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The Journal of Immunology, 1998, 161: 5712-5719.
Copyright © 1998 by The American Association of Immunologists

Human Complement Component C1q Inhibits the Infectivity of Cell-Free HTLV-I1

Fumihiro Ikeda*,{dagger}, Yuji Haraguchi{dagger}, Atsushi Jinno{dagger}, Yuichi Iino{ddagger}, Yasuo Morishita*, Hiroshi Shiraki§ and Hiroo Hoshino2,{dagger}

* Second Department of Surgery and Departments of {dagger} Hygiene and Virology and {ddagger} Emergency and Critical Care Medicine, Gunma University School of Medicine, Maebashi, Gunma, Japan; and § Fukuoka Red Cross Blood Center, Chikushino, Fukuoka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human T cell leukemia virus type I (HTLV-I) is a retrovirus that is not lysed by human serum or complement. It has not been determined, however, whether HTLV-I directly binds to complement components or whether it retains infectivity after incubation with human serum. We investigated the effects of human serum on the infectivity of cell-free HTLV-I produced by human and animal cells. Plating of vesicular stomatitis virus (HTLV-I) pseudotypes prepared in cat or human cells and formation of HTLV-I DNA after infection of cell-free HTLV-I produced by cat or human cells were markedly inhibited by treatment with fresh human serum, but not by heat-inactivated serum. HTLV-I infection was also inhibited by treatment with C2-, C3-, C6-, or C9-deficient serum, but not by C1q-deficient serum. Inhibitory activities of normal human serum against HTLV-I were neutralized by anti-C1q serum. Furthermore, purified C1q inhibited HTLV-I infection. The direct binding of C1q to HTLV-I was confirmed by comigration of C1q with HTLV-I virion upon sucrose density gradient ultracentrifugation of HTLV-I virion treated with C1q. Binding assay using synthetic envelope peptides indicated that C1q bound to an extramembrane region of the gp21 transmembrane protein. These findings indicate that the human complement component C1q inactivates HTLV-I infectivity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human T cell leukemia virus type I (HTLV-I)3 is a retrovirus that is the etiologic agent for adult T cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis. It is endemic in the Caribbean, Jamaica, South America, southern United States, equatorial Africa, and southern Japan (1, 2, 3). Epidemiologic studies have shown that the main routes of transmission of HTLV-I are mother-to-child, sexual, and iatrogenic (4, 5, 6). Mother-to-child infection of HTLV-I takes place in utero or through perinatal or postnatal pathways. In particular, milk-borne infection is implicated as a major pathway. HTLV-I is estimated to be transmitted from seropositive mothers to their children through breast milk at a rate of 20–30% (7). In addition, several studies have suggested the sexual transmission of HTLV-I is more frequent from males to females than from females to males. Kajiyama et al. estimated that >60% of female spouses of HTLV-I-positive husbands will be infected with HTLV-I over a 10-yr period, while <1% of husbands will seroconvert over a similar time period (8). For iatrogenic transmission due to blood transfusion, seroconversion rates of 63% in recipients of cellular components and of 0% in recipients of plasma have been reported (9). These epidemiologic findings suggest that infection of HTLV-I will not occur after mere exposure of an individual to the virus or virus-infected cells (5). There may be a nonspecific protection system against HTLV-I transmission in humans.

Complement is a component of the immune system that has a number of important functions in the absence or presence of Abs, such as opsonization and virolysis, and affects interactions with viruses and virus-infected cells (10, 11, 12). Many animal retroviruses activate the classical complement pathway independent of specific Ab. This activation is due to the direct binding of the first component of complement C1q to the viral envelope, resulting finally in the lysis of virions as detected by the release of virion reverse transcriptase by complement lytic system (13, 14, 15).

We previously showed that HTLV-I represents the first retrovirus that is resistant to lysis by the human complement system, unlike other animal retroviruses (16). HIV-1 is also resistant to virolysis by human complement (17). While the infectivity of HIV-1 is not inactivated by human serum (17, 18, 19, 20), it has not yet been determined whether HTLV-I, like HIV-1, retains infectivity after treatment with human complement. So far, it has been very difficult to detect infection of cells with cell-free HTLV-I quantitatively. Analyses of infection with cell-free HTLV-I have been done mainly using the pseudotype of vesicular stomatitis virus (VSV)-bearing HTLV-I envelope (Env) Ags (21, 22). The reason for the very low infection titer of HTLV-I is not clear, but may be due to a small amount of weakly infectious HTLV-I produced in culture supernatants.

