_Archives

Vfrology

Arch Virol (1992) 123:309-321

© Springer-Verlag 1992 Printed in Austria

Inhibition of human immunodeficiency virus (HIV) type 1 multiplication by an avian cellular factor M. Semmel 1, A. Alileche 1, D. Coulaud 3, J. Aguilar 2, P. Krief 1, and C. Jasmin 1 10ncogen~se Appliqu~e, INSERM Unit6 268, H6pital Paul-Brousse, Villejuif 2 I.R.S.C., Villejuif, and 3 Institut G. Roussy, Villejuif, France

Accepted September 3, 1991

Summary. A factor secreted from avian cells infected non productively with a non cytopathogenic mutant of vesicular stomatitis virus (VSV ts 1026) interferes with HIV replication in CEM cells and peripheral blood monocytes (PBL). Production of infectious particles is decreased and many virions lack cores and/ or spikes. In CEM cells the prmRNA is spliced into 7.5, 4, and 2kb mRNA. Residual virus contains less env encoded proteins and p 18; p25 appears as several bands. The processing of tat, rev, and nef proteins differs in treated cells and in controls.

Introduction HIV type 1 has in common with other retroviruses most features of reproduction: the RNA virus is introduced into the host cell, divested of its protein shell and the genome is transcribed by the enzyme reverse transcriptase contained in the virion into the corresponding DNA which is then integrated in the host cell genome. This DNA is transcribed into a large precursor mRNA of 9.6 kb. The prmRNA is either conserved as genomic RNA or spliced into mRNAs of 4 kb which encode structural viral proteins or 2 kb mRNAs which encode the regulatory proteins. These small regulatory proteins are a special feature of H I V - they have not been described in other retroviruses- and are not incorporated in the virions with the exception of vpr (HIV type 1) or vpx (HIV type 2). These mRNAs are predominant early in the multiplication cycle and are generated by alternative splicing of the prmRNA. One of the regulatory proteins, rev, has among other functions that of maintaining the balance between unspliced genomic RNA and spliced transcriptional mRNAs. Another, tat, increases HIV replication by binding to a region " T A R " of the HIV LTR, thus activating HIV transcription. Nef can downregulate HIV replication, vif is

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implicated in posttranslational processing of the viral proteins or in viral uncoating and vpr m a y play a role in virus m a t u r a t i o n (for review see [1]). In comparison, the p r m R N A of R o u s Sarcoma Virus (RSV) encodes the gag and pol precursor proteins; env and src m R N A s have to be spliced from the p r m R N A in order to be translated into the corresponding precursor proteins, which are then processed to m a t u r e structural proteins by the viral protease encoded by the gag gene (p 15). A transcriptional regulator, encoded by a sequence in the gag region, has been described [2]. We have shown that infection o f chick embryo fibroblasts (CEF) with the n o n cytopathogenic m u t a n t o f vesicular stomatitis virus (VSV) ts 1026 inhibits the multiplication of superinfecting RSV and transformation of the cells by this virus. The supernatant from cells infected n o n productively with VSV ts 1026 has the same effect. It contains no detectable interferon, progeny VSV or VSV proteins [3]. The inhibition is due to anomalies of RSV p r m R N A splicing and as a consequence, the p r o d u c t i o n o f a b n o r m a l a m o u n t s o f the different viral proteins [4]. We have studied the multiplication of H I V in C E M cells, a T cell line, and in peripheral blood lymphocytes (PBL) stimulated with phytohaemagglutinin, grown in the presence of supernatant of chick embryo fibroblasts (CEF) infected n o n productively with VSV ts 1026.

