VIROLOGY

181, 180-192

(1991)

Inhibition

of HIV and SIV Infectivity

by Blockade

of a-Glucosidase

Activity

LEE RATNER,’ NANCY VANDER HEYDEN, AND DOUGLAS DEDERA Departments

of Medicine

and Molecular

Microbiology,

Washington

Received August 6, 1990; accepted

University,

St. Louis, Missouri

63 110

October 3 1, 1990

Processing of HIV and SW envelope oligosaccharides is critical for proper intracellular trafficking and function. An inhibitor of ol-glucosidases I and II, N-butyl deoxynojirimycin (IV-BuDNJ). retards HIV-1 and SlVmac spread in lymphocytes and monocytes by diminishing virus infectivity, and also causes a reduction in syncytia formation between infected cells and uninfected lymphocytes. N-BuDNJ retards envelope processing from the precursor form to the mature surface (SU) and transmembrane proteins in HIV-l- and SIVmac-infected cells, as well as in cells infected with vaccinia-HIV-l envelope recombinant virus. However, no significant reduction is seen in the amount of SU in released virus particles, though the virus particle-associated SU from N-BuDNJ-treated cells has an altered electrophoretic mobility. In contrast, N-BuDNJ had no effect on GAG protein synthesis and processing. These findings demonstrate a critical requirement for oligosaccharide processing by a-glucosidases I and II for HIV-I and SlVmac envelope processing and fusogenicity. 0 1991 Academic Press, Inc.

ing, oligomerization, cleavage, and/or transport to the cell surface (Dewar et al., 1989; Dedera et a/., 1990). Third, glycan groups have been implicated to be important in the binding of the HIV-1 envelope to its receptor, CD4. Specific lectins block syncytium formation and infectivity of cell-free virus (Lifson et al., 1986; Robinson et a/., 1987; Ezekowitz et al., 1989; Hansen et a/., 1989; Chowdhury et a/., 1990). Specific anti-carbohydrate antibodies have a similar activity (Hansen et a/., 1990). Matthews and colleagues (1987) reported that envelope deglycosylation markedly impairs its ability to bind membrane-associated CD4 and to induce formation of syncytia. Similar results have been reported by Fennie and Lasky (1989). Fourth, glycan groups may also be critical for virus uptake after CD4 binding. Willey and colleagues (1988b) demonstrated that alteration of an asparagine residue at position 262 (numbering according to Ratner et al., 1985), to which oligosaccharide is presumed to attach, results in HIV-l particles which bind to CD4 normally, but which manifest impaired uptake. However, neighboring mutations that should not impair the N-glycosylation site had a similar phenotype. Fenouillet and colleagues (1989, 1990) have demonstrated that deglycosylation of envelope under nondenaturing conditions reduces CD4 binding about 5-to 1O-fold, but abolished uptake to a greater degree. Inhibitors of high-mannose-type oligosaccharide processing which block glucosidases or mannosidases, while not affecting HIV-l particle production, severely impair HIV-1 infectivity and cytopathogenicity (Gruters eta/., 1987; Walker eta/., 1987; Montefiori eta/., 1988; Dedera et a/., 1990). The current work extends the ac-

INTRODUCTION The envelope proteins are the predominant and perhaps only glycosylated proteins of human and simian lentiviruses, HIV-1 and SlVmac (Allan ef al., 1985). Approximately 50% of the mass of the envelope proteins is carbohydrate. The majority, if not all, of the carbohydrate is attached in the form of N-linked oligosaccharide; it is unclear whether there is any O-linked carbohydrate (Hansen et a/., 1989; Stein and Engleman, 1990). The structure of the envelope-associated oligosaccharide is highly heterogeneous (Mizouchi et al., 1990). About half of the glycan groups in the virus particle-associated envelope are of the high-mannose type and half are complex bi- and triantennary structures (Abel et a/., 1987; Geyer et al., 1988; Mizouchi et a/., 1988; Holschbach et al., 1990). In cell lines, the high-mannose oligosaccharides predominate (Kalyanaraman et a/., 1990; Wells and Compans, 1990). There are several findings indicating an important role of the oligosaccharide in the function of the retrovirus envelope protein, including that of HIV-l. First, glycan groups have been shown to be important for the interaction of envelope proteins with antibodies, including neutralizing antibodies (Portelle et a/., 1980; Robinson et a/., 1987; Montefiori et al., 1989; Moore et a/., 1990). Second, oligosaccharide addition and processing has been suggested to be important for envelope fold’ To whom reprint requests should be addressed at Box 8125, 660 S Euclid, Washington University, St. Louis, Missouri 631 10. FAX (314) 362-8859. 0042-6822191 Copyright All rights

$3.00

0 1991 by Academic Press, Inc of reproduction in any form reserved

180

INHIBITION OF GLUCOSIDASE I AND HIV AND SIV REPLICATION

tivity of such a glucosidase inhibitor, AI-butyl deoxynojirimycin (N-BuDNJ), to a useful animal lentivirus model system, SIVmac, and further defines the molecular mechanisms of viral infectivity which are impaired by N-BuDNJ treatment. MATERIALS

AND METHODS

Materials N-BuDNJ was provided by D. Tiemeier Mueller (Searle) and resuspended in media.

and R.

