HIV-1 pathogenicity and virion production are dependent on the metabolic phenotype of activated CD4+ T cells.

BACKGROUND: HIV-1, like all viruses, is entirely dependent on the host cell for providing the metabolic resources for completion of the viral replication cycle and the production of virions. It is well established that HIV-1 replicates efficiently in activated CD4+ T cells, whereas resting CD4+ T cells are refractory to infection with HIV-1. A hallmark of T cell activation is the upregulation of glycolysis to meet the biosynthetic and bioenergetic needs of cell proliferation and the execution of effector functions by the secretion of cytokines. To date, it has remained unknown if HIV-1 requires the high glycolytic activity of activated T cells to support its replication. RESULTS: We report that in primary CD4+ T cells, the flux through the glycolytic pathway is increased upon infection with HIV-1. This increase in glycolytic activity does not occur in T cell lines when infected with HIV-1. By providing cells with galactose instead of glucose, the former being a poor substrate for glycolysis, we monitored the effect of preventing glycolysis in CD4+ T cells on virus replication cycle and cell fate. We observed that HIV-1 infected primary CD4+ T cells cultured in galactose have a survival advantage over those cultured in glucose and this coincides with reduced caspase 3 activation and apoptosis in cultures with galactose. T cell lines do not recapitulate this difference in cell death. Finally, we demonstrate that virion production is dependent on glycolysis as cultures containing galactose yield reduced amounts of HIV-1 virions compared with cultures containing glucose. CONCLUSIONS: The replication of HIV-1 in primary CD4+ T cells causes an increase in glycolytic flux of the cell. Glycolysis is particularly required for virion production and additionally increases the sensitivity of the infected cell to virus-induced cell death.

The SOCS-box of HIV-1 Vif interacts with ElonginBC by induced-folding to recruit its Cul5-containing ubiquitin ligase complex.

The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif's BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function.

RNA-dependent oligomerization of APOBEC3G is required for restriction of HIV-1.

The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of retroviruses and transposable elements and is able to deaminate cytidines to uridines in single-stranded DNA replication intermediates. A3G contains two canonical cytidine deaminase domains (CDAs), of which only the C-terminal one is known to mediate cytidine deamination. By exploiting the crystal structure of the related tetrameric APOBEC2 (A2) protein, we identified residues within A3G that have the potential to mediate oligomerization of the protein. Using yeast two-hybrid assays, co-immunoprecipitation, and chemical crosslinking, we show that tyrosine-124 and tryptophan-127 within the enzymatically inactive N-terminal CDA domain mediate A3G oligomerization, and this coincides with packaging into HIV-1 virions. In addition to the importance of specific residues in A3G, oligomerization is also shown to be RNA-dependent. Homology modelling of A3G onto the A2 template structure indicates an accumulation of positive charge in a pocket formed by a putative dimer interface. Substitution of arginine residues at positions 24, 30, and 136 within this pocket resulted in reduced virus inhibition, virion packaging, and oligomerization. Consistent with RNA serving a central role in all these activities, the oligomerization-deficient A3G proteins associated less efficiently with several cellular RNA molecules. Accordingly, we propose that occupation of the positively charged pocket by RNA promotes A3G oligomerization, packaging into virions and antiviral function.

Restriction of retroviral replication by APOBEC3G/F and TRIM5alpha.

Pathogenic viral infections have exerted selection pressure on their hosts to evolve cellular antiviral inhibitors referred to as restriction factors. Examples of such molecules are APOBEC3G, APOBEC3F and TRIM5alpha. APOBEC3G and APOBEC3F are cytidine deaminases that are able to strongly inhibit retroviral replication by at least two mechanisms. They are counteracted by the lentiviral Vif protein. TRIM5alpha binds to sensitive, incoming retroviruses via its C-terminal PRY/SPRY domain and rapidly recruits them to the proteasome before significant viral DNA synthesis can occur. Both of these proteins robustly block retroviral replication in a species-specific way. It remains an open but important question as to whether innate restriction factors such as these can be harnessed to inhibit HIV-1 replication in humans.

The availability of the primer activation signal (PAS) affects the efficiency of HIV-1 reverse transcription initiation.

