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HIV-1 Tat protein induces viral internalization through Env-mediated interactions in dose-dependent manner

Poon, Selinaa,b,c; Moscoso, Carlos G.c; Yenigun, Onur M.c; Kolatkar, Prasanna R.b; Cheng, R. Hollanda,c; Vahlne, Andersa

doi: 10.1097/01.aids.0000432452.83604.59
Basic Science

Objective: To study the dose-dependent manner of HIV-1 Tat-induced effects on viral replication, internalization and spread, and to directly observe these effects on soluble Env immunogens and virus-like particles.

Design: In order to determine the manner through which Tat affects viral replication, we incubated cells, virions and soluble Env spikes with Tat at different concentrations, and directly visualized the effects of such incubation.

Methods: Cell-based infectivity assays were carried out to assay Tat dose-dependency of viral infectivity. Transmission electron microscopy of virus-like particles and soluble gp140 immunogens incubated with Tat at various concentrations was performed to directly observe Tat-induced effects.

Results: Treating virus with exogenous Tat increased infectivity in a dose-dependent manner. In the presence of anti-Tat antibodies, virus replication and spread were repressed, postulating Tat contributions to disease progression. When CXCR4 coreceptors were blocked, Tat treatment overcame the inhibition relative to absence of Tat treatment. Similarly, syncytium formation between chronically infected and uninfected target cells was also increased by exogenous Tat treatment. Inhibiting the CD4 receptor for virus entry abolished syncytium formation and Tat treatment was unable to overcome CD4 dependency. We show that Tat reduces virus infectivity at higher Tat concentrations through Env interactions resulting in viral aggregation.

Conclusion: Treating virions or chronically infected cells with exogenous Tat could enhance virus infectivity and spread through coreceptor tropism switch or through another undetermined mechanism. The aggregation potential of Tat suggests a mechanism of negative-feedback regulation of viral replication, providing another regulative function to control viral replication.

Supplemental Digital Content is available in the text

aDepartment of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institutet, Stockholm, Sweden

bLaboratory of Structural Biochemistry, Genome Institute of Singapore, Singapore

cDepartment of Molecular and Cellular Biology, University of California, Davis, California, USA.

Correspondence to Dr R. Holland Cheng, Professor, Molecular and Cellular Biology, University of California, Davis, CA 95616, USA. E-mail: Dr Anders Vahlne, Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institutet, Stockholm, Sweden. E-mail:

Received 31 December, 2012

Revised 29 May, 2013

Accepted 11 June, 2013

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (

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The HIV type 1 (HIV-1) viral protein Tat promotes stable HIV-1 mRNA transcription [1] by interacting with host transcription factors and the transactivation response element located within the long terminal repeat (LTR) of the HIV-1 genome [2,3]. Tat is released from infected cells [4,5] and exerts other effects on neighboring cells, such as activating uninfected primary T lymphocytes [6] and inducing expression of coreceptors CXCR4 and CCR5 [7].

A monoclonal antibody (MAb) targeting an N-terminal Tat domain (residues 6–14) inhibited replication of HIV-1IIIB and HIV-1MN isolates [8,9]. Analysis of HIV-1-positive patient sera revealed an inverse correlation between anti-Tat antibody titers and viral load magnitude [9]. Also, high antibody titers against Tat peptides containing residues 6–14, 36–50 and 46–60 correlated with undetectable viral load [10]. Furthermore, an inverse relationship between p24 antigen and natural anti-Tat antibodies in infected patients has been demonstrated [9,11].

In mice and monkeys vaccinated with Tat protein, humoral and cellular Tat-specific immune responses are induced [12–14] that prevent T-lymphocyte decline, controlling infection in cynomolgus monkeys upon challenge with highly pathogenic simian-HIV89.6P. Unvaccinated monkeys showed symptoms of infection and a profound decline in T-lymphocyte counts [13]. Long-term observation of vaccinated and challenged monkeys showed that protection against infection was prolonged up to week 104 after challenge, indicating that Tat-induced cellular immune responses may control infection.

