Virology 487 (2016) 59–67

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Virology

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Kinetics of the establishment of HIV-1 viral interference and comprehensive analysis of the contribution of viral

Azaria Remion a,b,c, Marc Delord b,c, Allan J. Hance a,b,c, Sentob Saragosti a,b,c, Fabrizio Mammano a,b,c,n a INSERM, U941, Paris, France b Univ Paris Diderot, Sorbonne Paris Cité, Paris, France c Institut Universitaire d’Hématologie, Hôpital Saint-Louis, Paris, France article info abstract

Article history: Viral interference defines the reduced susceptibility of an infected cell to reinfection. For HIV-1, both Received 9 June 2015 receptor-dependent and independent pathways were described. The relative importance of different Returned to author for revisions receptor-independent pathways has not been addressed. 1 July 2015 We have used reporter to quantify the percentage of single- and double-infected cells, as a Accepted 26 September 2015 function of the delay between the two . For co- experiments, the frequency of double Available online 23 October 2015 infected cells was higher than expected for independent events. By delaying the second infection, this Keywords: frequency progressively diminished, resulting in significant interference after 18 h. Interference mea- HIV-1 sured here was largely receptor-independent. By individually deleting viral genes or expressing them in Viral interference isolation, we demonstrate that the viral protein Rev plays a dominant role, while other viral proteins Super-infection contributes to optimal interference. Our study defines the kinetics of early HIV-1 interference, describing the transition from higher susceptibility to double-infection to viral interference, and identifies Rev as its dominant effector. & 2015 Elsevier Inc. All rights reserved.

Introduction Altfeld, 2003) is a clear and relevant proof that double-infection and recombination occur in infected patients. Infection of a single cell by two HIV-1 virions allows genetic Despite the advantages associated with double-infection, HIV- recombination, an important evolutionary strategy that generates 1, as many other viruses, has evolved viral interference mechan- chimeric genomes whose adaptation to the environment may isms to limit the occurrence of double-infections. It is assumed greatly exceed that of each parental genome (Burke, 1997). that the purpose of these strategies is to prevent excessive viral Recombination requires the infection of a single cell by distinct replication and premature cytolysis (Volsky et al., 1996). Mod- viruses, the subsequent incorporation of two different genomes ulating cell susceptibility to infection over time may allow HIV-1 fi into a single particle, and template switching during reverse to pro t from double-infection when it can lead to functional transcription following the infection of a new target cell (reviewed recombination, and avoid its negative effects if the second virus reaches a cell fully engaged in HIV-1 production. The kinetics of in Burke, 1997). Double-infection of target cells by HIV-1 in tissue the transition from susceptibility to interference has not been culture occurs more frequently than would be predicted if the two precisely characterized. infectious events were independent phenomena (Dang et al., Several distinct interference pathways have been described for 2004). There is also direct evidence of the presence of multiple HIV-1, and the expression of six of the nine viral genes has been HIV-1 genomes in cells obtained from infected patients. Although proposed to participate in viral interference. A common mechan- most infected circulating lymphocytes carry only one copy of viral ism of interference for different virus families consists in the genome, double-infected cells have been consistently detected occupation or down-modulation of the receptor by newly syn- (Josefsson et al., 2011, 2013). Also, the epidemiologic spread of a thesized viral proteins produced by the first virus infection (Potash wide variety of circulating recombinant forms of HIV-1 (Allen and and Volsky, 1998). Three HIV proteins (Env, Vpu and Nef) con- tribute to the reduction of CD4 expression at the cell surface by

n different mechanisms (Chen et al., 1996; Wildum et al., 2006). Env Correspondence to: Inserm U-941, Institut Universitaire d’Hématologie, Bâti- ment Hayem Hôpital Saint-Louis, 1, Avenue Claude Vellefaux, 75010 Paris, France. and Vpu interfere with CD4 transport to the plasma membrane; E-mail address: [email protected] (F. Mammano). Nef induces the internalization of CD4 molecules that had already http://dx.doi.org/10.1016/j.virol.2015.09.028 0042-6822/& 2015 Elsevier Inc. All rights reserved. 60 A. Remion et al. / Virology 487 (2016) 59–67