Recently, we have developed a sensitive assay system using PCR to detect infection of cell-free HTLV-I. There is a close correlation between results obtained from the PCR and the VSV(HTLV-I) pseudotype assay (23, 24). These two assays were used to investigate the effects of human complement on the infectivity of cell-free HTLV-I. It is known that human complement is activated much more efficiently by animal cells or by virus produced from animal cells (15). This is thought to be due mainly to the presence of natural Abs against Gal({alpha}1–3)Gal terminal carbohydrates in human serum (25, 26). Therefore, we used HTLV-I produced by cat as well as human cells.

In this report, we demonstrate the inhibitory effect of human complement, especially C1q, on the infection of cell-free HTLV-I and investigate a possible mechanism for this inhibition. To our knowledge, this report is the first to show that purified complement component C1q inactivates the infectivity of viruses, including retroviruses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and HTLV-I

c77 and S+L-HOS/SI-5 cells were used as HTLV-I-producing cells. c77 is a subclone of S+L-CCC (clone 8C) feline kidney cell line infected with HTLV-I derived from a Japanese ATL patient (HTLV-I2M) (27). S+L-HOS/SI-5 is derived from the human osteosarcoma cell line, HOS (28), and infected with Melanesian HTLV-I strain MEL5, which is produced by SI-5 cells (29). c77 cells were maintained in Eagle’s MEM supplemented with 10% FCS and S+L-HOS/SI-5 cells in DMEM at 37°C in a humidified, 5% CO2 atmosphere. For preparation of cell-free HTLV-I, c77 or S+L-HOS/SI-5 cells were seeded at 5 x 105 cells/ml and incubated for 2 days. Then the medium was replaced by fresh medium, which was harvested after incubation for another 24 h. Cells and debris were removed from the culture supernatants, and the supernatants were dispensed into small aliquots and stored at -110°C until use.

Human serum and complement sources

Fresh human sera were obtained from eight healthy, HTLV-I-seronegative Japanese. Fresh sera were measured for 50% complement-hemolytic activities (CH50) by the Mayer’s method (30). As for heat inactivation, sera were treated at 56°C for 30 min. The CH50 of each of the eight fresh serum samples was 36.0 ± 3.2; these hemolytic activities were completely inactivated by heat treatment. Sera that were deficient in one of the complement components (i.e., C1q-, C3-, C6- or C9-deficient sera) and complement components C1q, C3, C6, and C9 were purchased from Sigma (St. Louis, MO). C1q was also purchased from Chemicon (Temecula, CA). The CH50 of each serum sample deficient in one of the complement components was ~0.2 (0.5% of the activity level of normal fresh serum), and their activities regained up to 60% of the control level by addition of one of the deficient complement components.

ELISA for detection of HTLV-I p19 and C1q

A sandwich-type ELISA was used for detection of HTLV-I p19 (31) or C1q. Wells of enzyme immunoassay (EIA) high-binding plates (Costar, Cambridge, MA) were coated with mouse mAb against HTLV-I Gag protein p19 or C1q and incubated at 4°C overnight. Then the wells were washed three times with PBS(-), and nonspecific binding was blocked by incubation with PBS containing 3% BSA and 0.02% NaN3. Immunosorbent reaction was done at room temperature for 4 h. HTLV-I-seropositive human serum was diluted and added to each well. Then alkaline phosphatase-conjugated goat anti-human IgG (Sigma) was placed into the wells. After three washes with distilled water, alkaline phosphatase substrate pNPP (Bio-Rad, Hercules, CA) was added, and ODs at 405 nm were determined. Anti-C1q mouse mAb (Biogenesis, New Fields, U.K.), goat anti-C1q polyclonal Ab (Organon Teknika, Durham, NC), and alkaline phosphatase-conjugated rabbit anti-goat IgG (American Qualex, San Clemente, CA) were used to detect C1q.

VSV(HTLV-I) pseudotype assay

VSV pseudotype bearing HTLV-I Env Ags (HTLV-I pseudotype) was prepared and assayed as described (23). Briefly, HTLV-I-producing c77 feline cells or HOS/SI-5 human cells were infected with VSV (Indiana serotype) and culture medium containing VSV(HTLV-I) pseudotypes were harvested 12–15 h later and stored at -110°C until use. S+L-CCC (clone 8C) indicator cells were seeded at 2 x 105 cells/35-mm plate and infected with VSV pseudotype the next day. VSV pseudotypes were treated with serially diluted human serum or with serum deficient in one of the complement components at 37°C for 1 h before infection. Eagle’s MEM containing 1% agarose was overlaid on the cells, and plaques were counted 24 h later.