Cell lines H9, CEM, and MOLT3 cell lines, provided by R. C. Gallo (NIH), and BW5147 and an a-glucosidase Il-deficient cell line derived from it, PHAR2.7cells, provided by R. Kornfeld (Washington University) were cultured in RPMI-1640 (GIBCO) supplemented with 10% fetal calf serum (Hazelton), 4 mM glutamine, 50 pg/ml streptomycin, and 50 U/ml penicillin. Elutriated human macrophages were cultivated on plastic tissue culture plates in AIM-V medium (GIBCO) supplemented with 10% fetal calf serum, and 10 U/ml M-CSF. CVl cells were cultivated in DMEM medium (GIBCO) supplemented with 109/o human serum, 1 mlLl pyruvate, 50 pg/ml streptomycin, and 50 U/ml penicillin. Virus strains The HIV-1 strain used for all experiments except the monocyte studies was derived from H9 cells which were cocultivated with COS-1 cells transfected with molecular clone, HXB2gptX (Fisher et a/., 1987; Ratner et a/., 1987). HIV-1 strain BaL was used for monocyte infection experiments (Gartner et al., 1986). SlVmac strain 186 was derived from H9 cells chronically infected with this isolate (Kestler et al., 1988). Antisera The anti-HIV-l antiserum is a pooled serum sample derived from multiple HIV-l -infected patients with high titer and broad-spectrum reactivity by immunoblot. The anti-SIV antiserum is from a SIVmac-infected rhesus macaque. Virus replication

experiments

For acute infection assays, 1 ml HIV-1 or SlVmac [3-6 X lo5 cpm reverse transcriptase (RT) activity/ml or about lo4 TCID-50 units/ml] was added to the appropriate cell type in the presence of 2.5 pug/ml polybrene for 2-6 hr. The virus inoculum was then removed, and the cells were cultivated as described above. In drug-treated cultures, N-BuDNJ was added

181

at the same time as the virus, and with fresh medium at subsequent feeding. Syncytia

assays

In acute infection assays, syncytia were monitored by phase microscopy. Syncytia induction assays utilized 2 X 1O5 HIV-l, SIVmac, or vaccinia virus (VV)-infected cells which were incubated with 8 x 1O5T lymphoid cells for 16-24 hr in 2 ml duplicate cultures in a 24-well plate. Duplicate aliquots of 0.2 ml of cell suspension were transferred to a 96-well plate. All syncytia in the well (approximately 5 high power fields) were counted. Infectivity

assays

Aliquots of l.O-ml virus samples were tested undiluted, 5-fold, 25-fold, 125-fold, and 625-fold diluted in triplicate by addition to 1O6 H9 cells in the presence of polybrene in a 2-ml culture in a 24-well plate. The virus inoculum was removed after 6 hr and the cells cultivated as described above. Cultures were examined for syncytia formation after 8 days, and the infectivity was scored as the inverse of the lowest dilution capable of producing syncytia. Reverse transcriptase

assays

RT assays were performed by a modification of the procedure of Poiesz et al. (1980) with eightfold polyethylene glycol (PEG)-concentrated samples of conditioned media, poly(rA)-oligo(dT) as the template, [32P]TTP (ICN, spec. act. > 3000 Ci/mmole), and incubation for 2-l 6 hr. Cell toxicity

assays

Cell toxicity was monitored by cell counts performed with a hemocytometer, or by a 16-hr [3H]leucine incorporation assay (Dedera et al., 1990). lmmunoprecipitation

experiments

For these studies, 1O7cells were incubated in 2 ml of cysteine- and methionine-free RPMI medium supplemented with 10% dialyzed fetal calf serum and 1OO200 &i/ml of [35S]methionine and -cysteine (Translabel, ICN, spec. act. > 1000 Ci/mmole) for 4-16 hr. In some experiments, the cells were then sedimented and incubated at a concentration of 10” per ml in full medium. Cells were washed with phosphate-buffered saline (PBS) and resuspended in 1 ml of lysis buffer (10 mM Tris-Cl, pH 7.6, 150 m/l/l NaCI, 1 mM EDTA, 1% Triton X-l 00, 5 m/l/l 2-mercaptoethanol) for 10 min with vor-texing. The suspension was precleared by incuba-

RATNER, VANDER

182

HEYDEN,

AND DEDERA

Inhibition of Replication 50

to4 Concentration of N-BuDNJ QJM)

0

Syncytia

N-BuDNJ Concentration (pa

+ -I+

Reverse lOTranscriptase

Activity 5-

4

6

8

10

12

14

Time (days) FIG. 1. Inhibition of HIV-1 replication In H9 cells by /I-BuDNJ. H9 cells were acutely Infected with HIV-1 and cultured in the continuous presence of N-BuDNJ at the indicated concentrations. The presence or absence of syncytia during the course of the experiment is indicated to the right. The insert shows the level of inhibition of HIV-l replrcatron as a functron of N-BuDNJ concentration on a logarithmic scale, based upon reverse transcriptase assays at Day 6 (0) Day 11 (0) or Day 15 (W)

tion for 1 hr with 10 ~1 of a serum from a human not infected with HIV-l, and incubation on a rotating apparatus for 16 hr with 30 ~1 of a 50% (vol/vol) suspension in PBS of protein A-Sepharose (Sigma). The sample was placed in a microfuge for 1 min and the supernatant used for further analysis. Virus pellets were collected after precipitation with 10% polyethylene glycol (MW 8000) and were resuspended in 1 ml lysis buffer and used without preclearing. lmmunoprecipitation was performed with equivalent volumes (100-200 ~1)of cell lysate or virus pellets with 1 ~1 of the polyclonal SIVmac-infected macaque antiserum (a-SIV), or 1 ~1 of the polyclonal HIV-l-infected pooled human antiserum (a-HIV), incubated for 2 hr at 0”. Thirty microliters of the protein A-Sepharose suspension was added and the samples were placed on a rotator for 16 hr at 4”. The beads were washed three times with lysis buffer and resuspended in loading buffer (62.5 mM Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.02% bromphenol blue).

