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Portable Air Conditioner SVC MANUAL(Exploded View)
MODEL : GP120CE(TWC121ZBMM0)

CAUTION

Before Servicing the unit, read the safety precautions in Standard SVC manual. Only for authorized service personnel.

1. Specification

Models
Cooling Capacity Heating Capacity Electric Heater Capacity Power Input Cooling/Heating Running Current Cooling/Heating Starting Current Cooling/Heating Electric Heater Current EER COP Power Supply Power Factor Air Flow Rate Moisture Removal Sound Level Refrigerant & Charge kW Btu/h kW Btu/h kW Btu/h W A A A W/W Btu/h.W W/W / V / Hz % cmm(cfm) l/h dB(A)+3 g(oz) GP120CE 3.52 12,000 1,2.71 9./ 115V / 60Hz 97.1 7.0(247) 2.8 55/53/51 R22,500(17.7) Rotary(Non Tropical) K1-C176ABBC-A PSC SUNISO 4GSI(or NM56m) 315 MRA12151-12027 Blower/Blower AC Induction 50/135 3*2.1(AWG 14) 500*840*WZ Thermistor O O 3/3/Wireless simple 60F~86F(16C~30C) Micom 24h, On/Off Inclinded and Top B-Look -

Compressor

Fan Power Supply Cable Dimension Net Weight Tool Code(Chassis)
Type Model Motor Type Oil Type Oil Charge O.L.P Name Type(In/Out) Motor Type Motor Output W*H*L Indoor

W No.*mm2 mm kg

Functions
Temperature Control Energy Saver Switch Prefilter(washable/anti-fungus) Plasma Filter Steps, Fan/Cool/Heat Airflow Direction Control(up&down) Airflow Direction Control(left&right) Remocon Type Setting Temperature Cooling Range Heating Auto Operation (Micom Control) Panel Touch Type Timer Air Discharge Air Ventilation Deice Control(Defrost) Hot Start Look Cabinet Type(Chassis Type) Special Function
Note: O : Applied, - : No relation * For circuit breaker rating, please conform to local standards wherever necessary. Some of functions are slightly different depending upon models. The specification may be subject to change without notice for purpose of improvement.
2 Portable Air Conditioner
Copyright 2008 LG Electronics. Inc. All right reserved. Only for training and service purposes.

LGE Internal Use Only

2. Piping Diagrams

Capillary Tube

Heat Exchanger (Evaporator)
Heat Exchanger (Condenser)

Accumulator

Service Manual 3

3. Wiring Diagrams

CNPT CNRT CNRCV CNDISP

DOWN SWING CN-WAT FAN

UPER FAN
4 Portable Air Conditioner

4. Exploded View

359012B

346810B 731270 146811

352380A

147582A

354210 359012C 147582B

435300 131111

152500B

264110

145200A

152302

W0CZZB 346810A W50060C 152500A 268711B 261704 359012A 352380B

268711A

W0CZZC

145200B

35211A

554030

554160

552101

352113
147581 W50060B W50060A 267110

135511 W0CZZA 148000

144410
Note) * Please ensure GCSC since the replacement parts may be changed depending upon the buyer's request. Please check the correct parts in View RPL(Replacement Part List) on GCSC. (GCSC Website http://biz.Lgservice.com,)

