We then tested if the Tat-mediated increase in infectivity was due to Tat–virus interactions, or if Tat exerted a cellular effect. TZM-bl cells were treated with different concentrations of Tat for 1 h at 37°C prior to a 2-h incubation with virus at 37°C (Fig. 1b; TZM-bl/Tat + virus). Changes in infectivity were compared to the previous experiment, where virus was treated with Tat for 30 min at RT at different concentrations before incubation with TZM-bl cells for 2 h at 37°C (Fig. 1c; TZM-bl + virus/Tat). Pretreatment of cells with Tat did not increase infectivity.
To further show that the increased infectivity was not because of Tat affecting the cells, coreceptor expression on TZM-bl was analyzed by flow cytometry. Cell surface CCR5 and CXCR4 expression was not affected by Tat (Fig. 1c). Although CCR5 and CXCR4 expression level showed an increase at 1000 nmol/l Tat, infectivity did not increase at this concentration.
Tat-mediated increased HIV-1 infectivity was time-dependent on length of Tat-virus incubation. HIV-1LAI virions were treated with Tat for 30 min, 1 h, 2 h or 4 h before incubation with TZM-bl cells for 2 h at 37°C. Infectivity significantly increased at low Tat concentrations when incubation was carried out for 30 min or 1 h at RT (Fig. 1d). Infectivity did not increase when Tat was incubated for 2 or 4 h (Fig. 1d). In addition, infectivity decreased when high Tat concentration was used to treat virus before infection, regardless of incubation duration.
Tat-mediated effect on HIV-1 infectivity is reversed by an antibody recognizing the N-terminal Tat domain
1D9 is a MAb that recognizes the N-terminal Tat domain , though it does not abrogate Tat transactivation of HIV-1 LTR. Tat was incubated with 1D9 for 30 min at RT, and then used to treat HIV-1LAI virions as described previously. Pretreatment of Tat with 1D9 partially negated the Tat-mediated increase in infectivity (Fig. 1e). When Tat was first pretreated with 1D9, the previously observed reduced infectivity in viruses treated with 500 nmol/l and 1 μmol/l Tat protein became less pronounced. 1D9 pretreatment data were normalized to data of infection by HIV-1 without Tat treatment, in order to show the percentage change in infectivity as a result of pretreating Tat with 1D9 (Fig. 1f). Infectivity decreased in the absence of exogenous Tat. The increase in infectivity was partially negated at Tat concentrations between 1 and 100 nmol/l, with a less drastic reduction in infectivity at high Tat concentrations.
Tat at high concentrations increases the percentage of syncytia formed between ACH-2 and SupT1 cells
Formation of virological synapses between infected and uninfected cells may be increased by Tat. As a model for virological synapse formation between infected and noninfected cells, we used ACH-2 cells and SupT1, an uninfected T-lymphocyte line sensitive to infection and syncytium induction by HIV-1 . Induction of syncytia by infected ACH-2CFSE cells in the presence of Tat was studied by coculturing ACH-2CFSE cells with SupT1 cells.
There is a positive correlation between the percentage of syncytia formed in a coculture of ACH-2CFSE and SupT1 cells, and the concentration of Tat used to treat ACH-2CFSE cells before coculturing (Fig. 2a). Regression analysis yielded R 2 = 0.86. Syncytium formation increased significantly when Tat concentration exceeded 50 nmol/l. Labeling SupT1 cells with anti-hCD4 antibody before coculturing abolished the increase in syncytium formation (Fig. 2b), strongly suggesting that syncytium formation depends on gp120–CD4 interaction.
Cell surface expression of CCR5 and CXCR4 in SupT1 cells was also analyzed (Fig. 2c). CXCR4 expression did not increase as Tat concentration increased [Fig. 2c(i)]. CCR5 mRNA transcripts are present in SupT1 cells, but CCR5 protein concentration is very low or undetectable on the cell membrane . Treatment of SupT1 cells with Tat did not increase CCR5 expression on the cell membrane [Fig. 2c(ii)]. Therefore, Tat increased syncytium formation of ACH-2CFSE cells with SupT1 cells, most likely as a result of Tat–gp120 interactions on ACH-2 cell surfaces and not because of changes in coreceptor expression.
The gp120 protein of an X4-tropic virus, HIV-1LAI, can interact with both CCR5 and CXCR4 after interaction with Tat
To study how Tat–gp120 interaction might affect viral coreceptor tropism, TZM-bl cells were preincubated with antibodies against CXCR4 or CCR5 and then infected with Tat-treated HIV-1LAI. To distinguish Tat-induced changes in infectivity, the results were normalized against infection by untreated HIV-1LAI [Fig. 3a(ii) and b(ii)].