Endoglycosidase

H treatment

lmmunoprecipitated proteins released from protein A-Sepharose beads were dried at 100” for 15 min in loading buffer. Samples were resuspended in 40 ~1 50 mll/l sodium acetate, pH 5.5, with 1 mU endoglycosidase H (Boehringer-Mannheim). Incubations were performed for 3 hr at 37” and 40 ~1 of 2x loading buffer was added. Vaccinia

infection

experiments

A recombinant vaccinia virus expressing the entire HIV-l envelope protein (VVenv) was derived from plasmid pSC1 1.4, into which the HXB2 strain of HIV-1 env sequences were inserted. A mutation was made by oligonucleotide-directed mutagenesis (Kunkel, 1985) at nucleotides 5788-5790 of HXB2 (sequence numbering according to Ratner et al., 1985), substituting TCT for AGA, thus creating an Xbal restriction enzyme site. This clone was then digested with Xbal and Xhol, and

INHIBITION

OF GLUCOSIDASE

10 [3H]-Leucine Incorporation (cpmx

107

5

0 50 Reverse Transcriptase Activity

25

(cpm x 1 d3)

0

II-l-d IIJL 0

i

2b

200

2000

Concentration of N-BuDNJ (pW FIG. 2. Inhibition of HIV-1 replication in monocytes by N-BuDNJ. Monocytes were infected with HIV-1 as described under Materials and Methods and treated for 7 days with the indicated concentrations of N-BuDNJ, prior to determination of reverse transcriptase activity and f3H]leucine incorporation.

nucleotides 5788-8473 were cloned into the Asp7 18 and HindIll sites of pSC1 1.4, respectively, with the use of linkers. CVl cells were infected with 0.1 plaque forming units per cell parental vaccinia virus and were transfected with the resultant plasmid and parental VV (Wwt) DNA, and recombinant vaccinia virus obtained (Mackett et al., 1985). VVwt and VVenv were propagated and titered with CVl cells and stored in aliquots at -70”. The recombinant virus was used at a multiplicity of infection of 1 to infect CVI, H9, BW5147, or PHAR2.7 cells.

RESULTS

I AND HIV AND SIV REPLICATION

183

virus production between Days 8 and 15 was inhibited 35-709/o. Concentrations of 2-6 mll/l /V-BuDNJ also inhibited virus release at Day 6, more than 95%, and virus production between Days 8 and 15 by 60-85%. Concentrations of 20 &I /I/-BuDNJ or more completely inhibited syncytia formation. No toxic effects were noted in this experiment as determined by cell counts and [3H]leucine incorporation assays on Day 15 (not shown). HIV-l replication in primary monocytes was inhibited by 2 mn/l N-BuDNJ, but not lower concentrations (Fig. 2). Stimulation of HIV-l production at lower concentrations of IV-BuDNJ as seen in this experiment have been repot-ted previously (Montefiori et al., 1988; Dedera et al., 1990), and have been attributed to protection from virus-induced cytopathic effects. No significant inhibition of [3H]leucine incorporation was seen with NBuDNJ treatment. Similar results were obtained in studies of the effects of N-BuDNJ on the replication of SlVmac (Table 1). Acute infection and replication in H9 cells was inhibited by more than 80% at concentrations of 20 #--2 mM N-BuDNJ. Syncytia production was somewhat less sensitive to the inhibitory effects, and was manifested in the presence of 0.2-2.0 mM N-BuDNJ. Treatment of SIV chronically infected H9 cells with up to 2.0 mlVl N-BuDNJ did not have significant effects on either virus production or [3H]leucine incorporation (Table 1). However, with 2 pm-2 mll/l N-BuDNJ, infectivity of virus produced from these cells was reduced. At concentrations of 2-20 puM N-BuDNJ, syncytia production by these cells was reduced 28-34%, and with 200 PM-2 mM N-BuDNJ, syncytia production was reduced by 89-960/o. These results are similar to those obtained with N-BuDNJ treatment of HIV-1 chronically infected cells (Fig. 1 and Dedera et a/., 1990).