Initiation of reverse transcription of a retroviral RNA genome is strictly regulated. The tRNA primer binds to the primer binding site (PBS), and subsequent priming is triggered by the primer activation signal (PAS) that also pairs with the tRNA. We observed that in vitro reverse transcription initiation of the HIV-1 leader RNA varies in efficiency among 3'-end truncated transcripts, despite the presence of both PBS and PAS motifs. As the HIV-1 leader RNA can adopt two different foldings, we investigated if the conformational state of the transcripts did influence the efficiency of reverse transcription initiation. However, mutant transcripts that exclusively fold one or the other structure were similarly active, thereby excluding the possibility of regulation of reverse transcription initiation by the structure riboswitch. We next set out to determine the availability of the PAS element. This sequence motif enhances the efficiency of reverse transcription initiation, but its activity is regulated because the PAS motif is initially base paired within the wild-type template. We measured that the initiation efficiency on different templates correlates directly with accessibility of the PAS motif. Furthermore, changes in PAS are critical to facilitate a primer-switch to a new tRNA species, demonstrating the importance of this enhancer element.

Upregulation of Glucose Uptake and Hexokinase Activity of Primary Human CD4+ T Cells in Response to Infection with HIV-1.

Infection of primary CD4+ T cells with HIV-1 coincides with an increase in glycolysis. We investigated the expression of glucose transporters (GLUT) and glycolytic enzymes in human CD4+ T cells in response to infection with HIV-1. We demonstrate the co-expression of GLUT1, GLUT3, GLUT4, and GLUT6 in human CD4+ T cells after activation, and their concerted overexpression in HIV-1 infected cells. The investigation of glycolytic enzymes demonstrated activation-dependent expression of hexokinases HK1 and HK2 in human CD4+ T cells, and a highly significant increase in cellular hexokinase enzyme activity in response to infection with HIV-1. HIV-1 infected CD4+ T cells showed a marked increase in expression of HK1, as well as the functionally related voltage-dependent anion channel (VDAC) protein, but not HK2. The elevation of GLUT, HK1, and VDAC expression in HIV-1 infected cells mirrored replication kinetics and was dependent on virus replication, as evidenced by the use of reverse transcription inhibitors. Finally, we demonstrated that the upregulation of HK1 in HIV-1 infected CD4+ T cells is independent of the viral accessory proteins Vpu, Vif, Nef, and Vpr. Though these data are consistent with HIV-1 dependency on CD4+ T cell glucose metabolism, a cellular response mechanism to infection cannot be ruled out.

Evidence for Altered Glutamine Metabolism in Human Immunodeficiency Virus Type 1 Infected Primary Human CD4+ T Cells.

Glutamine is a conditionally essential amino acid that is an important metabolic resource for proliferating tissues by acting as a proteinogenic amino acid, a nitrogen donor for biosynthetic reactions and as a substrate for the citric acid or tricarboxylic acid cycle. The human immunodeficiency virus type 1 (HIV-1) productively infects activated CD4+ T cells that are known to require glutamine for proliferation and for carrying out effector functions. As a virus, HIV-1 is furthermore entirely dependent on host metabolism to support its replication. In this study, we compared HIV-1 infected with uninfected activated primary human CD4+ T cells with regard to glutamine metabolism. We report that glutamine concentrations are elevated in HIV-1-infected cells and that glutamine is important to support HIV-1 replication, although the latter is closely linked to the glutamine dependency of cell survival. Metabolic tracer experiments showed that entry of glutamine-derived carbon into the citric acid cycle is unaffected by HIV-1 infection, but that there is an increase in the secretion of glutamine-derived glutamic acid from HIV-1-infected cells. Western blotting of key enzymes that metabolize glutamine revealed marked differences in the expression of glutaminase isoforms, KGA and CAG, as well as the PPAT enzyme that targets glutamine-derived nitrogen toward nucleotide synthesis. Altogether, this demonstrates that infection of CD4+ T cells with HIV-1 leads to considerable changes in the cellular glutamine metabolism.

On the importance of the primer activation signal for initiation of tRNA(lys3)-primed reverse transcription of the HIV-1 RNA genome.

Initiation of reverse transcription is a complex and regulated process in all retroviruses. Several base pairing interactions have been proposed to occur between the HIV-1 RNA genome and the specific tRNA(lys3) primer. The tRNA primer can form up to 21 bp with the primer binding site (PBS), and an additional 8 bp interaction may form between the primer activation signal (PAS) in the HIV-1 RNA and sequences within the T(Psi)C arm of the tRNA. The latter interaction is further analyzed in this in vitro study with mutant RNA transcripts that were designed to preclude the PAS interaction. These mutant transcripts are able to efficiently bind the tRNA primer, but they exhibit a profound defect at initiating reverse transcription. This defect is specific for the tRNA primer because it is not observed for PBS-bound DNA oligonucleotide primers. These results reinforce the model of regulated reverse transcription in which the PAS-mediated interaction is critical for efficient initiation.