Infection with cell-free HTLV-I for PCR

HTLV-I DNA was detectable in 8C cells by PCR when 8C cells were infected with culture supernatants of c77 cells 1 day previously (24). Thus, 8C cells were seeded at 2 x 105 cells/35-mm plate, incubated for 1 day, and infected with cell-free HTLV-I produced by c77 or S+L-HOS/SI-5 cells. HTLV-I produced by S+L-HOS/SI-5 cells was centrifuged and concentrated 10-fold before infection. HTLV-I was treated with serum at 37°C for 1 h before infection. Inocula were removed, and the cells were washed once with PBS(+), then fresh medium was added to the dish. After incubation for 24 h, the cells were washed once with PBS(-) and lysed with 10 mM Tris-HCl (pH 8.3) containing 1 mM EDTA, 0.45% Nonidet P-40 (Sigma), 0.45% Tween 20 (Sigma), and 20 µg/ml proteinase K (Sigma). The lysates were incubated at 52°C for 2 h, heated for 10 min at 96°C to inactivate proteinase K, and stored at -20°C until use.

PCR to detect HTLV-I DNA

HTLV-I DNA in acutely infected 8C cells was amplified with thermostable Taq DNA polymerase in a thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). The sequences of oligonucleotides used as PCR primers were: pX 7302–7326 (5'-CCCACTTCCCAGGGTTTGGACAGAG-3') and pX 7504–7481 (3'-CTGTAGAGCTGAGCCGATAACGCG-5') (32). The cell lysates were mixed with 5 µM each of the pX primers. The mixture was first heated at 96°C for 6 min, then four kinds of deoxyribonucleoside triphosphates and Taq DNA polymerase were added to the mixture. The mixture was amplified for 35 PCR cycles as described (33). The amplified PCR reaction mixture was resolved by electrophoresis through gels containing 2% NuSieve agarose (FMC BioProducts, Rockland, ME) and 1% Takara H-14 agarose (Takara Shuzo, Kyoto, Japan) containing 0.5 µg/ml of ethidium bromide. The gels were photographed under UV light using Polaroid film. The length of the amplified DNA was calculated to be 203 bp.

Binding of C1q to HTLV-I virions

Virus particles produced by c77 cat cells or S+L-HOS/SI-5 human cells were concentrated by centrifugation at 27 K rpm for 2 h using a SRP-28 rotor (Hitachi Koki, Tokyo, Japan), and virus pellets were suspended with TNE buffer (20 mM Tris-HCl (pH7.5), 100 mM NaCl, 1 mM EDTA) (16). The viral suspension (50 µl) was mixed with C1q in TNE buffer (50 µl, 500 µg/ml) and incubated at 37°C for 1 h. Then the mixture was layered onto a continuous sucrose gradient (20–60% sucrose in TNE) and centrifuged in an SRP-28 rotor at 27 K rpm for 20 h. Each sample was fractionated into 14–15 tubes (34, 35). HTLV-I and C1q were detected by ELISA for HTLV-I Gag protein p19 and for C1q, respectively, as described above.

Binding assay of C1q to HTLV-I proteins

HTLV-I was lysed by addition of Nonidet P-40 (0.2% v/v) and placed into ELISA plates that had been coated with a 1:100 dilution of anti-HTLV-I Ab-positive human serum overnight. C1q in PBS was added to wells of these plates and incubated at 37°C for 1 h, after which time bound C1q was detected by ELISA.

Binding assay of C1q to HTLV-I Env oligopeptides

Fifteen overlapped synthetic peptides of gp46 and eight peptides of gp21 were produced as described elsewhere (36, 37, 38). Each synthetic peptide in distilled water (50 µl, 50 µg/ml) was placed into the wells of ELISA plates and incubated at 4°C overnight. C1q in PBS (50 µl, 10 µg/ml) was added and incubated at 37°C for 1 h. The binding of C1q to Env peptide-coated wells was detected by ELISA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lack of HTLV-I lysis by human serum

We previously showed that HTLV-I is not lysed by human serum, since reverse transcriptase is not released from virions (16). We confirmed this finding using HTLV-I strains and human serum in this study: ELISA for HTLV-I Gag protein p19 showed that p19 was not released from HTLV-I virions produced by c77 cat cells or S+L-HOS/SI-5 human cells even after treatment of HTLV-I with fresh human serum (Fig. 1Go). Nonidet P-40 was used as the virolysis control.