N-BuDNJ inhibition of HIV and SIV replication

Effects of N-BuDNJ on HIV and SIV envelope synthesis and processing

The effects of /V-BuDNJ on acute infection and replication of HIV-1 in H9 lymphoid cells were examined (Fig. 1). In the absence of N-BuDNJ, two phases of virus production were noted. At Day 6, a peak of RT activity followed syncytia production. With continued growth of the population of cells resistant to cytopathic effects, virus production increased from Day 8 through Day 15 of the culture. Concentrations of 2-60 @M A/BuDNJ had no significant effects on the first peak of virus production and inhibited subsequent HIV-1 release between Days 8 and 15, by O-20%. In contrast, concentrations of 200-600 PLIVIN-BuDNJ blunted HIV1 release at Day 6 by more than 95%, and subsequent

To examine the effects of N-BuDNJ on envelope synthesis and processing, chronically infected cells were pretreated with different concentrations of the drug for 6 days. Cells were then labeled with [35S]methionine and -cysteine, and labeled viral proteins immunoprecipitated with virus-specific antisera. In the absence of N-BuDNJ, SIV envelope precursor (Pr-gpl40) and HIV envelope precursor (Pr-gpl60), and mature surface SIV (SU-gpl 10) and HIV (SU-gpl20) envelope proteins are detected (Fig. 3). No significant alteration of the amounts or electrophoretic mobilities of these proteins were detected after treatment with 0.2-200 p/1/1NBuDNJ. In contrast, with 2.0 mM N-BuDNJ, the electro-

RATNER, VANDER

184

HEYDEN, AND DEDERA TABLE 1

SUMMARY OF EFFECTSOF N-BuDNJ ON SW REPLICATION Chronic infectionC

Acute infection

Concentration of N-BuDNJ (mM) 0 0 0.0002 0.002 0.02 0.2 2.0

Virus

Syncytiaa

-

-

+ + + + t t

+ + + + -

Reverse transcnptase activity (cpm)* 1,300 96,400 51,000 75,200 17,200 15,000 18,300

Reverse transcriptase activity hmV 3,100 195,000 149,000 104,000 102,000 77,600 139,400

BH-Leu incorporatron km)

Envelope expression’

51,000 59,000 53,000 39,300 41,500 53,300 39,000

+ + t t altered altered

-

Infectivity 0 125 125 25 25 5 0

Syncytra induction* 0 92 89 67 61 11 4

a Syncytia were scored as present (+) or absent (-) during the acute infectron assay, and the score was marntained throughout the trme course of assay. * The reverse transcriptase levels are listed for Day 1 1 of the acute infection assay, at which time the peak value was obtained in the absence of drug. c The chronic infection assay was performed by incubating HSKIV cells with or wrthout drug for 3 days, washing the cells, and incubating the cells with or without drug for an additional 3 days. d The reverse transcriptase assays were performed at the completron of the 6 days of cultrvation in the presence or absence of drug. e [3H]Leucine incorporation was measured at the completion of the 6 days of incubatron In the presence or absence of drug. ’ Envelope expression was measured by radiormmunoprecipitation analysis of [%]methronineand cysterne-labeled cultures after 6 days of rncubatron with or wrthout drug. Detectable bands for 120. and 160-kDa envelope products are lrsted as (+). Altered electrophoretic mobility of envelope bands is Indicated. B lnfectivrty assays were performed on virus obtained after 6 days of cultivation in the presence or absence of drug, by addition of virus to fresh H9 cells, and determining lowest dilution at which syncytia were present. Infectivity is scored as the inverse of that dilution. ’ Svncvtia induction assays were performed with cells cultured 6 days in the presence or absence of drug, by addition to a fivefold excess of fresh~H9~cells, and counting the number of syncytia Induced at 24 hr

phoretic mobility of the envelope precursor and surface proteins is reduced. Furthermore, the ratio of surface to precursor envelope proteins is reduced.

HP/SIV 880 c-4 @Jucucu@Jdo

H9 HP/HIV 000 ONr4NO

FIG. 3. N-BuDNJ-Induced alteration in electrophoretic mobility of envelope proteins of HIV-1 and SIVmac. Cells were incubated with [35S]methionine and cysteine for 16 hr. lmmunoprecipitates were obtained using polyclonal antisera from SIVmac- (HS/SIV) or HIV-1 infected H9 cells (HS/HIV), or uninfected H9 cells after 6 days treatment with the indicated concentrations of N-BuDNJ (PM). Positions of marker proteins are indicated to the right of the autoradiogram. Arrows designate envelope precursor proteins of SlVmac (Pr-gpl40) and HIV-1 (Pr-gpl60), and mature surface envelope proteins of SIVmat (SU-gpl 10) and HIV-l (SUgpl20). Triangles designate the altered electrophoretrc mobility of the envelope precursor and surface proteins of SlVmac and HIV-1 after 2000 PM N-BuDNJ treatment.

To further assess the effects of A/-BuDNJ on envelope processing, SIV and HIV-1 chronically infected cells were pulse labeled with [35S]methionine and -cysteine for 2 hr and then grown for a chase period of 16 hr in the absence of radioisotope (Fig. 4). Labeled proteins were then immunoprecipitated with virus-specific antisera. Immediately after the pulse period, most of the labeled envelope protein is in the form of the envelope precursor, Pr-gpl40 for SIV and Pr-gpl60 for HIV1. After treatment with 0.2 mM N-BuDNJ, a slightly lower electrophoretic mobility can be seen for each of these precursor bands. The failure to note the altered electrophoretic mobility of envelope protein from cells treated with 0.2 mM N-BuDNJ in the previous experiment (Fig. 3) is most likely due to the use in that experiment of a shorter gel with lower resolution. After treatment with 2 mM N-BuDNJ, the electrophoretic mobility of the precursor envelope protein is even more retarded. In contrast, the amounts and electrophoretic mobilities of GAG precursors (Pr55-gag) are not reduced by N-BuDNJ treatment. After the chase period, the envelope protein from SIVmac-infected cells not treated with N-BuDNJ is predominantly cleaved SU-gpl 10. With 0.2 or 2 mM N-