Fig. 1. Frequency of double-infected cells following co-infection and super-infection at 24 h. The 3 upper panels show the distribution of cells in different FACS quadrants in the following control conditions: left-no infection; center-infection by the virus expressing HSA; right-infection by the virus expressing GFP. The 2 lower panels show the distribution of cells in different quadrants following co-infection (lower left panel) and super-infection at 24 h (lower right panel). The number of cells in each quadrant of the 5 FACS plots is indicated in table. The OR of cells expressing both reporter genes (double-infection), was calculated by (B/A)/(D/C), as in (2). The distribution expected if the two infectious events were independent would result in an OR of 1. ORs above 1 indicate that double-infections are favored, and ORs below 1 indicate inhibition of double-infections, as expected in case of viral interference. reached the cell surface. The physiological role of CD4 down- involved are not fully understood, as reviewed in Potash and Volsky modulation in HIV-1 infection, however, does not seem to be the (1998) and Nethe et al. (2005). Volsky et al. (1996) reported viral prevention of super-infection. Rather, it ensures efficient Env interference in the first 24 hours post-infection, before detecting incorporation into budding virions, leading to increased infectivity any sign of CD4 down-modulation in infected cells. They excluded a (Argañaraz et al., 2003). Also, it was recently shown that CD4 role for Vpu, Vpr and Nef, because a mutant virus defective for the down-regulation by Nef and Vpu promotes escape from antibody- expression of these proteins displayed wild type level of inter- dependent cell-mediated cytotoxicity (Pham et al., 2014). Similar ference (Volsky et al., 1996). Federico et al. (1995) reported that cells to the effect mediated by modulation of CD4 expression, inter- chronically infected with a HIV-1 mutant that did not produce ference in HIV-1 may also result from down-modulation of the infectious virus particles were resistant to super-infection through a expression of the co-receptors CCR5 and CXCR4 by Nef (Venzke CD4-independent mechanism. This team went on to express indi- et al., 2006; Michel et al., 2005). vidual viral proteins of the mutant virus by transfection of HIV-1 Receptor/co-receptor-independent viral interference has also susceptible cells, and provided evidence that the viral proteins Gag, been described for HIV-1, but the viral genes and mechanisms Vif and Nef contributed to the CD4-independent viral interference A. Remion et al. / Virology 487 (2016) 59–67 61 observed in the original report, whereas expression of Tat, Rev or quadrant was used to calculate the ratio [B/(BþA)]/{1[B/ Vpu facilitated secondary infection (D’Aloja et al., 1998). In contrast (BþA)]}, which upon simplification becomes (B/A)/(D/C). If the to this finding, the viral protein Rev was reported to promote viral two infections are independent events, the ratio should be equal to interference in HIV-1-infected cells by Levin et al. (2010).Overall, 1. An OR41 indicates that double-infections occurred more fre- these reports support the existence of receptor-independent viral quently than expected for independent events, and an ORo1 interference in HIV-1, but in these studies only the potential corresponds to lower frequency. implication of subsets of genes was investigated. As a consequence, In the illustrated experiment, co-infection resulted in an OR of the relative importance of different viral genes in receptor- 2.47, indicating that the frequency of cells carrying the two independent HIV-1 interference has not been established. reporter genes is approximately 2.5-fold higher than expected for Here, using isogenic reporter viruses, we precisely quantified independent events. The OR of co-infection was significantly dif- the frequency of single- and double-infected cells, as a function of ferent from 1 using a Pearson's χ2 test (po0.0001). The higher the interval between the two infection events (from 0 to 24 h). We confirm the higher than random frequency of double-infected cells when cells are simultaneously exposed to the two viruses. By increasing the time between the infections, the frequency of double infected cells progressively decreased, reaching values below those expected for independent events, starting at 18 h post-infection. Using HIV-1 virions pseudotyped by the amphotropic-MLV (A-MLV) envelope glycoproteins, we show that viral interference was largely independent from receptor and co- receptor mediated mechanisms. By individually deleting or expressing viral genes in a sequential infection setting, we identify Rev as the key viral effector of the interference measured here, although other viral genes participate in the establishment of optimal interference.