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  		FIGURE 1. Lack of virolysis by human serum. HTLV-I virion suspension (25 µl) was mixed with an equal volume of fresh or heat-inactivated (56°C for 30 min) human serum and incubated at 37°C for 30 min. Anti-HTLV-I-seropositive serum and Nonidet P-40 (0.2%) in PBS(-) were used as a control. Virolysis was estimated by detection of HTLV-I p19 in the reaction mixture. p19 was quantified by ELISA. Each bar represents the mean ± SD of duplicate experiments.

 
Effect of human serum on plating of VSV(HTLV-I) pseudotypes

VSV(HTLV-I) pseudotypes prepared using c77 cat cells or HOS/SI-5 human cells were treated with fresh or heat-inactivated serum and inoculated onto the indicator 8C cat cells. Fresh serum at concentrations >20% completely inhibited the plating of VSV pseudotypes prepared in both cat and human cells. After treatment of the pseudotypes with even heat-inactivated serum at a concentration of 50%, about half numbers of plaques of the control were formed (Fig. 2Go). Fifty percent inhibitory concentrations (IC50) of the plating of VSV pseudotypes for serum were determined. The mean ± SD of the IC50 of eight fresh and heat-inactivated human serum samples for c77 pseudotypes were 0.40 ± 0.07 and 23.0 ± 3.7, respectively, and for HOS/SI-5 cells they were 1.2 ± 0.4 and 31.0 ± 3.7, respectively. Thus, treatment of VSV pseudotypes with fresh human serum markedly inhibited their plating. Next, the effect of human serum on plating of VSV was examined, because they might affect the pseudotype assay. Plating of VSV was not completely inhibited by either fresh or heat-inactivated serum: the mean ± SD of the IC50 of fresh serum and that of heat-inactivated serum were 3.1 ± 0.4 and 17.0 ± 3.0, respectively. That is, plating of VSV pseudotypes was more markedly inhibited by fresh human serum than that of VSV (Table IGo). Yet, the effect of human serum on HTLV-I infection was not clearly determined by the VSV pseudotype assay. Therefore, we examined the effect of human serum using PCR for detection of infection with cell-free HTLV-I.



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  		FIGURE 2. Inhibitory effect of fresh human serum on plating of VSV(HTLV-I) pseudotypes. The VSV pseudotype prepared in c77 cat cells (A) or HOS/SI-5 human cells (B) was treated with fresh serum (–{circ}–) or heated (56°C for 30 min) serum (–•–) at indicated concentrations (up to 50%) before infection. Relative numbers of plaques were calculated in comparison with the human serum-free control (100%). Results of one experiment representative of eight independent experiments are shown. Arrows indicate IC50s of fresh and heated serum.

 

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  		Table I. IC50 of human sera against plating of VSV pseudotypes

 
Inhibition of the infectivity of cell-free HTLV-I by human serum

Cell-free HTLV-I prepared using c77 cat cells was incubated with serially diluted fresh serum and inoculated onto 8C cat cells. DNA extracted from 8C cells was amplified by PCR for 35 cycles and analyzed by agarose gel electrophoresis. Discrete bands corresponding to expected 203-bp DNA fragments were detected at a serum concentration of 10% or lower but not at 20% or higher. However, bands of this size were detected after treatment of HTLV-I with heat-inactivated serum at concentrations up to 50%. Thus, fresh serum but not heat-inactivated serum markedly inhibited formation of HTLV-I DNA (Fig. 3Go). Similar results were also obtained after HTLV-I produced by S+L-HOS/SI-5 human cells was treated with human serum and analyzed as described above (data not shown). These findings suggested that human complement inactivated cell-free HTLV-I.



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  		FIGURE 3. Effect of treatment of c77 HTLV-I with fresh human serum or heated (56°C for 30 min) serum on its infectivity. c77 virus was incubated at 37°C for 1 h in the presence of human serum at a concentration of 0% (lane 1, Control) or 50–0.1% (lanes 3–11) or of anti-HTLV-I-seropositive serum (lane 2). Cell lysates were prepared for PCR amplification 24 h after infection.

 
To determine which complement pathway was responsible for the inhibition of HTLV-I infection, fresh serum was treated with EDTA, which is known to block both the classical and alternative pathways, or with ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid containing MgCl2 (EGTA-Mg2+), which blocks only the classical complement pathway (12). HTLV-I was mixed and incubated with human serum in the presence of 10 mM EDTA or EGTA-Mg2+. The effects of fresh serum treated in this manner were examined by PCR as described above. Its inhibitory effects were blocked by EGTA-Mg2+ as well as by EDTA, suggesting that this inhibition occurred via the classical complement pathway (data not shown).