INHIBITION

OF GLUCOSIDASE

I AND HIV AND SIV REPLICATION

a) Cells

b) Virus Pujse

I a- SIV H9 H9/SIV

185

Chase

I orHI’

-

H9/HIV

a-SIV H9

a-SIV

a-HIV

* H9

H9/SIV

I 0

0.2

2

Concentration N-8uDNJ (mtl)

- 67 67

- 43

29

FIG. 4. Effect of N-BuDNJ on SlVmac and HIV-l envelope synthesis and processing. Cells were treated with the indicated concentrations of N-BuDNJ (m&I) for 6 days, labeled with [%3]methionine and cysteine for 2 hr (pulse), and incubated for an additional 16 hr in the absence of radioisotope (chase). Conditioned media obtained after the chase period were precipitated with polyethylene glycol. lmmunoprecipitates were obtained from SlVmac (HS/SIV) or HIV-l -infected H9 cells (HS/HIV), or uninfected H9 cells using polyclonal antisera to SlVmac (&IV) or HIV-l ((r-HIV), respectively. Positions of marker proteins are shown to the right. The positions of envelope precursor(,). surface proteins (D), the gag precursor, Pr55 (+), and gag capsid CA proteins (0) are indtcated.

BuDNJ treatment, the ratios of SU-gpl 10 to Pr-gpl40 are reduced, as are the electrophoretic mobilities of both the precursor and mature surface envelope proteins (Fig. 4). Similar results were obtained with HIV-linfected cells (not shown). After the chase period, virus particles in polyethylene glycol-precipitated supernatant solutions from cells not treated with N-BuDNJ contained radiolabeled surface envelope protein, SU-gpl 10 for SIV, SUgpl20 for HIV-l, and capsid proteins (CA-p25 for SIV and CAp24 for HIV-l). With N-BuDNJ treatment, there was no reduction in the amount of virus-associated envelope or capsid proteins. Though the electrophoretic mobility of the capsid proteins was not altered by N-BuDNJ treatment, the virus-associated surface envelope proteins manifested slower electrophoretic mobilities. In order to assess the nature of the abnormality in electrophoretic mobility of SIV envelope proteins derived from /V-BuDNJ-treated cells, endoglycosidase H

treatment was performed (Fig. 5). After 4 hr of [35S]methionine and -cysteine incorporation in the absence of A/-BuDNJ treatment, 30% of the radioisotope is incorporated into Pr-gpl40, and 70% of the label is incorporated into SU-gpl 10. In the presence of 2 mM NBuDNJ, 90% of the isotope is found as a precusor protein of approximately 160 kDa, and 10% as a surface envelope protein of 120 kDa. Endoglycosidase H cleavage between the two N-acetyl glucosamine (NAcGlc) residues of oligomannosidic oligosaccharide groups yields a predominant product in each case of 90 kDa. Effect of N-BuDNJ on GAG synthesis

and processing

The effects of N-BuDNJ on HIV-1 GAG synthesis and processing were examined in chronically infected cells labeled for 6 hr with [35S]methionine and -cysteine and then incubated in the absence of radioisotope for 16 hr (Fig. 6). Immediately after the pulse period, the major

RATNER. VANDER

166 2000

0

A-+

+I- +

/JM N-BuDNJ endo

H

-200 kDa

FIG. 5. Endoglycosidase H cleavage products of envelope proteins from SIVmac-infected cells incubated in the presence or absence of N-BuDNJ. HS/SIV cells were incubated in the presence or absence of N-BuDNJ for 6 days and labeled with [%3]methionine and cysteine for 4 hr. lmmunoprecipitation was performed with a polyclonal antiserum from a W-infected macaque. Half of each sample was treated with endo H and the products were analyzed on a polyacrylamide gel. The positions of marker proteins are shown to the right of the autoradiogram. The positions of the envelope precursor (Prgpl40, F) and surface envelope protein (SU-gpl 10, D) are shown.

GAG product was the 55-kDa precursor protein. A smaller amount of 24-kDa capsid protein was present. At the completion of the chase period, the 55-kDa protein had been completely processed, and only CA protein was present. Treatment with 2-600 @I N-BuDNJ had no effect on the rate of GAG precursor synthesis or the rate or pattern of GAG processing.

HEYDEN.

AND DEDERA

line, PHAR2.7, derived from BW5147, which contains only 39/oof the parental level of cu-glucosidase II activity (Reitman et al., 1982). VVenv-infected BW5147 cells readily formed syncytia with MOLT 3 or CEM cells (Fig. 7b). In contrast, VVwt infection did not induce syncytia. Treatment with 2 mfl/l N-BuDNJ prevented VVenv syncytia induction. In PHAR2.7 cells, neither VVwt nor VVenv was capable of producing syncytia in the presence or absence of N-BuDNJ. The effects of N-BuDNJ on envelope synthesis and processing in BW5147 and PHAR2.7 cells were examined after treatment with [35S]methionine and -cysteine for a 2-hr pulse period, and chase period in the absence of radioisotope for 6 hr (Fig. 8). The predominant envelope protein seen is SUgpl20 in each cell type, and no significant differences are seen in the amounts of this species. With N-BuDNJ treatment, a form of SU is seen with a retarded electrophoretic mobility. DISCUSSION Inhibition of HIV and SIV replication glucosidase inhibitor