Results

Frequency of double-infected cells following co-infection or super-infection at 24 h

We have used two HIV-1 proviral clones each expressing a reporter protein, either green fluorescent protein (GFP) or heat- stable antigen (HSA), allowing quantification of the cells infected by one, the other, or both viruses. For this study, it was important to use isogenic viral clones expressing all viral proteins, and dif- fering only by the reporter . In the pNL4-3-derived plasmids used here (Imbeault et al., 2009; Levy et al., 2004), sequences coding the two reporter proteins were introduced before the nef gene along with an IRES sequence, allowing concomitant expres- sion of the viral and reporter proteins. To prevent virus spread in culture, we deleted the env gene from the proviral constructs. These Env-deficient mutants were co-transfected with a plasmid that allows HIV-1 Env expression in trans, and produced virus particles competent for single cycle infection. Precluding virus spread facilitates the study of the extent of super-infection as a function of time, and eliminated the Env-mediated pathway of CD4 down-modulation. As shown in Fig. 1 (upper panels), infection by each virus could be monitored by the distribution of cells into the four quadrants (A, B, C, and D) of the FACS plot, following the expression of the reporter genes (GFP or HSA). To compare the frequency of double infected cells in the setting of co-infection (infection with two different viruses added simultaneously) and super-infection at Fig. 2. Frequency of double-infected cells as a function of the time that separates 24 h, cells in parallel wells were infected by both viruses or the two infectious events. (A) The OR of double-infected cells is shown for co- infected only with either the GFP virus or the HSA virus. Cells were infections (exposure to both viruses at time 0) and for super-infections, in which washed 2 h post-infection. Wells infected by a single virus were exposure to the second virus was delayed by 3, 6, 12, 18 and 24 h. Both the initial exposed to the other virus 24 h after the initial infection. The and the super-infecting virus carry the HIV-1 Env. The bar graph represents the mean and SEM from 12 experiments (6 in which the first infection was performed results of a representative experiment comparing co-infections using the GFP virus, and 6 in which initial infection was performed with the HSA (t¼0) and super-infections at 24 h are shown in Fig. 1 (lower virus). *** indicates po0.0001 as compared to the frequency observed for t¼0. panels). From the distribution of the cell populations into the (B) CD4-independent viral interference. Experiments were conducted as in A, quadrants of the FACS plot, we measured the frequency of double- except that the second infection was performed using an HIV-1 particle pseudo- typed by the A-MLV Env. ORs of double-infections is shown for infections by A-MLV infected cells and compared it to the expected frequency if the two pseudotyped virus taking place at time 0, 6, 12, 18 and 24 h after initial infection by events were independent, by calculating the odds ratio (OR) as HIV-1. Data shown represent the mean and SEM (n¼4). *po0.05 as compared to previously described (2). Briefly, the number of cells in each the frequency observed for t¼0. At 24 h the p value was 0.07. 62 A. Remion et al. / Virology 487 (2016) 59–67 frequency of double-infections in the settings of co-infection has and nef) were evaluated. As shown in Fig. 3A, all clones still been previously described (Dang et al., 2004). In striking contrast, induced HIV-1 interference at 18 and 24 h post-initial infection, in the same experiment, delaying the second infection by 24 h indicating that no single accessory gene is responsible for inter- resulted in an OR of 0.46, indicating that the observed frequency of ference. The same conclusion could be drawn when A-MLV- double-infection is the half of what would be expected for inde- pseudotyped virions were used for super-infection (Fig. 3B). pendent events (po0.0001), thus showing potent viral inter- To study the potential involvement of the expression of the ference, particularly as compared to the frequency observed in the remaining viral genes gag, pol, tat and rev in HIV-1 interference (all co-infection conditions. These results confirm prior studies, which experiments were performed in an env-defective context), a dif- separately showed high frequency of double-infection at t¼0, and ferent strategy was required, because: i) preventing the expression interference at t¼24 h. of tat and/or rev would indirectly abolish the expression of several additional viral proteins in the infected cells, and ii) the production Dynamics of viral interference in HIV-1 infection of infectious virus particles after the abrogation of gag or pol expression requires complementation. Thus, lentiviral vectors We next compared the OR of double-infections as a function of were produced by co-transfection of a Gag-Pol-expressor plasmid, the time between the initial infection and super-infection (3 h, 6 h, an Env-expressor plasmid and a transfer vector carrying the 12h,18hor24h)(Fig. 2A). This analysis shows