Effect of human serum deficient in complement components on HTLV-I infection

To confirm the involvement of the classical complement pathway in inhibition of cell-free HTLV-I infection, we examined the effect of human serum deficient in complement components on HTLV-I infection. First, we investigated its effect by VSV pseudotype assay. Plating of VSV(HTLV-I) was >90% inhibited by treatment with 25% C6- or C9-deficient serum. C1q-deficient serum (25%) inhibited the plaque formation by only 40%, and even heat-inactivated C1q-deficient serum showed a similar level of inhibitory activity (Fig. 4GoA). One of the reasons for this inhibition by C1q may be the presence of natural Abs against VSV in the serum. Next, we also examined the effects of these complement component-deficient sera on cell-free HTLV-I infection by PCR. HTLV-I PCR bands were not detected after treatment of HTLV-I with fresh C2-, C3-, C6- or C9-deficient serum, but appeared on treatment with C1q-deficient serum (Fig. 4GoB), suggesting that C1q is responsible for this inhibition.



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  		FIGURE 4. Effect of complement component-deficient serum on HTLV-I infection. A, VSV(HTLV-I) pseudotype prepared in c77 cells was treated with human serum (25%) before infection. Relative numbers of plaques were calculated in comparison with human serum-free control (100%). The results present the mean ± SD of two independent experiments. B, c77 virus was incubated at 37°C for 1 h in the presence of anti-HTLV-I-seropositive serum, fresh or heated normal serum, or human serum deficient in one of the complement components (C1q-, C2-, C3-, C6-, or C9-deficient serum). Cell lysates were prepared for PCR amplification 24 h after infection.

 
To confirm the inhibitory activity of C1q on HTLV-I infection, fresh serum incubated with goat anti-C1q or anti-C3 serum (Organon Teknika) was mixed with HTLV-I. Fresh serum incubated with anti-C1q serum did not inhibit HTLV-Iinfection markedly at concentrations of up to 40%, and discrete PCR bands of HTLV-I DNA were detected, while fresh serum treated with anti-C3 serum still retained an inhibitory activity at a concentration of 20 or 40% (Fig. 5Go). These findings suggest that the complement component C1q plays a critical role in inhibition of HTLV-I infection.



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  		FIGURE 5. Abrogation of inhibitory activity of fresh human serum on HTLV-I infection by anti-C1q serum. A, c77 virus was incubated at 37°C for 1 h in the absence of anti-C1q serum (lane 1) or in the presence of anti-C1q serum (lane 2). Fresh serum was mixed with anti-C1q serum (lanes 3, 4, and 5). B, c77virus was incubated with anti-C3 serum in the absence of fresh serum (lane 1) or in the presence of fresh serum (lanes 2 and 3). Cell lysates were prepared for PCR amplification 24 h after infection.

 
Effects of complement components on cell-free HTLV-I infection

To further test the inhibitory effects of complement components C1q, C3, C6, and C9 on HTLV-I infection, HTLV-I was incubated with fresh or heated complement components before infection. As shown in Fig. 6GoA, no band was detected in lane 4 where HTLV-I had been treated with fresh C1q in PBS at 50 µg/ml. The other components showed little effect on HTLV-I infection. Fresh C1q inhibited HTLV-I infection even at 25 µg/ml, whereas heated C1q at 50 µg/ml lost its inhibitory activity (Fig. 6GoB). Plating of VSV(HTLV-I) pseudotype was >90% inhibited by fresh C1q at 25 µg/ml (data not shown). The C1q obtained from two different sources gave similar results (data not shown). From these lines of evidence, we concluded that human complement component C1q inhibited cell-free HTLV-I infection. Normal human serum contain C1q at a concentration of ~80 µg/ml, indicating that the infectivity of cell-free HTLV-I is almost completely inactivated under physiologic conditions.



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  		FIGURE 6. Effect of complement component C1q, C3, C6, or C9 on HTLV-I infection. A, c77 virus was incubated at 37°C for 1 h in the absence of components (lanes 1 and 2); or in the presence of fresh components (lanes 4-7), of heated (56°C for 30 min) components (lanes 8-11) at a concentration of 50 µg/ml, or of anti-HTLV-I-seropositive serum (lane 3). The virus was diluted 1:10 before infection (lane 2). B, c77 virus was mixed with C1q and incubated at 37°C for 1 h at concentrations of 0% (lane 1) or 12.5–50 µg/ml (lanes 3–10) or with anti-HTLV-I serum (lane 2). Cell lysates were prepared for PCR amplification 24 h after infection.