This study demonstrates that a potent inhibitor of both glucosidase I and II (R. Kornfeld, personal communication), N-BuDNJ, retards both HIV-1 and SlVmac replication. The dose-response relationship of the two viruses is similar. In acute infection studies in lymphoid cells and primary monocytes, N-BuDNJ inhibits HIV-l

Chase

Pulse

HS/HIV

Effects of N-BuDNJ on recombinant envelope processing

vaccinia-HIV-l

The effects of N-BuDNJ were also analyzed with a vaccinia HIV-1 envelope recombinant virus (Wenv). Infection of H9 cells with VVenv in the absence of NBuDNJ did not induce syncytia, but 24 hr after infection the cells were capable of forming syncytia with MOLT 3 cells (Fig. 7a). No syncytia were apparent when wildtype vaccinia virus (Wwt)-infected H9 cells were mixed with MOLT 3 cells. Treatment with 2 PM N-BuDNJ had no significant effects on the number of syncytia induced by VVenv, whereas 20-200 PM N-BuDNJ caused a 65-75% inhibition of syncytia formation, and 2 mM N-BuDNJ caused more than 95% inhibition of syncytia formation. To further correlate the effect of N-BuDNJ on syncytia formation by VVenv with the effect of a-glucosidase activity on VVenv processing, a murine thymoma cell line, BW5147, was utilized, as well as a mutant cell

by a

I-\\-\ 0

0

2

6 2060200600

0

0

2

Supsrnatant H9 HS/HIV I

6 20 602006000

0

@N-BuDNJ

FIG. 6. Effect of N-BuDNJ on HIV-l GAG synthesis and processing. HS/HIV cells were incubated in the indicated concentrations of NBuDNJ for 6 days. Cells were then labeled with [%]methionine and cysterne for 6 hr (pulse) and incubated in the absence of radrorsotope for an additronal 16 hr (chase). lmmunoprecipitation was performed with a polyclonal antrserum from an HIV-l-infected indrvrdual. The positions of marker proteins are shown to the right of the autoradiogram. The positions of the surface envelope protein (SUgpl20; D), gag precursor, Pr55 (+) and the mature gag capsid protern, p24 (c>) are indicated.

INHIBITION

OF GLUCOSIDASE

I AND HIV AND SIV REPLICATION

187

a)

Number

lo-

of

(perhigh Spowrr nstd)

o-

-I""Wt

O

-

vvwl Concmlmtlon

Used In Inlm.ted

BW5147

--

vvenv of N-BuDNJ

Cultwas

QIhl)

""en"

vvwt

vvenv

’ p”A

R2.7 ’

Syncytla with CEMCells

vvwt

vvenv

vvwt

I

N-BuDW vvenv

1

PHAR2.7 Syncytia wlth MOLT3 Cells

FIG. 7. Inhibition of vaccinia-envelope induced syncytia by N-BuDNJ. (a) H9 cells were pretreated with indicated concentration of N-BuDNJ for 2 days, and then infected with wild-type or HIV-l env recombinant forms of vaccinra virus at a m.o.i. of 1. After 24 hr, 2 X 1O5 infected cells were mixed with 10’ uninfected MOLT 3 cells. The number of syncytia in quadruplicate cultures was counted 24 hr later. No syncytia were seen in the cultures not cocultivated with MOLT 3 cells. Standard errors are shown for each determination. (b) VVwt or Wenv was used to infect BW5 147 or PHAR2.’ cells in the presence or absence of 2 mM N-BuDNJ. After 24 hr, duplicate aliquots of 2 X lo6 vaccinia virus-infected ceils were cocultivated for an additional 20 hr with 2 X 10’ MOLT 3 or CEM cells in 2 ml in a 24.well plate. Duplicate aliquots of 0.5 ml of cell suspension were transferred to a 96-well plate and the number of syncytia per well was determined.

replication as manifested by a suppression of virus production, and inhibition of syncytia formation. These effects occurred with concentrations of ALBuDNJ which produced no evident toxicity.

Chase I I WA%147 PHP2.7 BW5147 PwY7 -II III Pulse

I

In cells chronically infected by SlVmac or HIV-l, NBuDNJ had no significant effects on virus production. However, the ability of these infected cells to fuse with uninfected lymphoid cells was markedly diminished by N-BuDNJ treatment. Furthermore, the infectivity of virus released from N-BuDNJ treated cells was impaired.

I

FIG. 8. Effect of N-BuDNJ on recombinant vaccinia-HIV-l envelope synthesis and processing. BW5147 or PHAR’.7 cells were infected with VVwt or VVenv in the presence (+) or absence (-) of 2 mM N-BuDNJ, labeled for 2 hr with [35S]methionine and -cysteine (pulse), and then incubated in the absence of radioisotope for 6 hr (chase). lmmunoprectpitation was performed with a polyclonal antiserum from a HIV-l-infected human, The positions of marker proteins are shown to the right. The position of the surface envelope protein (SUgpl20, D) IS indicated.