that the frequency packaging signal. We constructed transfer vectors that allow the of double-infected cells gradually diminished as the interval expression in target cells only of Tat (Tþ), Tat and Rev (TþRþ), or between initial infection and super-infection events increased. The all viral proteins except Gag-Pol and Env (ΔGagPol). As shown in OR values remained above 1 for super-infection taking place in the Fig. 4A, cells initially infected by the vector encoding all proteins first 12 h post-initial infection. Significant viral interference, with except Gag-Pol and Env displayed an interference profile similar to OR lower than 1, was measured starting at 18 h and it further the one observed with the virus defective only for Env expression increased at 24 h post-initial infection. (WT), demonstrating that expression of Gag-Pol in infected cells is not required for interference. The expression of Tat alone did not Receptor-independent viral interference interfere with the second infection, resulting in a profile similar to that observed for empty (GFP-only) lentiviral particles. In contrast, We next determined the proportion of interference that was the expression of both Tat and Rev did result in viral interference not due to receptor and co-receptor down-modulation. We repe- at 18 h and 24 h, reducing the OR below 1. These results show that ated studies evaluating the kinetics of super-infection, but using the expression of Rev (together with Tat, but not Tat alone) was for the second infection virus particles pseudotyped with the A- required to reduce the cellular susceptibility to super-infection. As MLV Env glycoproteins, whose entry does not depend on CD4 and for the previous sets of data, we verified that this interference chemokine receptors. The OR of co-infection using viruses carrying profile was largely independent of effects on the expression of these different Env proteins was still higher than 1, but did not HIV-1 receptors, because the results obtained using A-MLV pseu- reach the same level as observed for HIV-1/HIV-1 co-infections dotyped HIV-1 virions as super-infecting particles gave a similar (Fig. 2B). As in the previous set of experiments, viral interference profile (Fig. 4B). (ORo1) was observed at 18 and 24 h post-initial infection. The The potency of interference observed after infection with potency of interference is lower than that observed in experiments viruses expressing only Tat and Rev, as measured by the OR, was in which the second virus also carried the HIV-1 Env proteins, lower than that observed when wild-type virus was used for initial suggesting that CD4 and/or co-receptor down-modulation con- infection. This suggests that although expression of Rev per se tributes to, but does not fully account for, a major proportion of induces significant viral interference, the expression of other viral the viral interference observed in Fig. 2A. proteins, many of which are Rev-dependent, is required for opti- mal interference. From Fig. 3, however, it appears that the deletion Role of viral genes in the establishment of HIV-1 viral interference of single accessory genes did not prevent the establishment of interference. Altogether these results suggest that, in addition to We next showed that the expression of viral genes is required Rev, multiple genes participate in the interference phenotype, but to induce viral interference. Indeed, initial infection performed no single protein other than Rev plays a predominant role. with HIV-1 particles carrying a lentiviral vector (LV) that expresses only GFP (pLenti-GFP) did not induce significant interference with the infection by a wild-type virus (expressing HSA) added 18 h or Discussion 24 h post-initial infection (Fig. 3A, sample LV). In this situation, the OR of double-infected cells remained above 1 at these late time Cellular susceptibility to virus infection and to super-infection points, although it decreased as compared to the co-infection depends on multiple parameters. Double-infection of target cells is context. Thus, completion of the early phases of HIV-1 replication clearly advantageous for HIV-1 by fostering the genetic mixing of does not induce interference if it does not lead to viral protein viral genomes through recombination, but appears to be dis- expression; rather, cells targeted by the first virus remain pre- favored during later steps of infection by viral interference. This ferential targets for the second infection at late time points. study offers new insights into several aspects of this process, To begin to evaluate the role of specific viral proteins in the including: i) the timing of the transition of HIV-1 infected cells interference with HIV-1 super-infection, we performed similar from a state of susceptibility to double-infections to that of resis- experiments to those detailed above, but using for the initial tance, ii) the identification of viral genes that contribute to the infection viruses that are defective for a single viral gene. In these inhibition of super-infection, and iii) the evaluation of the relative studies, infected cells were exposed 18 h and 24 h later to wild- importance of the receptor-dependent