Material and methods Cells and viruses

CEF were explanted from eleven days old lymphomatosis free embryonated eggs purchased from Station de Pathologie Aviaire, INRA, Nouzilly. HT 4 Lac Z-1 cells were a gift from J. P. Nicolas, Institut Pasteur, Paris. These cells were cultivated in Dulbecco's medium containing 100 units penicillin, 10 gg streptomycin, 0.25 gg amphothericin/ml, 2 mM glutamine, 10% fetal calf serum and 1 mM Na-pyruvate. CEM cells (clones 11 and 13) were a gift from F. Barr6-Sinoussi (Institut Pasteur, Paris). Peripheral blood lymphocytes came from a stock of lymphocytes obtained by plasmapheresis. They were purified by a Ficoll gradient, washed and kept frozen at - 170 °C. For experiments, the cells were thawed, treated for 3 days with phytohaemagglutinin and cultivated in RPMI 1640 medium containing 100 units penicillin, 10 gg streptomycin, 0.25 gg amphotericin/ml, 10% heat inactivated fetal calf serum, 2 mM glutamine, 2 units IL 2, and 20-40 units anti-interferon serum (Miles)/ml. Media were purchased from Gibco. CEM cells were cultured in the same medium, but without IL 2. Medium was changed twice weekly. Cell viability was estimated by trypan blue exclusion. HIV type 1 strain BRU was a gift from F. Barr6-Sinoussi, Institut Pasteur, Paris. HIV was grown on CEM cells. VSV ts 1026 was a gift from Dr. Poliquin, McGill University, Montreal. VSV was propagated on primary CEF. HIV replication was assayed by measuring reverse transcriptase activity in the supernatant using the micromethod described by Oysten Jonassen [5]. Briefly, 50 gl cell free supernatant were incubated for 1 h at 37°C in a mixture containing 0.05% Triton X100, 50 mM KC1, 5 mM dithiothreitol, 30 mM Tris pH 7.8, 0.3 mM EGTA, 0.13 units poly-rAdT as primer (Pharmacia) and 0.037 mCi [-3H]deoxythymidine triphosphate (Amersham). The reaction was stopped with 10% trichloracetic acid containing 2 mM Na-pyrophosphate. The precipitate was washed 6 times with 5% trichloracetic acid containing 12mM Napyrophosphate, using an Automash 2000 Harvester on Skatron filtermats. The filters were

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counted in scintillation liquid with a Beckmann counter. Infectious particles were determined by titrating the virus in the supernatant according to Rocancourt et al. [6].

Electron microscopy Cells were fixed in 4% glutaraldehyde and pelleted at low speed. The pellet was resuspended in S6rensen's buffer (67 mM phosphate buffer pH 7.4), then postfixed in 2% osmic acid, dehydrated with ethanol and propyl-oxide, and included in EPON resin with the usual techniques. Cell free supernatant containing virus was fixed in 4% glutaraldehyde and ultracentrifuged at 100,000 g for 1 h. The pellet was resuspended in S6rensen's buffer, filtered on Millipore filters (100 gm pore diameter) and postfixed as described by Barbieri etal. [7]. Sections of millipore filters or cells were coloured with uranyl acetate and Pb citrate and observed with a Zeiss EM 902 microscope. Optimal contrast was obtained by selecting the elastic electrons at the slit of the spectrophotometer.

Preparation of inhibitor To obtain the inhibitor, primary CEF were infected with VSV ts 1026 at a m.o.i, of 3-5, absorbed for 1 h at 40°C, washed for 30-45 seconds with KCt-HC1 buffer p H 2 100mM, then in phosphate buffered saline (PBS) and maintained at 40 °C for 48 h in Dulbecco's medium containing glutamine, antibiotics and 2% "ultroser" serum substitute (IBF). The supernatant was harvested, centrifuged to eliminate the cellular debris and precipitated with (NH4) 2 SO4. After centrifugation of the precipitate, the pellet was dissolved in 1/20 of the volume of the supernatant of Tris buffer, pH 7.4, 10 mM, dialyzed against the same buffer and filtered first through a 0.45 la membrane, then through a 0.1 g membrane (Millex). Protein content was estimated from absorbance at 280 nm. Aliquots of this solution were conserved at - 20 °C. The concentrated supernatant did not contain infectious VSV as estimated by titration on CEF, and VSV encoded proteins were absent as estimated by Western blotting with anti-VSV serum, prepared according to Rosen et al. [8]. Inhibitor was present during the whole period of culture.

RNA and protein assays RNA was extracted from 2-3 x 107 cells, and Northern blotting was performed according to Chomczynski and Sacchi [9] using a PBT1 probe corresponding to the entire genome of HIV (a gift from M. C. Lang, Institut Pasteur, Paris) and an actin probe or a GDPH probe as internal controls. Autoradiographs were scanned with a CCD camera and values were calculated by integration of the points constituting the channel. Values were adjusted to saturation value 100. To analyse the viral proteins in cell free supernatant and in cell lysates, either a viral pellet corresponding to 3 / 106cpm RT HIV or 2-2.5 x 106 cells washed twice in PBS were suspended in 50 gl Laemmli's buffer. 8-25% polyacrytamide gels were run using the Phast system (Pharmacia) and blotted on nitrocellulose membranes (Schleicher and Schuell) using the Phast transfer system. Membranes were saturated with 5% bovine serum albumine Fraction V (Boehringer) in PBS, incubated overnight with the primary antibody, washed and incubated with biotinylated anti-species antibody, washed and incubated with streptavidine alcaline phosphatase and revealed with the appropriate substrate (Amersham) according to the instructions from the supplier. Rainbow molecular weight markers were purchased from Amersham. Monoclonal antibodies were a gift from Dr. Traincard, Institut Pasteur Hybridolab [anti-p 18 (B 31-1 t), anti-gp4t (A9-11), anti gp 120 (A 105-34)]; from M. P. Kieny, Transgene Strasbourg (anti-nef, anti-vif) and recombinant tat and nefproteins; purchased from Miles Biotechnologies (anti-rev, anti-tat); from Genzyme (anti-reverse