Service Manual 5

P/NO : MFL38891311

JANUARY, 2008

high conservation of HR1 (around 65% in M group), in comparison with HR2 (34% in M group),[11] may be correlated with the localization of the RRE (Rev Responsive Element) within this region. This region is essential for the export of mono- and unspliced viral mRNA from the nucleus to the cytoplasm.[12] The fusion process is a complex phenomenon that depends on a wide range of structural, biochemical and biophysical interactions. In addition, the molecular and structural mechanisms of membrane fusion, mediated by HIV-1 gp41, are still only partially understood, but enough to allow the development of new molecules able to block this key step of the viral cycle. To this end, the first applications showed that the (FP) of HIV-1, and its analogues[13, 14] were able to inhibit HIV-1 envelope glycoprotein mediated fusion. So far, efforts have been focused on developing fusion inhibitor peptides that interfere with HR1 and HR2 interactions. The T20 (DP-178 or enfuvirtide/fuzeon) is a 36-amino-acid peptide sharing two-thirds of its N-terminal amino acids with HR2.[15] This peptide efficiently inhibits HIV-1 replication in vitro at nM levels,[16, 17] and in vivo.[18] Its inhibitory activity seems to be mediated by direct interactions with HR1 and by interaction of its C-terminal part, different from HR2, with membrane or the transmembrane domain of gp41.[19-23] T20 is now currently used as an entry inhibitor of HIV, essentially in patients who have developed resistance to the usual highly active antiretroviral therapy (HAART).[24] However, the growing problem of the emergence of new T20 resistance to HIV-1 strains[25] needs the development of new peptidic inhibitors including: i) analogues of T20 such as T-1249,[26] a 39 all L-amino acid peptide composed of sequences derived from HIV-1, HIV-2 and SIV. The great advantage of this peptide is its ability to remain active against T20-resistant viruses,[27] ii) C34 peptide, corresponding to 95
HR2, which inhibits HIV-1 infection in vitro at nM level. Several analogues of HR2 regions have also proved to be potent inhibitors both in vitro and in vivo, [26, 28, 29] iii) N36 peptide, a 36 amino acid peptide corresponding to HR1. In contrast to T20 and C34 peptides, N36 peptide inhibits HIV-1 with lower activity (IC50 of the order of a M ).[30] The use of T20 peptide is also limited essentially by its relatively short half-life in vivo. The introduction of D-amino acids in punctual positions may enhance the resistance of peptides or proteins to protease degradation. However, the structural stability, conformation and activity might be negatively affected.[31] The magnitude of the structural changes depends on the localization and the character of the modification.[32] Partial D-amino acid substitutions could break the structure, decreasing its stability, or, in contrast, restrain a favourable conformation with lower stability cost. For instance, the substitution of glycine residues by Dalanine satisfies the topological requirements for retaining structure and function[33] or even

forming a 6 helix bundle(29, 40). This hairpin structure is believed to be responsible for the fusion of HIV envelope with the host cell membrane. Understanding the mechanism of fusion of the viral envelope with the host membrane played a crucial role in the development of new generation of peptidic anti-retroviral drugs able to intefere with virus entry by blocking virus-cell fusion. Enfuvirtide, a 36 L amino acids, is based on the sequence of HRII region. It binds to the triple-stranded coiled coil formed by the three HRI domains, thereby preventing HRI-HRII interaction and the formation of the six-helix bundle and hence inhibiting membrane fusion(7). Thus, T-20 peptide seems to mediate its inhibitory action by binding to a transient envelope conformation induced by gp120-CD4-CCR5/CXCR4 binding(17, 23, 32). In the initial stages of the use of T-20, this molecule inhibits eficiently HIV-1 replication in various cell types and clinical trials(31). Because of its limited half-life, an efficient treatment with this peptide necessitates the
subcutaneous injection of great amount (90-100 mg) twice a day (25). Although the efficiency of this treatment is prooved, such treatment remained very heavy to support by patients, in addition to the side reactions observed at the site of injection like palpable nodules (1, 35). Several studies have reported the presence in HIV-1 infected patients of antibodies against HRI-HRII complex. In addition, antipeptide antibodies were generated in patients treated with T-20 (39). Despite the presence of ELDKWA which corresponds to the epitope recognized by the broadly neutralizing antibody 2F5, antibodies generated against T-20 in HIV-1 infected patients are not neutralizing. In addition it was reported that HRI sequence is not immunogenic in vivo (16, 20). We suggest that the presence of such antibodies in the HIV-1 infected patients may act negatively on the efficiency of the antiviral effect of the peptide by forming stable antigenantibody complexes.