Regulated HIV-2 RNA dimerization by means of alternative RNA conformations.

The dimer initiation site (DIS) hairpin of the HIV-2 untranslated leader RNA mediates in vitro dimerization through 'loop-loop kissing' of a loop-exposed palindrome sequence. Premature RNA dimerization must be prevented during the retroviral life cycle. A regulatory mechanism has been proposed for the HIV-1 leader RNA that can adopt an alternative conformation in which the DIS motif is effectively masked by long-distance base pairing with upstream leader sequences. We now report that HIV-2 RNA dimerization is also regulated. Sequestering of the DIS motif by base pairing interactions with downstream leader sequences mediates a switch to a dimerization-impaired conformation. The existence of two alternative conformations of the HIV-2 leader RNA is supported by UV melting experiments. Furthermore, the equilibrium between the two conformations can be shifted by annealing of antisense oligonucleotides or by deletion of certain leader regions. These measures have a profound impact on the dimerization properties of the transcript, demonstrating a mutual exclusivity between the alternative conformation and dimerization, similar to what has been described for the HIV-1 leader. The overall resemblance in regulation of HIV-1 and HIV-2 RNA dimerization suggests that a similar mechanism may be operating in other lentiviruses and perhaps all retroviridae.

Probing alternative foldings of the HIV-1 leader RNA by antisense oligonucleotide scanning arrays.

Scanning arrays of antisense DNA oligonucleotides provide a novel and systematic means to study structural features within an RNA molecule. We used this approach to probe the structure of the untranslated leader of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This 335 nt RNA encodes multiple important replication signals and adopts two mutually exclusive conformations. The poly(A) and the dimer initiation signal (DIS) sequences of the leader RNA are base-paired in the long-distance interaction (LDI) conformation, but both domains form distinct hairpins in the branched multiple hairpins (BMH) conformation. An RNA switch mechanism has been proposed to regulate the activity of the DIS dimerization signal that is masked in one, yet exposed in the other conformation. The two RNA conformations demonstrate discrete differences in the array-based hybridization patterns. LDI shows increased hybridization in the poly(A) region and decreased hybridization in the DIS region when compared with BMH. These results provide additional evidence for the structure models of the two alternative leader RNA conformations. We also found a correlation between the efficiency of oligonucleotide hybridization and the accessibility of the RNA structure as determined by chemical and enzymatic probing in previous studies. The array approach therefore provides a very sensitive method to detect structural differences in related transcripts.

Rationalisation of the differences between APOBEC3G structures from crystallography and NMR studies by molecular dynamics simulations.

The human APOBEC3G (A3G) protein is a cellular polynucleotide cytidine deaminase that acts as a host restriction factor of retroviruses, including HIV-1 and various transposable elements. Recently, three NMR and two crystal structures of the catalytic deaminase domain of A3G have been reported, but these are in disagreement over the conformation of a terminal beta-strand, beta2, as well as the identification of a putative DNA binding site. We here report molecular dynamics simulations with all of the solved A3G catalytic domain structures, taking into account solubility enhancing mutations that were introduced during derivation of three out of the five structures. In the course of these simulations, we observed a general trend towards increased definition of the beta2 strand for those structures that have a distorted starting conformation of beta2. Solvent density maps around the protein as calculated from MD simulations indicated that this distortion is dependent on preferential hydration of residues within the beta2 strand. We also demonstrate that the identification of a pre-defined DNA binding site is prevented by the inherent flexibility of loops that determine access to the deaminase catalytic core. We discuss the implications of our analyses for the as yet unresolved structure of the full-length A3G protein and its biological functions with regard to hypermutation of DNA.

Identification of amino acid residues in APOBEC3G required for regulation by human immunodeficiency virus type 1 Vif and Virion encapsidation.

