection were treated with 50 U/ml of RNAse or DNAse
with
three X205.
127
different
HIV1
strains.
BDM
cultures
infected
with
before amplification. The specific viral DNA was detected in both HIVl-LAV- and HIVl-NDK-infected BDM, although in a lower quantity than that found in the BDM infected with HIVl-PAR (Fig. 2). The results indicate that all three viruses were able to enter BDM and retrotranscribe their genomes. Higher levels of HIV1 provirus copy number were present in HIVl-PAR infected BDM than in BDM infected with T-lymphotropit viruses. In situ hybridization
analysis of viral RNA and DNA
Ten days after infection, the frequency of BDM productively infected with HIVl-PAR detected by in situ hybridization reached 15 and 66% in the cultures producing low and high RT levels, respectively(Table 2). In situ hybridization data obtained in BDM from different blood donors correlated well with results of ultrastruc-
128
FIG. 4. In situ hybridization analysis of viral infection of BDM with HIVl-LAV (A), HIVl-NDK
SCHMIDTMAYEROVA
ET AL.
DNA in BDM cultures after RNAse (B), and HIVl-PAR (C). Uninfected
tural analysis and with RT production. Variation of infectibility of macrophages from different donors was in agreement with the recent data by Olafsson e[ al, (1991). The infection of BDM with HIVl-LAVand HIVlNDK resulted in a low frequency of HIV1 -RNA-positive cells (0.3 and l%, respectively) and a much lower density of silver grains associated with BDM than that found in HIVl-PAR-infected cells (Fig. 3). Completely different distribution was obtained by in situ hybridization analysis of viral DNA (Fig. 4). In this case no significant differences between BDM infected with HIVlPAR, HIVl-LAV, or HIVl-NDK were found. A high frequency of HIV1 -DNA-containing cells was associated with all three HIV1 strains (Table 2). In contrast to significant difference in intensity of PCR signal between macrophage-tropic and T-lymphotropic viruses (Fig. 2) the results of in situ hybridization of DNA did not show a great difference in intensity.
treatment. The hybridization was BDM (D). Magnification, X205.
performed
10 days
after
This could be caused by a lower sensitivity of in situ hybridization, by differences in accessibility of HIV1 DNA target sequences in BDM, or by nonspecific hybridization. ln situ hybridization of viral RNA provided a control of specificity for DNA experiments: The same labeled viral probe was used and the same infected BDM target cells were analyzed by in situ hybridization of viral RNA and DNA. Therefore, the negative results of in situ hybridization of viral RNA in a great majority (>98%) of BDM infected with HIV1 -LAV and HIV1 -NDK (Fig. 3A and 3B) indicate that the signal obtained by in situ hybridization of viral DNA in these cells was specific. Electron
microscopy
examination
The BDM cultures were examined by electron microscopy 17 days after infection with the three HIV1
HIV1
INFECTION
OF
MACROPHAGES
g low RT FIG 5. Ultrastructure of HIV-PAR-infected BDM examined 17 days after infection. (A, B) HIVl-PAR-infected BDM cultures producrn level: ; show large number of secretory products (sp) processing from the Golgi apparatus. The higher magnification of the insert fr om A (B) show s the viral particles in the saccules of the cis and trans.Golgi complex (arrows) and the accumulation of viral particles (*) in the pros ;ecretoty (C) overall morphology of HIV1 -PAR-infected BDM producing high RT levels, with arrows indicating cytc oplasmic grant rle (pg) of the trans.Golgi. proce SSUS. Higher magnifications (D, E, F) of the insert from C shows the accumulation of viral particles (VP) in the hypertrophic tubular network (htn). Arrows indicate typical invagination of produced virus and the close association of some viral panrcles with secretory product: 3 (*). The relatic Inship of Golgi complex to the hypertrophic tubular network containing viral particles is shown (G). Peripheral portron of HIV1 -PAR -infected BDM (H, I) shows intracisternal viral partrcles and their presence also in the intercellular space (is; arrow). Magnification: A, Xl 1 ,200; B, D, X22,800; E, F, X58,400; G, I, X76,800; H, X9200. X28,1 100; C, X31,200;
130
SCHMIDTMAYEROVA
ET AL
FIG. 6. Ultrastructural alterations induced by HIV1 -NDK in BDM 17 days after infection. Cytoplasm of uninfected (A) and HIV1 -LAV-infected (B) BDM shows numerous mitochondria (m), endoplasmic reticulum (er) and lipidic droplets (Id). Ultrastructural changes induced by HIVl-NDK in the BDM are manifested by the accumulation of lysosomes (ly) and tuboreticular inclusions (tri) (C), which are exocyted (arrows) from BDM (D) as well as by cylindrical lamellar structures (E, arrows). In some lysosomes the presence of one or several internal dense “crystals” in the middle of a gray matrix (arrows) is shown (F, G). Intensive production of secretory vesicles (vs) and secretory granules (sg) by Golgi apparatus (g) (H) with the accumulation of secretory granules in the cytoplasm (I) is shown. Nucleus, n. Magnification: A, B, H, X31,200; C, D, Xl 6,800; E, I, Xl 2,400; F, G, X58,400.