Inhibition of envelope but not GAG processing Concentrations of N-BuDNJ which caused profound alterations in the ability of (1) HIV-1 and SlVmac to replicate in acutely infected cells and (2) chronically infected cells to induce fusion or release infectious particles were associated with abnormalities in the electrophoretic mobility of the envelope proteins. This included a retarded electrophoretic mobility of both the precursor and mature surface envelope proteins synthesized in N-BuDNJ-treated cells. The 41-kDa transmembrane protein (TM) was not examined in this study, due to limited reactivity with the available antisera. Endoglycosidase H-treated envelope proteins from cells grown in the presence or absence of N-BuDNJ had similar electrophoretic mobilities. The molecular

RATNER, VANDER

t

0

0

0

0

0

0

HEYDEN, AND DEDERA

0

0

.

. Glc OMM . NAcGlc a Fuc l Gal l Sal

FIG. 9. HIV envelope syntheses and processing. A scheme IS proposed for the synthesis and processrng of the HIV-1 envelope and the effects of N-BuDNJ. The primary translation product of env mRNA IS a 92-kDa protein to which oligosacchande IS transferred in the endoplasmic reticulum to produce the hrgh-mannose-containing gpl60. This is modrfied by the glucosrdases in the RER and mannosidases in the Golgi apparatus. N-BuDNJ IS thought to interrupt this step and retard further processing. The glycoprotern assumes the proper conformation and undergoes disulfide bond formation and oligomerization. In the absence of N-BuDNJ, about 85% of the envelope protein is targeted to the lysosome for degradatron, whereas the remanning envelope protern moves into the Golgi apparatus for proteolytrc processing to gpl20 (SU) and gp41 (TM) and further oligosaccharide modification. Envelope oligomers are transported to the cell surface where one of three possible events occurs. (1) Some gpl20 dissocrates from gp41 and is released from the cell. (2) Some gpl20 binds CD4 from uninfected lymphocytes to induce fusion. (3) Envelope protein is also incorporated into newly released vrrus particles together with virion RNA and other virion proteins. This scheme is modrfred from that of Kornfeld and Kornfeld (1985) and Bryant and Ratner (1990).

weight of the endoglycosidase H product of the envelope precursor protein is consistent with complete cleavage of a high-mannose glycoprotein. However, a product of similar molecular weight was obtained after endoglycosidase H treatment of SIV SU-gpl 10, suggesting that the oligosaccharide residues were only

partially susceptible to cleavage. This would suggest that at least some of the oligosaccharide moieties have been modified to a complex form. The failure to identify a difference in endoglucosidase H products from SUgpll0 synthesized in the presence or absence of NBuDNJ could be a result of the small amount of SU-

INHIBITION OF GLUCOSIDASE I AND HIV AND SIV REPLICATION

gpl 10 in the preparation of envelope protein from the /V-BuDNJ-treated cells. Alternatively, some of the envelope protein arrested by glucosidase blockade may undergo processing by an alternate pathway which utilizes an endo-cr-o-mannosidase (Lobas and Spiro, 1987). In addition to the effect on electrophoretic mobility, IV-BuDNJ-treated cells also manifested a higher ratio of precursor to mature envelope proteins. This suggested a decrease in the rate of proteolytic processing of the envelope precursor to the surface and transmembrane envelope proteins. This was confirmed by pulse-chase analysis in HIV-1 - and SIVmac-infected cells, as well as recombinant vaccinia HIV-l envelopeinfected cells. The similarities between the blockade of envelope processing by N-BuDNJ and that due to a genetic deficiency of glucosidase activity reaffirms that the effects of N-BuDNJ are specific. The scheme in Fig. 9 outlines a likely processing scheme for the HIV-l envelope and the role of glucosidase activity. The envelope protein is directed to the endoplasmic reticulum by its signal peptide, and the primary translation product is predicted to be 92 kDa. However, cotranslational addition of Glc,Man,NAcGlc, (where Glc is glucose, Man is mannose, and NAcGlc is N-acetyl glucosamine) occurs on up to 30 different asparagine residues (Kornfeld and Kornfeld. 1985; Ratner et al., 1985). a-Glucosidase I and II activities in the rough endoplasmic reticulum (RER) cleaves the outermost glucose residue and the next two glucoses, respectively, and are the major targets of N-BuDNJ action. Folding, disulfide bond formation, and oligomerization also likely occur in the RER (Rey et a/., 1990; Earl et al., 1990). Cleavage of envelope to oligomers of SU-gpl20 and TM-gp41 most likely occurs next in the Golgi apparatus. This is based on the finding that most retroviral envelope precursors, including that of HIV-l, are cleaved prior to modification of oligosaccharide to complex forms (England et al., 1977; Rosner et al., 1980; Chatterjee et a/., 1981; Bosch et a/., 1982; Polonoff et a/., 1982; Freed et a/., 1989; Pal et a/., 1989; Tsai et a/., 1990). Whereas inhibitors of N-linked glycosidation such as tunicamycin and inhibitors of glucosidase activity block gpl60 processing, deoxymannojirimycin and swainsonine, which inhibit a-mannosidases, do not block proteolytic cleavage. Proteolytic cleavage of envelope is mediated by a cellular trypsin-like enzyme and is essential for HIV infectivity (McCune et a/., 1988). Cleavage is determined by the primary amino acid sequence of the potential cleavage site (Freed et a/., 1989; Bosch and Pawlita, 1990), as well as more distant residues in the protein (Tschachler et a/., 1990). In related systems, cleavage