MATERIAL AND METHODS

Peptide synthesis Four peptides were synthetized and used in this study (table 1). The peptide sequences tested were derived from HIV-1 Lai and correspond to the Cterminal heptad repeat HR1 and HR2 also named N36 and C34 respectively. The peptides were automatically synthesized in parallel on 330 mg of Rink amide-Gly-MBHA resin with low loading (0.3 mmol/g) on an Applied Biosystems 433A peptide synthesizer by using Fmoc chemistry. The MBHA resin HL (loading: 0.77 mmol/g, Novabiochem) was solvated 134

9. 10.

15. 16.

21. 22.

Doms, R. W., and J. P. Moore. 2000. HIV-1 membrane fusion: targets of opportunity. J Cell Biol 151:F9-14. Earl, P. L., C. C. Broder, R. W. Doms, and B. Moss. 1997. Epitope map of human immunodeficiency virus type 1 gp41 derived from 47 monoclonal antibodies produced by immunization with oligomeric envelope protein. J Virol 71:2674-84. Gallo, S. A., A. Puri, and R. Blumenthal. 2001. HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4 in the HIV-1 Envmediated fusion process. Biochemistry 40:12231-6. Geffin, R. B., G. B. Scott, M. Melenwick, C. Hutto, S. Lai, L. J. Boots, P. M. McKenna, J. A. Kessler, 2nd, and A. J. Conley. 1998. Association of antibody reactivity to ELDKWA, a glycoprotein 41 neutralization epitope, with disease progression in children perinatally infected with HIV type 1. AIDS Res Hum Retroviruses 14:579-90. Geoghegan, K. F., and J. G. Stroh. 1992. Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alcohol. Application to modification at N-terminal serine. Bioconjug Chem 3:138-46. Golding, H., M. Zaitseva, E. de Rosny, L. R. King, J. Manischewitz, I. Sidorov, M. K. Gorny, S. Zolla-Pazner, D. S. Dimitrov, and C. D. Weiss. 2002. Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates. J Virol 76:6780-90. Greenfield, N. J. 2004. Analysis of circular dichroism data. Methods Enzymol. 383:282-317. Guichard, G., S. Muller, M. van Regenmortel, J. P. Briand, P. Mascagni, and E. Giralt. 1996. Structural limitations to antigenic mimicry achievable with retroinverso (all-D-retro) peptides. Trends Biotechnol 14:44-5. He, Y., R. Vassell, M. Zaitseva, N. Nguyen, Z. Yang, Y. Weng, and C. D. Weiss. 2003. Peptides trap the human immunodeficiency virus type 1 envelope glycoprotein fusion intermediate at two sites. J Virol 77:1666-71. Johnson, O. L., J. L. Cleland, H. J. Lee, M. Charnis, E. Duenas, W. Jaworowicz, D. Shepard, A. Shahzamani, A. J. Jones, and S. D. Putney. 1996. A month-long effect from a single injection of microencapsulated human growth hormone. Nat Med 2:795-9. Kilby, J. M., S. Hopkins, T. M. Venetta, B. DiMassimo, G. A. Cloud, J. Y. Lee, L. Alldredge, E. Hunter, D. Lambert, D. Bolognesi, T. Matthews, M. R. Johnson, M. A. Nowak, G. M. Shaw, and M. S. Saag. 1998. Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nat Med 4:1302-7. Klatzmann, D., E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J. C. Gluckman, and L. Montagnier. 1984. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 312:767-8. Leghmari, K., X. Contreras, C. Moureau, and E. Bahraoui. 2008. HIV-1 Tat protein induces TNF-alpha and IL-10 production by human macrophages: Differential implication of PKC-betaII and -delta isozymes and MAP kinases ERK1/2 and p38. Cell Immunol. Loomis-Price, L. D., J. H. Cox, J. R. Mascola, T. C. VanCott, N. L. Michael, T. R. Fouts, R. R. Redfield, M. L. Robb, B. Wahren, H. W. Sheppard, and D. L. Birx. 1998. Correlation between humoral responses to human immunodeficiency virus type 1 envelope and disease progression in early-stage infection. J Infect Dis 178:1306-16. Lu, M., S. C. Blacklow, and P. S. Kim. 1995. A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat Struct Biol 2:1075-82.