 
Treatment of 8C cells with fresh serum or fresh C1q before infection did not affect the infectivity of cell-free HTLV-I.The infection of cell-free HTLV-I to 8C cells was well neutralized by HTLV-I Ab-positive serum (data not shown). These findings indicate that our system using feline cells is effective for the analysis of cell-free HTLV-I infection in the presence of human complement, especially C1q.

Binding of C1q to HTLV-I virions

To clarify the interaction between C1q and HTLV-I, we examined whether C1q directly bound to HTLV-I virions. For this purpose, we used 20–60% sucrose density gradient ultracentrifugation to detect binding of C1q to HTLV-I. Preliminary experiments showed that sucrose density gradient assays of purified HTLV-I virions gave a peak at a density of 1.15 g/cm3, as reported previously (34), and that a peak of C1q was detected at a density of 1.05 g/cm3. Cell-free HTLV-I mixed with C1q at 37°C for 1 h was placed on the top of the sucrose gradient and ultracentrifuged at 27 K rpm for 20 h. Then the sucrose gradient was fractionated into 15 tubes. Each fraction was examined for HTLV-I Ag p19 and C1q (Fig. 7GoA). Peaks of C1q and HTLV-I were detected in the same fraction at a density of 1.15 g/cm3. As for C1q, another peak was also detected at a density of 1.05 g/cm3 as described above. Comigration of C1q with HTLV-I suggested the direct binding of C1q to HTLV-I virions. Similar results were obtained when another HTLV-I strain produced by S+L-HOS/SI-5 human cells was used: a large peak of C1q was detected at a density of 1.05 g/cm3 and a small peak at a density of 1.15 g/cm3 (data not shown). Judging from the sizes of the C1q peaks, we estimated that ~20–30% of C1q bound to HTLV-I virions under our assay conditions. We also examined the binding of C1q to HTLV-I using HTLV-I lysates and ELISA. C1q bound to ELISA plates coated with HTLV-I lysates (Fig. 7GoB).



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  		FIGURE 7. Binding of C1q to HTLV-I virions. A, c77 HTLV-I suspension (50 µl) was mixed with fresh C1q (50 µl, 500 µg/ml) and incubated at 37°C for 1 h. The reaction mixture (100 µl) was ultracentrifuged through a 20–60% sucrose density gradient. Samples were fractionated into 15 tubes. HTLV-I in each fraction was detected by p19 ELISA (–{circ}–). C1q was detected by C1q ELISA (–•–) B, HTLV-I lysates were added into wells of 96-well microplates and incubated with 50 µl of fresh or heated C1q at 37°C for 1 h. Bound C1q was detected by C1q ELISA. Relative binding of C1q was calculated in comparison with control absorbance of blocking buffer alone. Each value represents the mean ± SD of two independent experiments.

 
Identification of a C1q-binding region on HTLV-I Env glycoproteins

A C1q-binding region on HTLV-I Env glycoproteins was examined by using 23 overlapping synthetic peptides and ELISA. As shown in Fig. 8Go, an absorbance peak was detected around the peptide corresponding to 400–429 amino acids of Env glycoprotein, which belongs to gp21. We further confirmed this by a competition assay; namely, the binding of purified C1q to ELISA plates coated with HTLV-I lysate was readily detectable by ELISA for C1q. The binding of C1q (10 µg/ml) to the plates coated with the HTLV-I lysate was competitively inhibited by addition of the gp21 400–429 synthetic peptide at 25 µg/ml (Fig. 9Go). We have reported that this peptide inhibits syncytia formation induced by HTLV-I (36, 37). It is probable that C1q will bind to this region of the Env protein of HTLV-I virion to inhibit its infection.



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  		FIGURE 8. Identification of a C1q-binding region of HTLV-I. ELISA plates were coated with one of the synthetic peptides of gp46 or gp21 of HTLV-I. C1q (50 µl, 10 µg/ml) was added to each well and incubated at 37°C for 1 h. C1q bound to each peptide was detected by ELISA using the monoclonal anti-C1q Ab. The relative amount of C1q bound to each peptide was calculated by dividing each absorbance value by that of the control, i.e., blocking buffer alone. Each value represents the mean ± SD of three independent experiments.