and receptor-independent type HIV-1 virus (Fig. 3A) or to A-MLV-pseudotyped HIV-1-parti- mechanisms implicated in viral interference. cles (Fig. 3B), permitting evaluation of both receptor-dependent and receptor-independent mechanisms. Loss of interference in Frequency of double infected cells during co-infection experiments cells initially infected by a mutant virus would suggest the parti- cipation of the missing viral protein to viral interference. Viral In our studies, the simultaneous exposure of cells to two dis- clones individually defective for each accessory gene (vif, vpr, vpu tinct viral populations resulted in a higher frequency of double- A. Remion et al. / Virology 487 (2016) 59–67 63 infected cells than would be predicted for independent events. We not stable and must evolve over time, but with kinetics that cannot observed frequencies 2.5-fold higher than predicted for random be attributed, for example, to a specific phase of the cell cycle. distribution, using two viruses pseudotyped by HIV-1 Env glyco- Similarly, the viral entry pathway appears to modulate the pro- proteins, while if one of the two viruses carried the A-MLV Env pensity to double-infection, but the levels of expression of CD4 complex, the frequency was somewhat lower (less than 2-fold). and/or co-receptors are unlikely to explain this, because the gra- Theses phenomena have been observed in previous studies, but dual decay in the probability of double-infection was also seen for the mechanisms responsible have not been firmly established. cells super-infected by A-MLV pseudotyped viruses. It has also Two observations in our study provide additional information. been suggested that the disproportionate frequency of doubly- First, we found that the frequency of double infected-cells infected cells during co-infection could result from increased remained higher than expected for independent events for up to detection of otherwise silent infection events, triggered by the 18–24 h, provided that the first virus did not express viral proteins, additional Tat expression induced by the second virus (Bregnard but only the reporter GFP protein (Fig. 3). Second, however, the et al., 2012). In our experimental system, this potential artifact did propensity for double-infection was always highest when the not play a role, since co-infection using lentiviral vectors that only viruses were added simultaneously, and then slowly decayed, express GFP resulted in similarly high frequencies of double- beginning at very early time points (3–6 h post-infection), before infected cells as those obtained using vectors that also encode any viral protein could be synthesized. Tat (Fig. 3). It has been proposed that the increased frequency of double- Our results support the existence of an alternative, non- infections was strictly a cellular phenomenon, reflecting the pre- mutually exclusive mechanism, in which viral infection actively sence of distinct cell subpopulations that are more or less sus- promotes additional infectious events when the cell is simulta- ceptible to infection (Dang et al., 2004). If this is the case, however, neously exposed to the two viruses, but the advantage would be our results indicate that the susceptible/resistant cell phenotype is progressively reduced by increasing the time between the two

Fig. 3. Effect of the deletion of viral genes on the establishment of interference. (A) Initial infection was performed using the indicated viruses: wild type HIV-1 (WT), a lentiviral vector expressing only GFP (LV), a series of viruses defective for the expression of the indicated viral proteins (ΔX). Cells were then challenged by HIV-1 particles at 0, 18 h or 24 h. The ORs of double-infections were calculated as described in Fig. 1 legend. Data shown represent the mean and SEM from at least 3 independent experiments. (B) Experiments were conducted as in A, except that the second infection was performed using an HIV-1 particle pseudotyped by the A-MLV Env. *po0.05, **po0.01, and ***po0.001, as compared to the frequency observed for t¼0. 64 A. Remion et al. / Virology 487 (2016) 59–67

Fig. 4. Effect of the expression of viral proteins on the establishment of viral interference. (A) Initial infection was performed using the indicated viruses: wild type HIV-1 (WT), a lentiviral vector expressing GFP (LV), a vector expressing all HIV-1 proteins except Gag-Pol and Env (ΔGagPol), a vector expressing only Tat (Tþ), or Tat and Rev (TþRþ). Cells were coinfected (t¼0) or superinfected 18 h or 24 h later by wild type HIV-1. The ORs of double-infections were calculated as described in Fig. 1 legend. (B) Experiments were conducted as in A, except that the second infection was performed using an HIV-1 particle pseudotyped by the A-MLV Env. *po0.05, **po0.01, and ***po0.001, as compared to the frequency observed for t¼0. infectious events. Among potential stimuli that might be expected genes were tested in parallel in the setting of successive virus to follow such kinetics are cell signaling and activation induced by infections, allowing to identify Rev as a dominant effector of Env-receptor interactions (Pace et al., 2011; Weissman et al., 1997), receptor-independent