Simian immunodeficiency virus (SIV) is a non-human primate lentivirus related to the retroviridae familly. It was initially isolated from a captive rhesus monkey [1]. Like human immunodeficiency viruses, HIV-1 and HIV-2, this virus induces an AIDS-like disease only in non-natural monkey hosts, such as Asian monkeys. Although naturally infected, African monkeys develop only an asymptomatic chronic infection [2, 3]. HIV and SIV genomes present similar organization, except for the presence of vpx and Vpu genes in SIV and HIV genomes respectively [4]. SIV also shares several additional properties with HIV including tropism for CD4 positive cells, oligomeric structure of the surface glycoprotein (gpSU) and the transmembrane glycoprotein (gpTM) which are assembled as trimers on the surface of the
viral particles. The physiopathology of the infection is also comparable. All these properties make SIV/macaque a valuable model for evaluating candidate vaccines and antiretroviral drugs. In contrast to HIV-1, the native form of SIV precursor gp140 seems to be essential for the generation of appreciable titers of neutralizing antibodies. Denatured gpSU or synthetic peptides corresponding to that of the HIV-1 principal neutralizing determinant, also named the V3 region, are unable to induce or adsorb measurable amounts of neutralizing antibodies [5]. In line with the latter report, the same group has also shown that SIV-infected monkeys produce neutralizing antibodies that continue to interact with a 45 kDa fragment that includes both V3 and V4 variable loops of SIV gpSU [6]. However, several groups have also described linear neutralizing epitopes in the SIV V2 region between amino acids 170 and 190 [7] and in the V4 loop as shown by the capacity of this peptide to adsorb neutralizing antibodies from the sera of SIV infected macaques [8]. This suggests the presence of a linear epitope of neutralization within this region. In accordance with these results, our group produced and characterized monoclonal antibodies (Mab1B9) which recognized a linear epitope in the V4 region (aa 411-430) of the gpSU and neutralized SIV replication[9]. Several lines of evidence have also shown the crucial role of the conformational structure of gp120 from HIV-1 in the induction of protective immune responses. At least four categories of neutralizing antibodies have been purified from HIV-1 infected human sera, including those directed against the linear epitope within the V3 region, the conformational CD4 binding site, the carbohydrate moieties and the quaternary epitopes present only in the