189

of influenza hemagglutin is affected by a specific neighboring oligosaccharide (Kawaoka and Webster, 1989). Cleavage of prosomatostatin, a hormone precursor, by a related cellular enzyme, is affected by the secondary structure of the cleavage site (Gomez et al,, 1989). After transfer to the cis Golgi compartment, a-mannosidase I further trims the HIV-1 envelope oligosaccharide to a Man,NAcGlc, structure. After transfer to the medial Golgi cisternae, NAcGlc is added by NAcGlc transferase I, two additional mannose residues are cleaved by a-mannosidase II, and another outer chain NAcGlc residue is added by NAcGlc transferase Il. At this stage fucosyl transferase may add a fucose residue to the innermost NAcGlc residue. Further synthesis of complex oligosaccharide then occurs in the trans Golgi cisternae and the trans Golgi network with the activities of galactosyl and sialyl transferases. These later steps are inhibited by weak acids such as chloroquine, primaquine, and ammonium chloride as well as by monensin, which block intracellular transport mechanisms (Tsai et al., 1990). Approximately 85% of the envelope protein is targeted for destruction in the lysosomes as a result of a defect in one or more of the previous steps (Willey et a/., 1988a). Alternatively, intracellular binding between precursor or surface envelope proteins and CD4 may contribute to lysosomal degradation (Chowdhuryetal., 1990). The remaining 15% of the envelope is transported to the cell surface. These steps require 0.5-3 hr (Willey et al., 1988a; Stein and Engelman, 1990). At this point, one of three possible events may occur. (1) The envelope may interact with GAG and GAGPOL precursors and a dimer of a 9.0-kb viral RNA to form a virus particle which matures with viral protease activity during the budding process (Bryant and Ratner, 1990). (2) The envelope protein may also dissociate with the release of SU-gpl20 from gp41-TM into the culture medium (Kowalski et a/., 1987). (3) Alternatively, the envelope protein may contact the CD4 protein either during intracellular processing, on the surface of same infected cell, or on the surface of a neighboring uninfected CD4+ cell (Hoxie et a/., 1986; Lifson et a/., 1986; Maddon et a/., 1986; Sodroski et a/., 1986; Kowalski et a/., 1987; Bosch et a/., 1989; Felser et al., 1989; Kawamura et al., 1989). This interaction leads to an unmasking of the N-terminal domain of TM-gp41, perhaps mediated by an endoproteolytic cleavage of SU-gpl20 after residue 315 (Stephens et a/., 1990). This results in membrane fusion and cytopathic effects. The current studies further demonstrate the specificity of /V-BuDNJ by the lack of effects on GAG synthesis and processing. This finding supports the contention that GAG and GAG-POL membrane targeting occur

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independent of viral envelope trafficking (our unpublished data). However, mechanisms that regulate the stoichiometry of GAG, POL, and envelope proteins in the virus particle, and the apparent exclusion of host cellular proteins remain to be deciphered.

Why’ does glucosidase I blockade alter HIV- and SIV-induced cell fusion and virus infectivity? Though glucosidase inhibitors, such as N-BuDNJ, retard envelope precursor processing, there is no significant reduction in the amount of processed SU envelope protein in the virus particle, and only a small reduction in cell-surface expression of SU (Dedera et a/., 1990). Virus particle-associated SU from N-BuDNJtreated cells has a decreased electrophoretic mobility, suggesting that the oligosaccharides are incompletely processed. Thus, abnormalities in SU function, not alterations in the amount of SU, are likely to account for the effects on virus infectivity and fusogenicity. These effects may be due to (1) effects on proper envelope folding, disulfide bond formation, or oligomerization, (2) altered binding affinity of SU to CD4, or (3) changes in post-CD4-binding events such as exposure or activation of the TM-gp41 fusion sequence. Though not examined in this study, alterations in TM structure may also affect fusogenicity.

ACKNOWLEDGMENTS We thank T. Braciale and C. Rice for the gifts of the vaccinia expression clone, wild-type vacclnla virus, and CVl cells, and advice on the experimental methodology, S. Gartner for HIV-1 strain BaL. H. Gendelman for advice on macrophage cultures, M. Hanamoto (Cetus) for M-CSF, R. Brown for elutriated macrophages, A. Cantor for advice on the endoglycosidase H experiments, S. and R. Kornfeld for advice and encouragement In these studies as well as the BW5 147 and PHAR2.’ cell lines, D. Tiemeier and R. Mueller for the gift of NBuDNJ (SC-48334), R. Desrosiers for the gift of SlVmac antisera, M. Arens for the HIV-l antisera (collected with support from Al25903, Washington University AIDS ClInIcal Trials Unit), and NIAID AIDS Repository for SlVmac186-Infected H9 cells. This work was supported by NIH Grants Al24745 and Al27302, and a Washington Unlversity Searle/Monsanto Corporation Research Agreement. L.R. is an American Cancer Society Research Professor. Note added in proof. Moore and Spiro (1990, /. f?io/. Chem. 265, 13,104-l 3,1 12) have recently demonstrated that Golgl endo-a-omannosldase provides a pathway for the formation of complex Nlinked oligosaccharides of glycoproteins In HepG2 cells treated with 1-deoxynojinmycin or castanospermlne.

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