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transcriptase); from Tebu (anti-mouse IgM, normal mouse serum); from Immunotech (antiCD 4, iot 4, clone BL 4; anti-CD 8, iot 8, clone B 9-2; anti-mouse IgG linked to ftuoresceine). Results The presence of inhibitor (2 gg protein/105 cells) in the medium did not interfere with the growth of C E M and PBL (not shown). H I V infected C E M (clone 13) cells survived 1 week longer when the inhibitor was present. Up to 9 days p.i. the ratio CD 4 + / C D 8 + cells was the same in uninfected control cells and in infected PBL grown in the presence of the inhibitor. In HIV infected controls this ratio decreased 3 days p.i. and increased 9 days p.i. (Table 1). The latter result could be the consequence of the selection of a subpopulation: more than half of the cells were excluded from the analysis since they showed autofluorescence typical for damaged cells. At still later times so many cells were damaged that FACS analysis was meaningless. In any case, uninfected controls and infected cells grown with inhibitor had the same CD 4 + / C D 8 + ratios which were different from that of infected controls. At low multiplicities of infection, no virus was found in the supernatant of PBL grown in the presence of inhibitor when controls produced maximal amounts (Fig. 1 A). The values obtained 2 and 6 days p.i. do not correspond to HIV, since these supernatants failed to infect C E M (clone 13)cells. Treated C E M (clones 13 and 11)cells did produce virus but with a delay compared to the controls (Fig. 1 B). At high multiplicities of infection (Fig. 1 C) or when inhibitor was added during the first 4 days after infection at low multiplicity (not shown), C E M (clones 13 and 11) cells produced Table 1. Expression of CD 4 and CD 8 antigens on PBL treated with supernatant of VSV infected CEF (% positive cells, FACS) Cells

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CD 8

days p.i. HIV

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* Mean standard deviation: 3.53, 2000 cells counted ** Mean standard deviation: 2.58, 5000 cells counted

Fig. 1. Reverse transcriptase activity in supernatant of HIV infected cells grown with 0-0 and without A- Zk inhibitor (2 gg protein/105 cells). A PBL, infected with 500 cpm RT(HIV)/ 105 cells. B CEM clone 13 cells, infected with 500cpm RT(HIV)/10 s cells; C CEM clone 13 cells, infected with 5000 cpm RT(HIV)/105 cells

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significantly more virus than controls as estimated by reverse transcriptase activity in the supernatant. However, when the same supernatants were assayed for infectious centers, cells grown in the presence of inhibitor produced less beta-galactosidase positive colonies (Fig. 2), but more syncytia than the controls. The ratio cpm RT(HIV)/infectious centers illustrates this paradox: the more inhibitor is added, the less infectious centers and the more cpm RT(HIV) are found in the supernatant (Fig. 3). Growth of cells was not affected even at the highest concentration of inhibitor. Since the assay of infectious centers is based on the activation of HIV LTR linked to beta-galactosidase by the tat gene product synthesized in cells replicating HIV, the absence of infectious centers in monolayers of HT 4 Lac Z-1 cells infected with supernatant from infected cells grown in the presence of inhibitor suggests that this progeny virus is either replication defective or incapable of producing functional tat protein. As shown in Fig. 4, virions from treated cells lack either cores or spikes more frequently than virions from control cells. When counted (100 virions per sample), 2% of the controls were "empty" and 17% lacked spikes; In samples from treated cells, 27% of the virions were "empty" and 38% lacked spikes. In both samples some but not all "empty" virions lacked spikes, and some virions with cores had no or irregular spikes. We tested the effect of inhibitor on HIV mRNA transcription. Early during the infectious cycle more HIV mRNA was synthesized by cells grown in the presence of inhibitor than by controls. The cells continued to produce HIV mRNA at later times (Fig. 5). Northern blotting showed that the majority of