(Macaca mulatta) against the infection with SIVmac32H grown on T-cells or derived ex vivo Virology 1996; 216: 444 Hoxie, J. A. Hypothetical assignment of intrachain disulfide bonds for HIV-2 and SIV envelope glycoproteins AIDS Res Hum Retroviruses 1991; 7: 495 Chavez, L. G., Jr. and Scheraga, H. A. Intrinsic stabilities of portions of the ribonuclease molecule Biochemistry 1980; 19: 1005 Furie, B., Schechter, A. N., Sachs, D. H. and Anfinsen, C. B. An immunological approach to the conformational equilibrium of staphylococcal nuclease J Mol Biol 1975; 92: 497 Sachs, D. H., Schechter, A. N., Eastlake, A. and Anfinsen, C. B. An immunologic approach to the conformational equilibria of polypeptides Proc Natl Acad Sci U S A 1972; 69: 3790 Neurath, A. R., Strick, N., Fields, R. and Jiang, S. Peptides mimicking selected disulfide loops in HIV-1 gp120, other than V3, do not elicit virus-neutralizing antibodies AIDS Res Hum Retroviruses 1991; 7: 657 Schulze-Gahmen, U., Klenk, H. D. and Beyreuther, K. Immunogenicity of loopstructured short synthetic peptides mimicking the antigenic site A of influenza virus hemagglutinin Eur J Biochem 1986; 159: 283 Satterthwait, A. C., Arrhenius, T., Hagopian, R. A., Zavala, F., Nussenzweig, V. and Lerner, R. A. Conformational restriction of peptidyl immunogens with covalent replacements for the hydrogen bond Vaccine 1988; 6: 99 Christodoulides, M., McGuinness, B. T. and Heckels, J. E. Immunization with synthetic peptides containing epitopes of the class 1 outer-membrane protein of Neisseria meningitidis: production of bactericidal antibodies on immunization with a cyclic peptide J Gen Microbiol 1993; 139: 1729 Palker, T. J., Matthews, T. J., Langlois, A., Tanner, M. E., Martin, M. E., Scearce, R. M., Kim, J. E., Berzofsky, J. A., Bolognesi, D. P. and Haynes, B. F. Polyvalent human immunodeficiency virus synthetic immunogen comprised of envelope gp120 T helper cell sites and B cell neutralization epitopes J Immunol 1989; 142: 3612 Wang, C. Y., Looney, D. J., Li, M. L., Walfield, A. M., Ye, J., Hosein, B., Tam, J. P. and Wong-Staal, F. Long-term high-titer neutralizing activity induced by octameric synthetic HIV-1 antigen Science 1991; 254: 285 Ahlers, J. D., Dunlop, N., Pendleton, C. D., Newman, M., Nara, P. L. and Berzofsky, J. A. Candidate HIV type 1 multideterminant cluster peptide-P18MN vaccine constructs elicit type 1 helper T cells, cytotoxic T cells, and neutralizing antibody, all using the same adjuvant immunization AIDS Res Hum Retroviruses 1996; 12: 259 Kobs-Conrad, S., Lee, H., DiGeorge, A. M. and Kaumaya, P. T. Engineered topographic determinants with alpha beta, beta alpha beta, and beta alpha beta alpha topologies show high affinity binding to native protein antigen (lactate dehydrogenase-C4) J Biol Chem 1993; 268: 25285 Merrifield, B. Solid phase synthesis Science 1986; 232: 341 Chakrabarti, L., Guyader, M., Alizon, M., Daniel, M. D., Desrosiers, R. C., Tiollais, P. and Sonigo, P. Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses Nature 1987; 328: 543 Benjouad, A., Babas, T., Montagnier, L. and Bahraoui, E. N-linked oligosaccharides of simian immunodeficiency virus envelope glycoproteins are dispensable for the interaction with the CD4 receptor Biochem Biophys Res Commun 1993; 190: 311 Le Grand, R., Vogt, G., Vaslin, B., Roques, P., Theodoro, F., Aubertin, A. M. and Dormont, D. Specific and non-specific immunity and protection of macaques against SIV infection Vaccine 1992; 10: 873

isozyme activation. The PKC pathway is crucial for many cellular events and its involvement has been reported in the step of entry of a number of envelopped viruses like rhabdoviruses, alphaviruses and herpesviruses (Constantinescu, Cernescu et al. 1991). PKCs are also involved in the viral life cycle of non-envelopped viruses. It has, for example, been demonstrated that type 2 adenovirus entry is PKC-dependent. Indeed, when infection is performed in presence of calphostin, a conventional and novel PKC inhibitor, viral particles do not reach endosomes anymore and accumulate in the cytoplasm (Otaka, Nakamura et al. 2002). Additionally, influenza virus interaction with its receptor induces activation of the PKC pathway. Selective inhibition of PKC- II isozyme was accompanied by the blocking of viral infection which aborts following the block of viral trafficking at the level of late endosomes (Sieczkarski, Brown et al. 2003). Activation of PKCs enhances HIV-1 gene expression in latently infected T-cells and in
cell lines (Hu, Frank et al. 2004). Indeed, PKCs stimulate NF-B (Hamamoto, Matsuyama et al. 1990; Meichle, Schutze et al. 1990; Wei, Ghosh et al. 1995), via the phosphorylation of IB. NF-B binds to the HIV promoter and is involved in initiation and elongation of transcription (Nabel 2005). In addition, PKCs activate other transcription factors like AP-1 and NF-AT (Siekevitz, Josephs et al. 1987; Lamph, Wamsley et al. 1988) which have specific binding sites on the HIV-1 promoter. Moreover, PKCs have been suggested to phosphorylate a number of viral proteins such as p17gag (Burnette, Yu et al. 1993), Nef (Guy, Kieny et al. 1987; Guy, Riviere et al. 1990; Baur, Sass et al. 1997) and Rev (Malim, Hauber et al. 1989). They are also involved in cytoskeleton rearrangements required in human macrophages during the early steps of reverse transcription (Bukrinskaya, Brichacek et al. 1998). A number of reports have demonstrated the major role of PKC- in the maintenance of cytoskeleton integrity. Indeed, the C2 domain of PKC- contains an actin binding site. This binding could be involved in the redistribution of actin in neutrophils (Lopez-Lluch, Bird et al. 2001; Smallwood, Hausman et al. 2005). PKC- also plays a central role in differentiation of monocytes, resistant to infection by HIV-1 (Sonza, Maerz et al. 1996; Sonza, Mutimer et al. 2001), into macrophages, that are susceptible to infection (Fantuzzi, Conti et al. 2000; Weiden, Tanaka et al. 2000). Furthermore, macrophage differentiation induced by M-CSF (Mischak, Pierce et al. 1993; Junttila, Bourette et al. 2003) or by PMA (Mischak, Pierce et al. 1993; Tan, Liu et al. 1997) is dependent on PKC-. This is mainly because of its action on the NF-kB transcription factor (Biswas, Ahlers et al. 1994; Kang, Park et al. 2004) and, maybe, because of its association to vimentin in the cytoskeleton (Owen, Johnson et al. 1996). PKC- and I could also be involved in this differentiation (Kim, Gollapudi et al. 1996). 194