HIV1 TABLE
INFECTION
3
ULTRASTRUCTURAL ANALYSIS OF HIV1 -INFECTED BLOOD DERIVED MACROPHAGES Detected
lntratubular viral particles ilV1 -PARb ilV1 -PAR” IIV-LAV Wl-NDK
cytopiasmic
structures
Viral particles associated with hypersecretion
-
10
80
-
-
-
(Yo)
TRI
CLS
HS
L
LCDC
-
-
100
+
t
-
-
+
10
-
10
5
20
Note. TRI, tuboreticular inclusions; CLS, cytoplasmic lamellar structures; HS, hypersecretion; L, lysosomes; LCDC, lysosomes containing dense “crystals.” a Over 100 cell sections from each infected culture were examined by electron microscopy. The numbers indicate the percentage of positive cell sections. f, few indicated structures were observed; -, no structures. b HIV1 -PAR-infected BDM producing low RT levels. c HIV1 -PAR-infected BDM producing high RT levels.
strains studied. Over 100 cell sections from each culture were analyzed. Different ultrastructural changes were observed in BDM prepared from the blood of two donors after infection with HIV1 -PAR in vitro. Surprisingly, hypersecretion was seen in 100% of cell sections of low RT level producing BDM, in which were found a relatively low number of viral particles in the saccules and prosecretory granules of Golgi complex. No hypersecretion was seen in BDM producing high amounts of RT, where high accumulation of viral particles in the hypertrophic tubular network (HTN) was observed in 80% of the cell sections (Fig. 5). HIV1 not only accumulated within HTN, but also assembled and budded from the vacuolar membranes producing typical invaginations. Only rare viral particles associated with the plasma membrane were observed in the infected macrophages (Orenstein et a/., 1988). In contrast to the lack of cytopathic changes in BDM infected with HIVl-LAV and HlVl-NDK as observed in optical microscopy, the ultrastructural analysis of BDM infected with HIVl-NDK revealed alterations represented by tuboreticular inclusions in more than 10% of cell sections analyzed and the accumulation of lysosomes and hypersecretion in 20% of cell sections. These structures were not observed in the noninfected controls or in the BDM infected with HIV1 -LAV (Fig. 6b; Table 3).
DISCUSSION Our results characterized further the biological properties of macrophage-tropic virus HIV1 -PAR (Cher-
OF
MACROPHAGES
131
mann, 1990). A striking difference was demonstrated in BDM between production of HIVl-PAR and that of two laboratory strains adapted to T-cell lines, HIVlLAV and HIV1 -NDK. In contrast to the virus production, no significant difference in the proportion of infected macrophages containing retrotranscribed HIV1 genomes was found between the macrophage-tropic and T-lymphotropic viruses. A vast majority of macrophages infected with any of the three HIV1 viruses tested contained HIV1 DNA, as detected by in situ hybridization. These results indicate that the strains that are poorly tropic for macrophages can enter these cells and retrotranscribe their genomes. The quantitative difference in amount of HIV1 DNA detected by PCR could be explained by a higher amount of macrophage-tropic virus particles entering individual cells. Alternatively, the difference between both groups of viruses occurs after their cell entry, perhaps at the level of retrotranscription or integration. These mechanisms are still poorly understood in macrophages and are the center of present investigation in this laboratory. A difference in the quantity of HIV1 DNA in macrophages infected with macrophage-tropic strain HIV1 -JR-FL and T-lymphotropic strain HIVl-NL4-3, as determined by PCR amplification, was recently demonstrated by O’Brien et a/. (1990). Comparison of a greater number of HIV1 strains by in situ hybridization studies is necessary to determine more generally the efficacy of HIV1 entry into macrophage cells. Both viruses adapted to T-cell lines, HIVl-LAV and HIVl-NDK, replicated poorly in BDM cultures. Although both of them produced comparably low amounts of p24gag into culture fluid, only HIVl-NDK was detected in the form infectious for T-cell lines after multiplication in BDM. Further experiments are necessay to decide whether HIV1 -LAV lost its T lymphotropism after passage in BDM or whether this difference resulted from a lower infectivity of HIV1 -LAV for T lymphocytes (Spire et al,, 1989). HIVl-NDK also induced ultrastructural alterations in BDM. This indicates that significant difference between the growth and cytopathic properties of HIV-LAV and HIVl-NDK demonstrated in T-lymphocytes (Spire et a/., 1989; Hirsch er al., 1992) have been reflected also in BDM cultures. Pathological changes very similar to those found in BDM infected with HIVl-NDK, and characterized by apparent tuboreticular inclusions, were detected in PBMC isolated from patients with AIDS and AIDS-related complex (Yoffe et al., 1989). The chimeric viruses between HlVl-LAV and HIV1 -NDK (Hirsch et a/., 1992) could be used to elucidate the genetic nature of these phenotypic differences. The correlation between development of disease and emergence of HIV1 variants with highercytopathic-
132
SCHMIDTMAYEROVA
ity in vitro was clearly documented in several laboratories (Cheng-Mayer eta/., 1988; Tersmette eta/., 1988). Very few data are available on evolution of macrophage-tropic viruses in infected humans and their evolutionary relationship to T-lymphotropic HIV1 viruses. The partial permissivity of macrophages for the Zairian virus HIV1 -NDK, highly cytopathic for T lymphocytes, might be of special interest from this point of view. Both HIVl-LAV and HlVl-NDK retained their infectivity for T-cells when rescued from BDM by direct cocultivation. This indicates that macrophages could efficiently spread HIV1 infection to secondary susceptible targets cells. ACKNOWLEDGMENTS We are grateful to Dr. C. Cotte (Regional Center of Blood Transfusion, Marseille) for kindly providing leukopheresis. We thank J. BailIon for his critical comments. I. Hirsch is a recipient of ANRS senior research fellowship. This work was financially supported by INSERM.
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