interference. virus-induced cortical actin depolymerization (Spear et al., 2012), Our results provide several insights into the mechanisms and/or saturation of antiviral factors by an increased number of through which Rev induces viral interference. First, throughout virions penetrating each cell (Neil and Bieniasz, 2009). our study, we compared the potency of interference using viruses pseudotyped with the HIV-1 and A-MLV Env glycoproteins for the Viral interference second infection. The extent of interference appeared generally less potent using A-MLV, indicating the possible participation of The high cell susceptibility to double-infection declined as the receptor-mediated interference when using HIV-1 pseudotypes. intervals between the two infection events increased, and double- The patterns of interference, however, were very similar for HIV-1 infections become significantly disfavored (with ORo1) 18 h and and A-MLV pseudotypes; in both cases active interference began 24 h after initial infection. At these time points, several viral pro- 18 h post-infection, and the results of experiments evaluating the teins are expressed, and we clearly show here that expression of role of viral gene expression were indistinguishable in both set- Rev and Tat, but not Tat alone, is sufficient for this active form of tings. These results demonstrate that the Rev-induced inter- interference. It is important to stress that while several HIV-1 ference, and the overall interference observed within the first 24 h genes were previously shown to participate in viral interference, post-infection, are both largely independent of HIV-1 receptor/co- this is to our knowledge that the first study in which all HIV-1 receptor modulation. A. Remion et al. / Virology 487 (2016) 59–67 65

Second, the comparison of the interference induced after interference is established 18–24 h post-infection. Interference infection by the Rev-encoding vector and by the wild type HIV-1 requires the expression of viral genes and, using an unbiased (Fig. 4A) shows that optimal interference required the expression approach, we show that Rev is the dominant factor for this phe- of other viral genes, in agreement with previous reports (Volsky notype. Rev-induced interference appears to be largely indepen- et al., 1996; Potash and Volsky, 1998; Venzke et al., 2006; Michel dent of effects on the expression of HIV-1 receptors/co-receptors. et al., 2005; Nethe et al., 2005; Federico et al., 1995; D’Aloja et al., Optimal efficacy of viral interference also requires the expression 1998). Most viral genes require Tat and Rev to be expressed. of accessory genes, none of which, individually, plays a Abrogating the expression of individual accessory genes did not predominant role. prevent the induction of robust interference at 18–24 h (Fig. 3). Altogether these results suggest that multiple genes contribute in inducing optimal interference, but that no single accessory protein Methods plays a predominant role. Several viral genes have been previously proposed to participate to receptor-independent HIV-1 inter- Cells and proviral plasmids ference (Volsky et al., 1996; D’Aloja et al., 1998; Levin et al., 2010), and based on our results, expression of individual genes by viral HEK293T cells were maintained in Dulbecco modified Eagle's vectors competent for Tat and Rev expression, could allow the medium (DMEM) supplemented with 10% heat-inactivated fetal determination of their relative potency. calf serum (FCS) and antibiotics (100 IU/ml penicillin and 100 μg/ Third, our observation that Rev triggers viral interference even ml streptomycin). MT4R5 cells (Amara et al., 2003) were grown in in the absence of other viral proteins (with the exception of Tat, RPMI-1640 medium supplemented with 10% heat-inactivated FCS, whose expression was necessary to induce the reporter gene) 100 IU/ml of penicillin, 100 mg/ml of streptomycin and 0.25 μg/ml shows that interference does not require the interaction of Rev of amphotericin B. All cultures were maintained at 37 °Cina with viral proteins expressed following the initial infection. Two humidified atmosphere with 5% CO2. possible pathways would be compatible with this observation. The proviral constructs used here were derived from previously First, Rev could interact with cellular factors in a way that published plasmids based on the pNL4-3 construct, and carrying increases their ability to restrict the second infection. The HIV-1 sequence coding either for GFP or HSA reporter proteins cloned Rev protein is indeed able to interact with several cellular ele- before the nef gene, with an IRES sequence allowing concomitant ments, and accomplishes different functions in the nucleus and expression of the viral and reporter proteins (Imbeault et al., 2009; cytoplasm of the infected cell, beyond its original role in viral RNA Levy et al., 2004). To prevent virus spread in culture, we have export (Blissenbach et al., 2010; Pollard and Malim, 1998; HCT modified these constructs by deleting 1.3 kb of the env gene et al., 2009). Alternatively, Rev could directly bind elements of the (between the KpnI and BglII sites). The expression of the individual