cytoplasm towards the exterior of the cell which are the result of actin cytoskeleton rearrangements (figure 5A-D). In these untreated macrophages, like in all adherent cells, actin microfilaments organize in stress fibers (figure 5A, arrow). However, macrophages treated by rottlerin (5 M) 24 h before observation are composed of very few pseudopodes (figures 5EH) and do not seem to contain stress fibers (figure 5E). Rottlerin, within range of the concentrations used in this study, has no significant effect on cell viability (figure 1 and data not shown). Thus, these data suggest that rottlerin interferes with actin cytoskeleton rearrangements in macrophages, thus inhibiting pseudopode formation. Since the reverse-transcriptase complex from the incoming virus interacts with actin microfilaments (Campbell, Nunez et al. 2004), we hypothesized that inhibition of PKC delta could result in loss of association between this complex and actin cytoskeleton. To address this question, membrane, cytoskeleton and nucleus fractions were obtained from macrophages preincubated or not with rottlerin or cytochalasin D (CCD, a compound that disrupts actin cytoskeleton) and infected with HIV-1 BaL. Presence of the matrix protein (Gag-MA) was followed by western blotting. In control non-treated cells, Gag-MA was found in the membrane, cytoskeleton and nucleus fractions (Figure 6). As expected, Gag-MA was not found in the cytoskeleton and nucleus fractions following pre-treatment with CCD. Similar results were obtained after pre-treatment of cells with rottlerin. Taken together these results demonstrate that PKC- is required for cytoskeleton integrity in human macrophages and suggest that the reverse transcription complex is not able to associate with the cytoskeleton in these conditions.
In this study, we demonstrated that inhibition of PKC- blocks viral replication of HIV-1 BaL in human macrophages. This inhibition blocked replication at the level of early reverse transcription. In addition, receptors and co-receptors were expressed at normal levels and syncitia formation was not impaired, suggesting that there is no blockade at the level of virus entry. Inhibiting PKC- affected cytoskeleton integrity. Moreover, the complex of reverse transcription was not associated with the cytoskeleton after inhibition of PKC-. Thus, our studies strongly suggest that inhibition of PKC- disrupts the actin cytoskeleton and thus prevents completion of reverse transcription. A virus pseudotyped with enveloppe glycoprotein G from VSV, penetrating by endocytosis, is not affected by rottlerin anymore. This PKC- inhibitor blocks reverse