 


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  		FIGURE 9. Competitive inhibition of the binding of C1q to HTLV-I lysate by gp21 400–429 animo acids. Cell-free HTLV-I produced by c77 cells (•) or S+L-HOS/SI-5 cells ({triangleup}) was lysed with Nonidet P-40 (0.2% v/v). The HTLV-I lysate was added into wells of 96-well microplates and incubated at 4°C for 2 h. Then, 50 µl of purified C1q (10 µg/ml) was added to the wells together with the gp21 313–334 (- - -) or gp 400–429 (—) peptide at the indicated concentrations. The binding of C1q to the HTLV-I lysate was detected by ELISA for C1q. The binding of anti-C1q Ab to the wells, which had been coated with the HTLV-I lysate, in the presence of the gp 400–429 peptide but in the absence of C1q was measured as a control ({diamond}). Each point is the mean of duplicate assays.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Human complement system plays an important role in the host defense against virus infection (11). Retroviruses isolated from avian, feline, murine, and simian sources have been found to be lysed and neutralized by the Ab-independent activation of the classical complement pathway induced by direct binding of C1q to viral Env proteins (12, 13, 14, 15). Thus, C1q, which circulates with C1r and C1s in the form of a calcium-linked C1 complex, has a virus recognition function (39).

We and others have reported that HTLV-I (16) and HIV-1 are not lysed by human complement (17, 18, 19, 20). In the case of HIV-1, it has been reported that human complement component C1q binds to HIV-1 virions, which will not lead to virolysis, due to the presence of inhibitory molecules such as CD46 (membrane cofactor protein (MCP)), CD55 (decay-accelerating factor (DAF)), or CD59 (homologous restriction factor (HRF)) of the complement activation pathway on the surface of HIV-1 virions (40, 41, 42, 43). Their infectivities are not affected even after partial activation of the complement pathway. On the other hand, it has not been determined whether HTLV-I retains infectivity upon treatment with human complement or whether C1q binds to HTLV-I virions.

In this paper, we describe the inhibitory effect of human complement, especially C1q, on cell-free HTLV-I infection and also that this inhibition was suggested to be induced by direct binding of C1q to HTLV-I virions ( Figs. 2–7GoGoGoGoGoGo). Previous reports have shown that C1q directly binds to HIV and HTLV-I coinfected cells (44) and that animal retroviruses activate the human complement classical pathway, which is caused by direct binding of C1q to the retroviral Env proteins, especially, transmembrane proteins. For example, human C1q binds to murine leukemia virus through the transmembrane Env protein p15E (45).

In this study, we have identified the putative C1q-binding region of HTLV-I Env protein (Fig. 8Go). We have reported that the transmembrane glycoprotein gp21 400–429 peptide, as well as the gp46 197–216 peptide, inhibits the formation of syncytia (36, 37). While the 400–429 region does not overlap with the putative fusion domain for membrane fusion, which is assigned around the gp21 313–333 peptide (36), it is probable that this region is also necessary for infection with cell-free HTLV-I. The treatment of HTLV-I with C1q would interfere in the interaction of gp21 with cellular membrane.

The heat-stable inhibitory activity presented in Fig. 2Go or 4 could be due to natural Abs against VSV and/or HTLV-I in human serum. With regard to natural Abs, it has been reported that there is a novel mechanism of retrovirus inactivation mediated by anti-{alpha}-galactosyl Ab in human serum (25, 26). This natural Ab may have interfered with VSV pseudotype assays. The plating of VSV pseudotypes prepared in human HOS cells was, however, still inhibited by heat-inactivated human serum (data not shown), suggesting that other substances including Abs may have interfered with the pseudotype assay. In contrast, the HTLV-I PCR assays showed that infection of cell-free HTLV-I produced by cat or human cells was hardly inhibited by heat-inactivated serum (Fig. 3Go). Thus, anti-{alpha}-galactosyl Ab may not play an important role in the infection of cell-free HTLV-I produced by cat cells.

The syncytium formation induced by cocultivation of HTLV-I-producing C91/PL cells with indicator MOLT-4 cells (46) was not inhibited by treatment with even 40% fresh human serum or C1q at 25 µg/ml (data not shown). Human serum and C1q were toxic to cells at concentrations that effectively inhibited the infection of cell-free HTLV-I. It remains to be examined whether human serum or C1q at physiologic conditions (~80 µg/ml) inhibits cell-to-cell infection of HTLV-I in a suitable assay system. In contrast, heated sera from HTLV-I-infected individuals inhibited syncytium formation as described (47). Almost all seropositive serum diluted several hundredfold markedly inhibited the plating of cell-free HTLV-I (23, 48), whereas fresh human serum hardly inhibited it at a dilution of 1:20 or higher (Figs. 2Go and 3Go).The inability of fresh human serum to inhibit syncytium formation induced by HTLV-I upon cocultivation may be explained by the lower inhibitory titers of human complement, especially C1q, than Abs against HTLV-I. Another possibility is that C1q may inhibit an infection step after the process required for cell fusion. C1q may bind not only to Env peptides 400–429 but also to 426–460 of gp21 (Fig. 8Go). We have reported that the Env peptide 400–429 but not peptide 426–460 inhibits syncytium formation (36, 37). It remains to be examined whether the peptide domains required for inhibition of syncytium formation and for C1q binding are overlapping or dissociable.