Membrane-associated Tat has been suggested to bind to gp120, the surface unit of the HIV-1 envelope glycoprotein (Env), and mediate virus entry via its basic domain [15], insofar the only report of Tat enhancing HIV-1 internalization, and also suggesting that entry was receptor and coreceptor-independent. Using cryo-electron microscopy, we have shown that interactions between Tat and trimeric gp140 induce an intermediate quaternary conformational change when compared with native gp140 or CD4-liganded gp140, partially priming Env toward the triggered conformation [16]. Here we corroborate these structural observations with in-vitro infectivity, neutralization and aggregation assays to investigate Tat effects on infectivity.

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Materials and methods

Cell lines, plasmids and antibodies

ACH-2, SupT1 and TZM-bl cell lines, HIV-1 Tat hybridoma, and BL21 bacteria transformed with HIV-1HXB2 Tat expression vector were obtained through the NIH AIDS Reagent Program [National Institutes of Health AIDS Reagent Program (NIH ARP, Germantown, Maryland, USA)]. Green monkey kidney (GMK) cells were obtained from the Clinical Virology Laboratory of Karolinska Hospital. ACH-2 and SupT1 were cultured in RPMI; TZM-bl and GMK were cultured in DMEM; HIV-1 Tat hybridoma was cultured in Hybridoma SFM. All media were supplemented with 10% fetal bovine serum (FBS), 100 U penicillin and 100 μg/ml streptomycin, except for Hybridoma SFM, which was supplemented with 1% FBS. All cell lines were cultured at 37°C in a humidified CO2 incubator.

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Tat protein production

Tat was produced and purified as described by the NIH ARP. Protein concentration was quantified with Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, California, USA). Purity of Tat was analyzed on a 15% SDS–PAGE, and biological activity of Tat was assayed by transactivation of HIV-1 LTR in TZM-bl cells (Figures S1 and S2, respectively;

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Preparation of virus

ACH-2 cells carry a single integrated inducible copy of the HIV-1 LAI strain (HIV-1LAI) [17]. Briefly, ACH-2 cells at a density of 1 × 106 cells/ml were cultured in growth media containing 100 nmol/l phorbol 12-myristate 13-acetate (PMA) (Sigma–Aldrich, St. Louis, Missouri, USA) for 3 days. The culture supernatant was collected, cleared by centrifugation and passed through a 0.45-μm pore filter. Virus concentration was determined by p24 ELISA [18]. Herpes simplex virus type 1 (HSV-1) obtained from the Clinical Virology Laboratory of Karolinska Hospital was propagated in GMK cells. Plaque titrations were performed as previously described [19].

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Virus and cell treatment with Tat

Unless otherwise stated, virus (200 ng) or cells used in the study were treated with Tat for 30 min at room temperature (RT) away from light. To study coreceptor expression, TZM-bl cells (1 × 105 cells/well in 24-well plate) and SupT1 cells (1 × 106 cells per tube) were incubated with Tat at 37°C for either 2 or 24 h, and CCR5 and CXCR4 expression was analyzed by flow cytometry.

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Virus infectivity assay

Single-cycle infectivity assays were performed in TZM-bl cells seeded in 48-well cell cluster plates 24 h before infection at a density of 2 × 104 cells/well. TZM-bl cells were incubated with Tat-treated HIV-1LAI for 2 or 24 h at 37°C (hereinafter, virus incubated with Tat refer to Tat-treated virus). Subsequently, cells were washed twice with 1× PBS and incubated in fresh media containing 5 μmol/l indinavir at 37°C. The cells were lyzed 48 h postinfection with Glo-lysis buffer (Promega, Madison, Wisconsin, USA) and luciferase production was measured.

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Cell labeling

ACH-2 cells were stained with CellTrace carboxylfluorescein diacetate, succinimidyl ester (CFSE; Molecular Probes, Invitrogen, Grand Island, New York, USA). Cell surface CD4 was labeled with allophycocyanin (APC)-conjugated anti-hCD4 antibody (Molecular Probes). Cell surface CXCR4 and CCR5 were labeled with APC-conjugated anti-hCXCR4 and phycoerythrin-conjugated antihCCR5 antibodies, respectively (BD Pharmingen, San Jose, California, USA). TZM-bl cells in studies investigating receptor involvement during virus entry after Tat treatment were labeled with five times the amount of antibodies used for flow cytometry overnight at 37°C.