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HIV specific RNA synthesized by treated CEM cells (clones 13 and 11) 7 days p.i. migrated as a 7.5kb species (Fig. 6A, lane 4, and B) not found in control cells. These cells had synthesized 9.6, 4, and 2 kb RNAs (Fig. 6 A, lane 2, and B). 10 days p.i. only 7.5 and 4kb RNAs were detectable in material extracted from treated cells (Fig. 6 A, lane 5, and C), whereas controls'contained 9.6, 4, and 2 kb RNAs (Fig. 6 A, lane 3, and C). Western blot analysis of HIV infected CEM cells (clone 11) and virus grown on these cells showed that most of the HIV structural proteins were the same whether cells had been grown with or without inhibitor (Fig. 7). We found, reproducibly, less p 18 (Fig. 7B), gp41 (Fig. 7C), and gp 120 (Fig. 7D) and more material migrating more slowly than p 25, reacting with anti-p 25 serum (Fig. 7 A) in progeny virus from treated cells than in control virus. The amount and species of structural viral proteins appeared to be identical in treated and untreated cell lysates. More material reacting with anti-nef (Fig. 7 F), anti-rev (Fig. 7 H), and anti-tat (Fig. 7 I) was found in treated cells than in controls. Except for one protein which reacted with anti-nef serum, there was no reaction with uninfected control cells. On the other hand, the bands did not correspond to most of the bands revealed in recombinant proteins; however, these proteins were produced on E. coli, and may be the result of cross-reactions between E. coli proteins and the antiserum. No virus specific proteins were detected in treated and infected PBL.

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Fig. 4. Electron micrographs of HIV produced by CEM clone 11 infected with 1000 cpm RT(HIV)/105 cells. A, C, and E Controls; B, D, and F grown with inhibitor, 1.1 ~tg protein/ 10s cells. A - D Sections of millipore filter; E and F sections of cells. Bar: 0.1 l.tm

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Fig. 5. Dot blots of RNA extracted from CEM clone 11 cells and PBL infected with HIV (5000 cpm RT(HIV)/105 cells) and grown with inhibitor (2 gg protein/105 cells), at decreasing concentrations. 1 CEM cells, uninfected control; 2 CEM cells, infected control, 7 days p.i. 3 CEM cells, infected control, 10 days p.i. 4 CEM cells, infected and grown with inhibitor, 7 days p.i. 5 CEM cells, infected and grown with inhibitor, 10 days p.i. 6 PBL, uninfected control; 7 PBL, infected, 6 days p.i. 8 PBL, infected and grown with inhibitor, 6 days p.i. A Hybridized with HIV (PBT 1) probe; B the same blot, after dehybridization and rehybridization with actin probe

Discussion Avian cells infected with VSV ts 1026 under non permissive conditions secrete a cytokine which interferes with retrovirus multiplication. In the homologous system, RSV multiplication is severely inhibited and this inhibition is correlated with anomalies of p r m R N A splicing and impaired RSV protein synthesis, in particular of the env and src products [4]. In a heterologous system, HIV and human cells, there is also inhibition of virus multiplication. Fewer infectious particles and more "empty" virions and particles lacking spikes are produced by cells grown with inhibitor. Several reports mention that lack of spikes is correlated with loss of infectivity [10-12]. On a molecular level, R N A transcription is perturbed. Early in the infection more HIV specific R N A and later, less is produced by treated CEM cells than by controls. This R N A is not spliced normally. In particular, a 7.5 kb species is produced by treated cells and the 9.6 kb R N A is undetectable. In PBL, HIV m R N A levels were very low so that analysis was inconclusive. No virus specific proteins and no progeny virus were detected in treated an infected cells and the CD 4/CD 8 ratio was the same in these cells and in uninfected controls. It is possible that the inhibitor acts differently on CEM and PBL, but it may also

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Fig. 6. A Northern blot analysis of RNA extracted from CEM clone 11 cells (infected with HIV 5000 cpmRT(HIV)/105 cells) and grown with inhibitor (2 gg protein/ 105 cells). 1 Uninfected cells; 2 infected cells, 7 days p.i. 3 infected cells, 10 days p.i. 4 infected cells, grown with inhibitor, 7 days p.i. 5 infected cells, grown with inhibitor, 10 days p.i. The inferior bands were revealed with a GDPH probe after dehybridization and rehybridization. B and C Scans of autoradiograph with a CCD camera; values were adjusted to saturation value 100 (ordinate). The abscissa represents the distance from the origin of the gel, in arbitrary units. B Comparison of treated (dark line) and control CEM cells 7 days p.i.; C comparison of treated (dark line) and control CEM cells 10 days p.i.