since gagMA, contained within the reverse transcriptase complex is associated to actin whereas the capsid, main component of the virus core, would not interact significantly. The infectious viral particle does not contain all the components necessary to terminate cDNA synthesis, since only the products of the early reverse transcription are obtained in in vitro reverse transcription reactions, using viral extracts (Arts and Wainberg 1996). Thus, it is also possible that the cytoskeleton is a specialised site for reverse transcription since the cellular factors required to the process of reverse transcription could localize at the level of the actin cytoskeleton. Studies by Mc Donald et al., (McDonald, Vodicka et al. 2002), suggest that after entry into the cytoplasm, HIV-1 genome uses the actin cytoskeleton to move in peripheral regions of the cell. Actin could be used to access the microtubular network (Taunton 2001). The reverse transcription complex then associates to microtubules to get in close proximity to the nucleus. Recent studies found that the reverse transcription complex was also able to bind actin around the nucleus, before nuclear import (Arhel, Souquere-Besse et al. 2007). This step is followed by uncoating after formation of the DNA flap, upon completion of viral DNA synthesis. Reverse transcription probably occurs in an integral capsid structure (Arhel, SouquereBesse et al. 2007). Some morphological changes in the capsid may be involved in the process of reverse transcription, as suggested by the impaired reverse transcription observerd in viruses with hyperstable capsids or unstable cores (Forshey, von Schwedler et al. 2002). It is thus possible that interaction with actin plays an important role in promoting correct stability of the viral core. In normal conditions, the transport of the viral core to the nucleus is more rapid than the RT. RT thus finished near the nuclear pores and triggers uncoating once the 204
DNAflap is synthesized. Most probably in the absence of actin cytoskeleton at both cell membrane and around the nucleus, the viral core cant be transported to the nuclear pore. In support of our conclusions, activation of the actin polymerization nucleator Arp2/3 was demonstrated to be involved in the transport of the viral core from plasma membrane to the microtubule network (Komano, Miyauchi et al. 2004). Importantly, in agreement with our results, membrane fusion was not impaired and VSV-G pseudotyped vectors that use endosomes to enter cells were not affected by such treatment. This study suggested that cortical actin is actively remodeled by Arp2/3. In our studies, inhibition of PKC- impaired the integrity of the cytoskeleton, preventing such remodeling from occurring. Rottlerin inhibits PKC- and PKCs are involved in cytoskeleton rearrangements and differenciation from monocyte to macrophage. PKC- would be capable to bind to the actin cytoskeleton in the neutrophil and induce its redistribution (Lopez-Lluch, Bird et al. 2001;

plasmid. Two days later, cells were observed with a confocal microscope. Images are representative of three independent experiments.
Figure 4 : Analysis of the products of reverse transcription by HIV-1 BaL in macrophages pretreated or not wih rottlerin. Macrophages (5.105 cells/well) were pretreated by rottlerin (5 M) for 30 minutes and then infected with HIV-1 BaL (1 ng p24) for 3 h at 37C. Cells were then lysed at 4, 12, 24, 48 h after infection, and 100 ng of DNA were analyzed by PCR. Amplification of the early (R-U5), intermediate (pol) and late (U3-gag) products of reverse transcription was performed by PCR. PCR products were analyzed after electrophoresis in an agarose gel. Three independent experiments realised with macrophages from three different donors gave similar results. C :
Control cells ; R : cells pretreated with rottlerin ; I : cells infected without pretreatment with rottlerin. Figure 5 : Actin cytoskeleton is altered by pretreatment of macrophages by rottlerin (5M) Human macrophages were treated for 24h by rottlerin (5 M, E-F), or not treated (controls) (A-D) and then fixed with formaldehyde (3,7 %). Macrophages were then permeabilised with triton X-100 (0,2 %) and stained with rhodamine-phallodine before mounting using moviol. Preparations were then observed under a confocla microscope. Confocal images are representative of three independent experiments. Figure 6 : Macrophages (5.105/ml) were pretreated or not with rottlerin or cytochalasin D and then infected with HIV-1 BaL (1ng p24). Membrane (M), Cytosqueleton (C) and Nucleus (N) fractions were obtained and presence of Gag-MA was assessed by Western Blotting.
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