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Cell–cell fusion assay

ACH-2 cells stained with CFSE (ACH-2CFSE) were incubated at 37°C for 24 h in media containing 10 μmol/l indinavir prior to treatment with Tat protein for 30 min at RT and then cocultured with SupT1 cells. After coculturing for 24 h, syncytia were stained with APC-conjugated mouse MAb antihuman CD4.

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Flow cytometry analysis

Cells labeled with both CFSE and fluorescent dye-conjugated antibodies or either of them were fixed with 4% paraformaldehyde before being analyzed on a FACScalibur flow cytometer (BD Biosciences, San Jose, California, USA). To analyze coreceptor expression, 30 000 events were collected and represented by median fluorescent intensity value. To analyze syncytium formation, 100 000 events were collected and gated using control cells in single cell culture. Syncytia were identified by double-positive staining with CFSE and APC, and represented as percentage of gated cells. Data from flow cytometry were analyzed by FlowJo (Tree Star, Inc., Ashland, Oregon, USA).

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Herpes simplex virus type 1 propagation assay

Herpes simplex virus type 1 at multiplicity of infection (MOI) of 0.1 was treated with Tat for 30 min at RT. The virus inoculum was incubated with TZM-bl cells for 2 h at 37°C. Virus inoculum was replaced with DMEM supplemented with 2% FBS containing Tat. Media were harvested 48 h postinfection and virus titers were determined by plaque assay with GMK cells.

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Negative stain of soluble gp140SF162 trimers and HIV-1NL4-3 virus-like particles incubated with Tat

Clade B gp140 from strain SF162 (NIH ARP) was incubated with Tat at different concentrations. Clade B HIV-1NL4-3 VLPs (Dr Joseph Anderson, UC Davis, California, USA) were also incubated with Tat. Three hundred-mesh copper grids were covered with continuous carbon film. Trimeric gp140SF162 at 0.1 mg/ml (309 nmol/l) was incubated with Tat at concentrations of 100, 500 and 1000 nmol/l at 4°C overnight. Three microliters of gp140SF162-Tat were blotted on grids, then imaged using a 2100JEOL 200 kV transmission electron microscope at 80 000× magnification (1.25 Å/pixel). Similarly, a 1.2-ml sample of VLP at a p24 concentration of 2240 pg/ml, containing approximately 3.54 × 107 VLPs [20], was ultracentrifuged at 50 000g for 90 min at 4°C, resuspended in 100 μl of 1× PBS and incubated with Tat at concentrations of 100 nmol/l, 500 nmol/l and 1 μmol/l for 1 h at RT away from light. Three microliters of Tat-VLP incubated solutions were blotted on grids, and imaged at 15 000× magnification (7.8 Å/pixel).

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Single-cycle infectivity assay of virus treated with Tat

To test whether preincubation of HIV-1 with Tat increases infectivity, HIV-1LAI virions produced from ACH-2 cells were incubated with Tat before infection of TZM-bl cells. The protease inhibitor indinavir was added to the culture medium to ensure single-cycle infection [21]. At both 2 and 24 h infection, Tat causes an increase in virus infectivity at concentrations up to 1 nmol/l, whereas infectivity plateaus were between 1 nmol/l and 100–500 nmol/l, and was reduced at higher Tat concentrations (Fig. 1a). Thus, whereas low concentrations of Tat were favorable for viral infection, higher levels of Tat protein had a detrimental effect on infectivity.

Fig. 1

Fig. 1

Fig. 1

Fig. 1

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.

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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 [22], 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.

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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 [23]. 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.

Fig. 2

Fig. 2

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 [24]. 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.

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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)].

Fig. 3

Fig. 3

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)].

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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.

Fig. 4

Fig. 4

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 [25], 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).

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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).

Fig. 5

Fig. 5

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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 [28]. 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 [15]. Prior incubation of Tat with the 1D9 antibody [22] 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 [29], 1D9 interaction with Tat had no effect on its transactivation function [22]. Hence, the data suggest that 1D9 prevents Tat interaction with Env, and that 1D9 and Env competitively bind to the same Tat epitope [30]. 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,

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 [34], occurs within minutes [35] and depends on Env–CD4 interaction between effector and target cells [32]. 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 [36]. Using CD4-negative ACH-2 cells [17], 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 [7]. 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 [7]. 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 [25].

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.

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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.

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Conflicts of interest

There are no conflicts of interest.

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aggregation; HIV; infectivity; neutralization; Tat

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