The binding of C1q to HIV-1 transmembrane Env glycoprotein gp41 and the activation of the human C1 complex by HIV-1 have been reported (18, 49); and with regard to the binding sites of HIV-1 and HIV-2, it has been demonstrated that the sequence 601–613 (GIWGCSGKLICTT) of the HIV-1 immunodominant region, which makes the Cys605-Cys611 S-S bridge, is important (49). However, a possible Cys-Cys loop exists on the gp21 393–400 peptide but not on the 400–429 peptide of HTLV-I or on the HTLV-II Env glycoprotein. It is necessary to further investigate the role of the Cys-Cys loop in the binding of C1q to HTLV-I and its inhibition of infection.

HIV-1 is not lysed by human complement in the absence of specific anti-HIV-1 Ab (50, 51), although C1q binds to HIV-1 virions. One possible explanation for this resistance is that complement control proteins CD46, CD55, and CD59, which protect cells from complement lysis by preventing the formation of the membrane attack complex of complement, are incorporated into virions as HIV-1 buds from the surface of infected cells and provide resistance to complement-mediated lysis (40, 41, 42, 43). With regard to activation of complement by HTLV-I, Saiffudin et al. have reported that HTLV-I treated with human serum binds to CR+ cells more efficiently than to CR- cells, suggesting that the complement pathway may be activated to C3 levels by cell-free HTLV-I (53). However, HTLV-I is not lysed by human complement (Ref. 16; Fig. 1Go), indicating that the terminal complement pathway is not activated by cell-free HTLV-I due to complement control proteins on the surface of the virion. While it is probable that HTLV-I is also protected from virolysis by these complement control proteins (52, 53), the mechanism by which HTLV-I resists complement-mediated lysis is unknown.

It is generally postulated that the transmission of HTLV-I in humans is mediated through virus-infected lymphocytes (cell-to-cell infection). In this sense, HTLV-I is a cell-associated virus. HTLV-I may have become transmissible by avoiding contact with human complement. On the other hand, it has been reported that the transmission of HTLV-I in vivo can be established through cell-free infection in rabbits (54, 55). Our preliminary experiment shows that HTLV-I virion is also not lysed by rabbit serum, and its infectivity determined by VSV pseudotype assays and PCR assays using cell-free HTLV-I is not inhibited by rabbit complement in vitro (data not shown). These findings suggest that the complement system does not contribute to preventing the transmission of cell-free HTLV-I in rabbits.

In this experiment, we have demonstrated that infection of cell-free HTLV-I was markedly inhibited by human complement, especially C1q. C1q was effective at a level of 25 µg/ml or higher ( Figs. 2–7GoGoGoGoGoGo). Because a normal concentration of C1q in human serum is ~80 µg/ml, C1q would be expected to inhibit the infectivity of HTLV-I in vivo. Cessation of feeding with breast milk from seropositive mothers has strongly contributed to the prevention of mother-to-child HTLV-I transmission, but ~3% of bottle-fed babies will still seroconvert (4). The amount of C1q in the serum of newborn babies is lower than in adults (56). It is probable that concentrations of C1q may become too low to inhibit the transmission of cell-free HTLV-I in some babies. It remains to be determined whether C1q inhibits the cell-to-cell transmission of HTLV-I in vivo. The possibility of administering C1q to newborn babies whose C1q concentrations are low could be considered as one means of reducing the rate of HTLV-I transmission from mother to child, which would be a new prophylaxis system against HTLV-I transmission in humans.


   Acknowledgments
 
We thank Ms. T. Nakamura for technical assistance.


   Footnotes
 
1 This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan. Back

2 Address correspondence and reprint requests to Dr. Hiroo Hoshino, Department of Hygiene and Virology, Gunma University School of Medicine, Schowa-machi, Maebashi, Gunma 371-8511, Japan. E-mail address: Back

3 Abbreviations used in this paper: HTLV-I, human T cell leukemia virus type I; VSV, vesicular stomatitis virus; Env, envelope; ATL, adult T cell leukemia; CH50, 50% complement-hemolytic activity; IC50, 50% inhibitory concentration; HOS, human osteosarcoma cell line. Back

Received for publication February 2, 1998. Accepted for publication July 8, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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