Blocking CXCR4 reduced infectivity by virus in the absence of Tat [Fig. 3a(i) and c], but Tat treatment resulted in 20–25% increase in infectivity [Fig. 3a(ii)]. Unexpectedly, blocking CCR5 resulted in a slight increase in infectivity without Tat [Fig. 3b(i) and c]. In the presence of Tat, infectivity increased to levels similar to cells without antibody treatment [Fig. 3b(i)]. As a result, infectivity in cells with antihCCR5 antibody was slightly lower than in cells not treated with antibodies [Fig. 3b(ii)].
Tat does not increase virus production or propagation
We investigated whether the rate of virus production by PMA-stimulated ACH-2 cells was different when Tat was added. Supernatant concentration of p24 remained constant in the presence of exogenous Tat (Fig. 4a), indicating that increasing Tat concentration does not result increase the rate of virus production.
The propagation of an enveloped DNA virus, HSV-1, in the presence of Tat, was investigated as a control for the Tat-mediated delivery system used to deliver drugs , and to observe if high concentrations of Tat had a detrimental effect on virus propagation. The rate of HSV-1 propagation did not significantly increase even after incubation with Tat at 500 nmol/l (Fig. 4b).
Tat induces aggregation in soluble gp140SF162 trimers and HIV-1NL4–3 virus-like particles
To probe how increased Tat concentration attenuates viral infectivity, and to determine whether this effect is mediated through interactions with Env and can aggregate viral particles, soluble gp140SF162 trimers and HIV-1NL4-3 VLPs were incubated with Tat and visualized by transmission electron microscope. A large extent of gp140 aggregation was observed when the concentration of Tat was increased (Fig. 5a–c). At 100 nmol/l Tat, a relatively monodisperse distribution of Tat-gp140 conjugates was evident (Fig. 5a). At 500 nmol/l Tat, several aggregates were visible (Fig. 5b), whereas at 1 μmol/l Tat, the effect was exacerbated (Fig. 5c). Similarly, aggregation of VLPs was observed as Tat concentration increased (Fig. 5d–f). At a Tat concentration of 100 nmol/l, there were relatively dispersed VLPs on the grid and no aggregates on 0 of 16 micrographs (Fig. 5d). At 500 nmol/l Tat, small aggregates of two or three VLPs were visible in 9% of micrographs (3/33) (Fig. 5e). At Tat concentration of 1 μmol/l, 30% (12/40) of micrographs exhibited aggregates, some encompassing more than 10 VLPs (Fig. 5f).
In HIV-1 patients, the serum concentration of Tat released by infected cells reaches the nano-molar range [4,26,27]. Patients identified as slow progressors have high amounts of anti-Tat antibodies compared to fast progressors, suggesting that Tat contributes to disease development [10,11]. Tat has been shown to mediate aggregation of cerebellar neurons in vitro in a dose-dependent manner . Here, we studied whether Tat has an effect directly on the virus and hence on its infectivity in vitro.
Indeed we found that Tat increased infectivity at low concentrations and the effect was dependent on the length of virus incubation time with Tat before cell incubation. Short virus-Tat incubations of 30 min to 1 h increased infectivity, suggesting that the Tat effect occurred over a short time span, since increasing the time of Tat-virus incubation beyond 1 h did not further increase infectivity. Tat increased infectivity only when HIV-1LAI was treated with Tat before infection, but not when cells were treated with Tat before infection, indicating that Tat-induced effects on the virus and not the cell were responsible for the observed changes in infectivity. This disagrees with study showing prior incubation of cells with Tat increased the entry of HIV-1 or HIV-1-derived lentiviral vectors . Prior incubation of Tat with the 1D9 antibody  partially negated the Tat-mediated increase in infectivity, which is further evidence that exogenous Tat increased the infectivity of the virus. In the absence of Tat, 1D9 also caused a partial decrease in infectivity, an unexpected result likely due to the interaction between 1D9 and Tat present in the media of stimulated ACH-2 cells. Although the N-terminal region is essential for Tat activity , 1D9 interaction with Tat had no effect on its transactivation function . Hence, the data suggest that 1D9 prevents Tat interaction with Env, and that 1D9 and Env competitively bind to the same Tat epitope . An obvious concern was that exogenous Tat could activate the LTR promoter and lead to increased luciferase expression in TZM-bl cells or structural protein expression in ACH-2 cells. However, we found that micromolar rather than nanomolar concentrations of exogenous Tat was required to activate luciferase production in TZM-bl cells (Figure S2, http://links.lww.com/QAD/A375).