The human immunodeficiency virus type-1 (HIV-1) accessory protein Vif serves to neutralize the human antiviral proteins apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G [A3G]) and A3F. As such, the therapeutic blockade of Vif function represents a logical objective for rational drug design. To facilitate such endeavors, we have employed molecular genetics to define features of A3G that are required for its interaction with Vif. Using alanine-scanning mutations and multiple different substitutions at key residues, we confirm the central role played by the aspartic acid at position 128 and identify proline 129 and aspartic acid 130 as important contributory residues. The overall negative charge of this 3-amino-acid motif appears critical for recognition by Vif, as single lysine substitutions are particularly deleterious and a double alanine substitution at positions 128 and 130 is far more inhibitory than single-residue mutations at either position. Our analyses also reveal that the immediately adjacent 4 amino acids, residues 124 to 127, are important for the packaging of A3G into HIV-1 particles. Most important are tyrosine 124 and tryptophan 127, and mutations at these positions can ablate virion incorporation, as well as the capacity to inhibit virus infection. Thus, while pharmacologic agents that target the acidic motif at residues 128 to 130 have the potential to rescue A3G expression by occluding recognition by Vif, care will have to be taken not to perturb the contributions of the neighboring 124-to-127 region to packaging if such agents are to have therapeutic benefit by promoting A3G incorporation into progeny virions.

Cytidine deamination and resistance to retroviral infection: towards a structural understanding of the APOBEC proteins.

The human apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G, or hA3G) protein, provides cells with an intracellular antiretroviral activity that is associated with the hypermutation of viral DNA through cytidine deamination. Indeed, hA3G belongs to a family of vertebrate proteins that contain one or two copies of a signature sequence motif unique to cytidine deaminases (CTDAs). We have constructed secondary structure models of the APOBEC proteins through a combination of structure prediction and subsequent alignment with nucleotide CTDAs whose structures have been solved to high resolution. Secondary structure elements common to all CTDAs are predicted, in addition to structural features that are apparently unique to the APOBEC family of proteins. Most notably, a putative looped-out helix abuts an amino acid that modulates the susceptibility of A3G proteins to the antagonistic action of the human and simian immunodeficiency virus (HIV and SIV) Vif proteins. Using the structure models as a guide, we reflect on mutagenesis studies of the APOBEC1 (A1), hA3G and activation induced deaminase (AID) proteins, with emphasis on the determinants of cytidine deamination and antiviral activities.

Multiple secondary structure rearrangements during HIV-1 RNA dimerization.

HIV-1 RNA dimerization is a complex process that involves a series of RNA refolding events. The monomeric RNA can adopt two alternative conformations that largely determine the efficiency of dimerization. The dimeric RNA also exists in two different conformations, an initial kissing-loop complex and a stable dimer with extended intermolecular base pairing. We describe an ordered RNA folding pathway that incorporates this multitude of HIV-1 RNA conformers. Analysis of mutant transcripts designed to block distinct steps of the refolding cascade supports this model. The folding properties of the wild-type RNA and the defects caused by the mutations can be fully understood in terms of the free energy changes associated with secondary structure rearrangements.

Evidence for a base triple in the free HIV-1 TAR RNA.

We propose the existence of a novel base triple in the HIV-1 TAR hairpin. This triple is supported by covariation of loop residue 31 with residue 22, which is part of an unusual base pair with U40 below the 3-nucleotide bulge. A set of mutants was constructed to test the involvement of bases A22, U31, and U40 in a triple interaction. RNA structure probing, trans-activation assays, and structure modeling are consistent with the existence of this base triple in a bent conformation of the free TAR element. However, disruption of the base triple does not affect binding of a Tat-derived peptide. We therefore compared the structure of free and Tat-bound TAR RNA by footprinting and site-specific cross-linking analyses. These studies indicate that the Tat arginine-rich motif, in addition to its known binding site at the bulge, is in close contact with U31 in the TAR loop. Because binding of Tat to TAR is known to coincide with the formation of a base triple with residues U23, A27, and U38, we hypothesize that Tat binding and the associated straightening of TAR triggers the disruption of the (A22-U40)U31 triple.

A riboswitch regulates RNA dimerization and packaging in human immunodeficiency virus type 1 virions.