second virus and prevent their function. This pathway was sug- accessory genes was abolished in different proviral constructs gested in the previous report describing a role for Rev in inter- inserting previously described mutations. These mutations consist ference (Levin et al., 2010). The authors showed that Rev expres- either in large deletions in the 50 region of the gene: vif (deletion sion from an integration defective virus led to reduced integration nt 5128-5303, according to pNL4-3 numbering) (Cao et al., 2005), of a super-infecting virus (Levin et al., 2010). Based on previous vpu (deletion nt 6065-6112) (Schubert et al., 1995), vpr (deletion nt work from this group, describing an interaction between Rev and 5622-5742) (Bouhamdan et al., 1996); or in a frameshift induced integrase that can inhibit integration (Rosenbluh et al., 2007; Levin by filling-in the XhoI site at position 8887 in the nef gene et al., 2009), the authors suggested that the Rev protein expressed (Schwartz et al., 1995). To complement these proviral constructs by the first virus might directly intercept and disable the viral we used an HIV-1 Env-expressor plasmid, in which HIV-1 (pNL4-3) integrase of the superinfecting virus (Levin et al., 2010). The report Env, Tat and Rev expression is under the control of a chimeric SRα also confirmed the existence of other mechanisms causing viral promoter (SV40-early promoter, and LTR from HTLV-I), or a pre- interference, but the relative importance of Rev, as compared to viously described A-MLV Env expressor plasmid pSV-A-MLV other HIV-1 genes, was not explored (Levin et al., 2010). (Landau et al., 1991). Of note, in the study by Levin et al. (2010) viral interference was To study the effect of Tat, Rev and Gag-Pol on viral interference, established with faster kinetics than in our analyses. Indeed, the we produced lentiviral vectors by cotransfection of a Gag-Pol- efficiency of second infections was reduced by 90% within 8 h from expressor plasmid (R8.74, from Addgene), the HIV-1 Env-expressor the first infection, a time at which the authors could detect Rev plasmid and a transfer vector (carrying the packaging signal). As expression from the non-integrated genomes following the first transfer vector we used a previously described pNL4-3 derivative virus infection. In our study, interference could not be detected in which the expression of most viral genes was prevented, until 18 h post-infection, a time at which Rev would be pre- allowing the expression in target cells only of Tat and Rev (TþRþ) dominantly expressed from the integrated genomes. This suggests (Sakai et al., 2006). By introducing a stop codon at position 5983 that at least the source of Rev-expression was different in the two (Sadaie et al., 1988) by site-directed mutagenesis in this plasmid, studies. The difference in the kinetics could be due to the amount we obtained a transfer vector that would only express Tat. We also of virus used. In Levin et al. the MOI ranged from 1 to 10, infecting constructed a transfer vector that expresses all viral proteins virtually all cells in the culture with both viruses, while we except Gag-Pol (ΔGagPol) by deleting the fragment between the ensured that a majority of uninfected targets cells were available two SwaI sites in NL4-3. Finally, as a negative control, we used a for the second infection, to preserve an unbiased distribution of vector expressing only GFP under the CMV early promoter (pLenti- the viruses. Rev-mediated viral interference may conceivably GFP, from ABM Inc.). result from more than one mechanism, in which different levels of Rev expression could drive either direct or cell-factor mediated Preparation of virus stocks and infection inhibition of the superinfecting virus. Further studies on the mechanisms by which Rev induces viral interference may provide Virus stocks were prepared by transfecting sub-confluent 293-T additional insights on the capacity of HIV-1 to hijack cellular fac- cells (in T75 flasks) by JetPei (Polyplus transfection), following the tors necessary for optimal infectivity. manufacturer's instructions. Medium was changed 16 h later, and The results described here show that co-infection is favored the virus-containing supernatant was collected 40 h post-trans- early after an initial infection by HIV-1, while potent viral fection, overlaid on a 20% sucrose cushion in a Beckman SW28 66 A. Remion et al. / Virology 487 (2016) 59–67 tube, and particles were pelleted by centrifugation (50,000 g; Acknowledgments 4 °C) for 90 min. Viral pellets were resuspended in RPMI medium with FCS to obtain a 10-fold concentration as compared to the We thank Michel Tremblay and David Levy for providing the initial production in the culture supernatant, separated into sev- HIV-1 proviral plasmids expressing HSA and GFP, respectively; eral aliquots and frozen at 80 °C. To evaluate the frequency of Michael J. Lenardo for the pNL4-3 construct in which expression of infection, it was important to infect sufficient cells to provide a all viral genes except tat and rev was inactivated. A.R. was sup- fi signal permitting precise quanti cation, but to avoid infecting so ported by a doctoral fellowship from “Région Martinique” (grant fi many cells with the rst virus that infection of cells with only the 2011). second virus would be disfavored. To accomplish this, we infected cells with serial 2-fold dilutions of virus, but excluded all results in which the proportion of cells infected with the first virus was either o10% or 440%. If in one experiment more than one virus References input resulted in the appropriate percentage of infected cells (between 10% and 40%) the individual OR values (see below) were Allen, T.M., Altfeld, M., 2003. HIV-1 superinfection. J. Allergy Clin. Immunol. 