The transmission of HIV-1 by cell–cell adhesion through the formation of virological synapses has been documented [31–33]. Cell-associated transfer of virus is more efficient than infection by cell-free virus , occurs within minutes  and depends on Env–CD4 interaction between effector and target cells . Similar to virological synapse formation, cell–cell fusion in HIV-1 infection forms syncytia and is initiated by Env–CD4 interaction. However, although syncytia are readily observed in vitro, they are not often observed in vivo except in the central nervous system . Using CD4-negative ACH-2 cells , virological synapse formation was studied indirectly by studying syncytium formation with CD4+ SupT1 cells. Our data indicated that Env–CD4 interaction between cells was affected by Tat in the extracellular environment, but required high Tat concentrations (above 50 nmol/l) to significantly increase syncytium formation. Blocking CD4 on SupT1 with anti-hCD4 antibody abolished syncytium formation, agreeing with previous studies [31,37,38]. Our observations led us to postulate that Tat could positively contribute to virus transmission by influencing virological synapse contacts.
Tat has previously been shown to up-regulate cell surface expression of Env coreceptors, increasing infection . We exposed target cells to Tat during incubation with virus and virus-producing cells, which did not significantly alter CCR5 and CXCR4 expression levels on SupT1 cells, diverging from previous observations . Although increased expression levels of CXCR4 and CCR5 were observed in TZM-bl cells incubated with 1000 nmol/l Tat for 24 h, infectivity did not increase. The observed changes in infectivity and syncytium formation in our studies were thus not due to effects on target cells, but on virus and virus-producing cells.
Blocking CD4 on SupT1 blocked syncytium formation, and blocking CXCR4 on TZM-bl cells immediately reduced infectivity by HIV-1, which are expected results since HIV-1 entry is dependent on interaction of Env with CD4 [39,40] and then a chemokine receptor [41–43]. Unexpectedly, a slight increase in infectivity was observed when virus was treated with low concentrations of Tat and when CXCR4 was blocked. Conversely, when CCR5 was blocked, virus infectivity in the absence of Tat increased, reaching levels similar to infection in cells without antibody treatment.
Comparing infectivity by untreated virus in cells blocked by different antibodies suggested that X4-tropic gp120 interacts with CCR5 after CD4-induced conformational changes, but arrests at entry because further necessary structural changes are not elicited (Fig. 3c). The change in infectivity by Tat-virus in CXCR4-blocked cells seemed to suggest entry via other mechanisms, possibly including Tat-induced coreceptor tropism switch and Tat-mediated delivery via its transduction domain .
In single-cycle infectivity assays of Tat-treated virus, we observed that high Tat concentrations reduced infectivity, further indicating that the effect on luciferase activity obtained with Tat-treated virus was not owing an effect of exogenously added Tat on the LTR in the TZM-bl cells, but rather to Tat's effect on the virus itself. Both negatively-stained gp140SF162 trimers and HIVNL4–3 VLPs displayed aggregation induced by Tat at concentrations above 500 nmol/l, suggesting that the Tat-induced effect is mediated by interactions with Env, and that virions similarly undergo aggregation. HSV-1, an enveloped virus, was propagated in low (5 nmol/l), intermediate (50 nmol/l) and high concentrations (500 nmol/l) of Tat to study if Tat could reduce propagation of another enveloped virus by causing aggregation between adjacent viruses. HSV-1 propagation at high Tat concentration did not decrease, instead supporting our observation that the aggregative effect is Env-mediated and specific. Another possibility is that Tat aggregation can occur as a result of thiol oxidation between cysteines on different Tat copies, as previously reported [44,45].
In conclusion, the data presented here indicate that Tat increases infectivity and spread in part by an Env-specific effect at or near the coreceptor binding site, and pose a significant advancement in understanding specific Tat effects on Env function and virus infectivity. Combination vaccine design based on Env and Tat proteins has shown the ability to protect macaques against simian-HIV [46,47]. Such vaccines encompassing structural and regulatory proteins of HIV-1 could work synergistically to control acute virus infection and to protect from progression, providing a promising avenue for rational vaccine design.
We thank Dr Barbara Ensoli and Dr Paolo Monini for sharing prepublication data with us.
The study was funded by NIH NIAID (AI095382), NIH NCI pilot, UC Discovery Programs, Swedish Research Council (K2000-06X-09501-10B), SIDA (HIV-2006-050), A*STAR, Istituto Superiore di Sanità and Karolinska Institutet.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2013 Lippincott Williams & Wilkins, Inc.
aggregation; HIV; infectivity; neutralization; Tat