be that the amount of inhibitor present is sufficient to suppress HIV in PBL but not in CEM: at most, 20% of PBL but more than 90% of C E M produce virus, and PBL multiplication decreases as time in culture increases, thus slowing virus production further. The in vivo effect of the inhibitor could also be the result of a mixed population of defective and normal viruses from treated cells, competing with each other, different subpopulations having different defects canceling each other out so that the overall profile of the viral proteins does not differ much from that of control virus. In favour of this hypothesis is the observation of syncytia in H T 4 Lac Z-1 cells infected with progeny virus from treated cells where there is no corresponding activation of beta-galactosidase. Such syncytia can be caused by infectious but non replicating virus or by the HIV glycoproteins secreted into the medium [1]. The precise mode by which the cytokine interferes with R N A splicing has to await its isolation, which is underway. An attractive hypothesis is that the

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cytokine is a transcription factor with a particular affinity for the splicing sites of retrovirus prmRNA. Alternately it could be a factor acting at the cell membrane, triggering a mechanism which interferes with p r m R N A splicing in general and with retrovirus prmRNA splicing in particular.

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Fig. 7 A-I. Western blot analyses of proteins in CEM clone 11 cells 11 days p.i. and virus from cells infected with HIV (5000cpm RT(HIV)/105 cells) grown with inhibitor (2gg protein/105 cells). I Uninfected control cells; 2 infected control cells; 3 infected cells grown with inhibitor; 4 virus from cells grown with inhibitor; 5 virus from control cells; 6 recombinant protein. Revealed with A anti-p24 IgG; B anti-p 18 IgG; C anti-gp41 IgG; D antigp 120 IgG; E anti-reverse transcriptase IgM; F anti-nef antibody; G anti-vif antibody; H anti-rev IgG; I anti-tat IgG

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Acknowledgements We would like to thank Francoise Barr~-Sinoussi for critical revision of this manuscript and Marc Nadal for the scanning of the autoradiographs.

References 1. Cann AJ, Karn J (1989) Molecular biology of HIV: new insights into the virus life cycle. AIDS 3 [Suppl 1]: 19-34 2. Broome S, Gilbert W (1985) Rous sarcoma virus encodes a transcriptional activator. Cell 40:537 546 3. Semmel M, Sathasivam A (1983) Inhibition of Rous sarcoma virus induced transformation by preinfection with rhabdoviruses. J Gen Virol 64:275----284 4. Semmel M, Mercier G, Pavloff N, Dambrine G, Gay F, Biquard JM (1988) Viral products in cells infected with vesicular stomatitis virus and superinfected with Rous sarcoma virus. Arch Virol 100:121-129 5. Oysten-Jonassen T (1986) Direct measurement of reverse transcriptase activity in the medium from HTLV-II LAV infected cells: an application of the Skatron Harvester. Technical note, Skatron 6. Rocancourt D, Bonnerot C, Jouin H, Emerman M, Nicolas JF (1990) Activation of a beta galactosidase recombinant provirus: application to titration of human immunodeficiency virus (HIV) and HIV infected cells. J Virol 64:2660-2668 7. Barbieri D, Delain E, Lazar P, Hue G, Barski G (1970) Method of virus particle counting using millipore filters. Virology 42:544-547 8. Rosen CA, Cohen PS, Ennis HL (1983) Identification of a new protein present in vesicular stomatitis virus infected chinese hamster ovary cells as a degradation product of viral M protein. Virology 130:131-139 9. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159 10. Mc Keating J, Mc Knight A, Moore JP (1991) Differential loss of envelope glycoprotein gp 120 from virions of human immunodeficiency virus type 1 isolates: effects on infectivity and neutralization. J Virol 65:852-860 11. Gelderblom HR, Reupke H, Pauli G (1985) Loss of envelope antigens of HTLV-III/ LAV, a factor in AIDS pathogenesis? Lancet ii: 1016-1017 12. Gelderblom HR, Hausmann HS, Osel M, Pauli G, Koch MA (1987) Fine structure of human immunodeficiency virus (HIV) and immunolocalization of structural proteins. Virology 156:171-176 Authors' address: Dr. M. Semmel, U268 INSERM, 14-16 Avenue P. V. Couturier, F-94800 Villejuif, France. Received June 10, 1991