112, calculated and the mean value was used in the compilation of 829–835. Argañaraz, E.R., Schindler, M., Kirchhoff, F., Cortes, M.J., Lama, J., 2003. Enhanced results. CD4 down-modulation by late stage HIV-1 nef alleles is associated with 5 For each experiment, 2 10 MT4R5 cells in parallel wells of increased Env incorporation and viral replication. J. Biol. Chem. 278, the same 96-well plate were “co-infected” (two viruses at the 33912–33919. fi Amara, A., Vidy, A., Boulla, G., Mollier, K., Garcia-Perez, J., Alcami, J., Blanpain, C., same time) or infected by the rst virus (e.g. expressing HSA) and Parmentier, M., Virelizier, J.L., Charneau, P., Arenzana-Seisdedos, F., 2003. 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(Becton Dickinson) with CellQuest software, and analyzed using Cao, J., Isaacson, J., Patick, A.K., Blair, W.S., 2005. High-throughput human immu- nodeficiency virus type 1 (HIV-1) full replication assay that includes HIV-1 Vif FlowJo software (Treestar). FACS analysis was performed to as an antiviral target. Antimicrob. Agents Chemother. 49, 3833–3841. determine the kinetics of expression of the reporter proteins, Dang, Q., Chen, J., Unutmaz, D., Coffin, J.M., Pathak, V.K., Powell, D., KewalRamani, V. which reached a plateau 48 h post-infection (data not shown). Cell N., Maldarelli, F., Hu, W.S., 2004. Nonrandom HIV-1 infection and double infection via direct and cell-mediated pathways. Proc. Natl. Acad. Sci. USA 101, viability was measured in parallel, and found to be stable for at 632. least 72 h post-infection (data not shown). We verified that the D’Aloja, P., Olivetta, E., Bona, R., Nappi, F., Pedacchia, D., Pugliese, K., Ferrari, G., kinetics of induction of viral interference were independent of the Verani, P., Federico, M., 1998. gag, vif, and nef genes contribute to the homo- logous viral interference induced by a nonproducer human immunodeficiency order of infecting viruses (i.e., GFP followed by HSA vs. HSA fol- virus type 1 (HIV-1) variant: identification of novel HIV-1-inhibiting viral lowed by GFP) (data not shown). protein mutants. J. Virol. 72, 4308–4319. Federico, M., Nappi, F., Bona, R., D’Aloja, P., Verani, P., Rossi, G.B., 1995. Full expression of transfected nonproducer interfering HIV-1 proviral DNA abro- Frequency of double infected cells gates susceptibility of human He-La CD4þ cells to HIV. Virology 206, 76–84. HCT, Groom, Anderson, E.C., AML, Lever, 2009. Rev: beyond nuclear export. J. Gen. The frequency of double infected cells were quantified by cal- Virol. 90, 1303–1318. Imbeault, M., Lodge, R., Ouellet, M., Tremblay, M.J., 2009. Efficient magnetic bead- culating the odds ratios (O.R.) as previously described (Dang et al., based separation of HIV-1-infected cells using an improved reporter virus 2004). Briefly, according to the expression of GFP and/or HSA, cells system reveals that p53 up-regulation occurs exclusively in the virus- can be divided in the FACS graph into four quadrants A, B, C, and D expressing cell population. Virology 393, 160–167. Josefsson, L., King, M.S., Makitalo, B., Brännström, J., Shao, W., Maldarelli, F., Kear- in Fig. 1. Cells in quadrant A express HSA, those in B are positive ney, M.F., Hu, W.-S., Chen, J., Gaines, H., Mellors, J.W., Albert, J., Coffin, J.M., both for HSA and GFP (double-infected cells), cells in quadrant C Palmer, S.E., 2011. Majority of CD4þ T cells from peripheral blood of HIV-1- are uninfected, while cells in D are GFP positive. We calculated the infected individuals contain only one HIV DNA molecule. Proc. Natl. Acad. Sci. 108, 11199–11204. distribution of the two infection events in the cell population to Josefsson, L., Palmer, S., Faria, N.R., Lemey, P., Casazza, J., Ambrozak, D., Kearney, M., determine if they were independent (OR¼1), or if double- Shao, W., Kottilil, S., Sneller, M., Mellors, J., Coffin, J.M., Maldarelli, F., 2013. infection occurred more frequently (OR41) or less frequently Single cell analysis of lymph node tissue from HIV-1 infected patients reveals þ o that the majority of CD4 T-cells contain one HIV-1 DNA molecule. PLoS (OR 1) than expected for independent events. The number of Pathog. 9, e1003432. cells in each quadrant was obtained by multiplying the percentage Levin, A., Hayouka, Z., Friedler, A., Brack-Werner, R., Volsky, D.J., Loyter, A., 2010. A of cells in that quadrant by the total number of cells analyzed, and novel role for the viral Rev protein in promoting resistance to superinfection by fi – þ þ human immunode ciency virus type 1. J. Gen. 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