The genome of retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNA strands that are packaged as noncovalently linked dimers. The core packaging and dimerization signals are located in the downstream part of the untranslated leader of HIV-1 RNA-the Psi and the dimerization initiation site (DIS) hairpins. The HIV-1 leader can adopt two alternative conformations that differ in the presentation of the DIS hairpin and consequently in their ability to dimerize in vitro. The branched multiple-hairpin (BMH) structure folds the poly(A) and DIS hairpins, but these domains are base paired in a long distance interaction (LDI) in the most stable LDI conformation. This LDI-BMH riboswitch regulates RNA dimerization in vitro. It was recently shown that the Psi hairpin structure is also presented differently in the LDI and BMH structures. Several detailed in vivo studies have indicated that sequences throughout the leader RNA contribute to RNA packaging, but how these diverse mutations affect the packaging mechanism is not known. We reasoned that these effects may be due to a change in the LDI-BMH equilibrium, and we therefore reanalyzed the structural effects of a large set of leader RNA mutations that were presented in three previous studies (J. L. Clever, D. Mirandar, Jr., and T. G. Parslow, J. Virol. 76:12381-12387, 2002; C. Helga-Maria, M. L. Hammarskjold, and D. Rekosh, J. Virol. 73:4127-4135, 1999; R. S. Russell, J. Hu, V. Beriault, A. J. Mouland, M. Laughrea, L. Kleiman, M. A. Wainberg, and C. Liang, J. Virol. 77:84-96, 2003). This analysis revealed a strict correlation between the status of the LDI-BMH equilibrium and RNA packaging. Furthermore, a correlation is apparent between RNA dimerization and RNA packaging, and these processes may be coordinated by the same LDI-BMH riboswitch mechanism.

In vitro evidence that the untranslated leader of the HIV-1 genome is an RNA checkpoint that regulates multiple functions through conformational changes.

The HIV-1 RNA genome forms dimers through base pairing of a palindromic 6-mer sequence that is exposed in the loop of the dimer initiation signal (DIS) hairpin structure (loop-loop kissing). The HIV-1 leader RNA can adopt a secondary structure conformation that is not able to dimerize because the DIS hairpin is not folded. Instead, this DIS motif is base-paired in a long distance interaction (LDI) that extends the stem of the primer-binding site domain. In this study, we show that targeting of the LDI by either antisense oligonucleotides or specific mutations can induce the conformational switch to a branched multiple hairpin (BMH) structure, and this LDI-to-BMH switch coincides with increased RNA dimerization. Another interesting finding is that the extended LDI stem can resist a certain level of destabilization, indicating that a buffer is created to prevent a premature conformational switch and early dimerization. Because the tRNA(Lys3) primer for reverse transcription anneals to multiple sequence elements of the HIV-1 leader RNA, including sequences in the LDI stem, we tested whether tRNA-annealing can destabilize the LDI stem such that RNA dimerization is triggered. Using a combination of stem-destabilizing approaches, we indeed measured a small but significant effect of tRNA-annealing on the ability of the RNA template to form dimers. This observation suggests that HIV-1 RNA can act as a checkpoint to control and coordinate different leader functions through conformational switches. This in vitro result should be verified in subsequent in vivo studies with HIV-infected cells.

Requirements for RNA heterodimerization of the human immunodeficiency virus type 1 (HIV-1) and HIV-2 genomes.

Retroviruses are prone to recombination because they package two copies of the RNA genome. Whereas recombination is a frequent event within the human immunodeficiency virus type 1 (HIV-1) and HIV-2 groups, no HIV-1/HIV-2 recombinants have been reported thus far. The possibility of forming HIV-1/HIV-2 RNA heterodimers was studied in vitro. In both viruses, the dimer initiation site (DIS) hairpin is used to form dimers, but these motifs appear too dissimilar to allow RNA heterodimer formation. Multiple mutations were introduced into the HIV-2 DIS element to gradually mimic the HIV-1 hairpin. First, the loop-exposed palindrome of HIV-1 was inserted. This self-complementary sequence motif forms the base pair interactions of the kissing-loop (KL) dimer complex, but such a modification is not sufficient to permit RNA heterodimer formation. Next, the HIV-2 DIS loop size was shortened from 11 to 9 nucleotides, as in the HIV-1 DIS motif. This modification also results in the presentation of the palindromes in the same position within the hairpin loop. The change yielded a modest level of RNA heterodimers, which was not significantly improved by additional sequence changes in the loop and top base pair. No isomerization of the KL dimer to the extended duplex dimer form was observed for the heterodimers. These combined results indicate that recombination between HIV-1 and HIV-2 is severely restricted at the level of RNA dimerization.

A human immunodeficiency virus type 1-infected individual with low viral load harbors a virus variant that exhibits an in vitro RNA dimerization defect.

We investigated the in vitro RNA dimerization properties of the untranslated leader RNA derived from human immunodeficiency virus type 1 variants circulating in an individual with a low viral load and slow disease progression. The leader sequences of these viruses contain highly unusual polymorphisms within the dimerization initiation site (DIS): an insert that abolishes dimerization and a compensatory substitution. The dimerization of leader RNA from late stages of infection is further improved by additional mutations outside the DIS motif that facilitate a secondary structure switch from a dimerization-incompetent to a dimerization-competent RNA conformation.