Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein.

We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag.     

Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays.

Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the psi or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the psi riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral psi sequences almost as well as to the HIV-1 psi riboprobe.     

Retrovirus-Specific Differences in Matrix and Nucleocapsid Protein-Nucleic Acid Interactions: Implications for Genomic RNA Packaging

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.     

Amino acid sequence divergence of Tat protein (exon1)of subtype B and C HIV-1 strains: Does it have implications for vaccine development?

Functional genes of HIV-1 like the tat express proteins essential for viral survival and propagation. There are variations reported in levels of Tat transactivation among the different subtypes of HIV-1. This study looked at the amino acid differences in the different regions of Tat protein (exon 1) of subtype B and C strains of HIV-1 and tried to observe a molecular basis for protein function. HIV-1 sequences of subtype B (n=30) and C (n=60) strains were downloaded from HIV-1 Los Alamos data base. Among the 60 subtype C strain sequences, 30 each were from India and Africa. A HIV-1 Tat protein (exon 1) sequence, the consensus B and C sequence was obtained from the 'sequence search interface' in the Los Alamos HIV-1 sequence data. The sequences were visualized using Weblogo and the RNA binding regions of the three consensus sequences were also determined using BindN software program. Compared to subtype B, there was a high level of divergence in the auxiliary domain of tat exon 1 (amino acid positions 58- 69). The net charge of the subtype C (Indian) Tat protein (exon 1) auxiliary domain was -1.9 at pH 7 and it had an isoelectric point of 4.1. The net charge of the subtype C (African) auxiliary domain was -2.9 at pH 7 and it had an isoelectric point of 3.7 while the net charge of same region in subtype B was -0.9 at pH 7 with an isoelectric point of 4.9. The ratio of the hydrophilic residues to the total number of residues was 60% in the in both the Indian and African subtype C in the auxiliary domain while this was 50% in subtype B. The consensus subtype B sequence was found to have 36 RNA binding sites while subtype C (India) had 33 and subtype C (Africa) had 32 RNA binding sites. The HIV-1 Tat-TAR interaction is a potential target for inhibitors and being considered for its potential use in HIV-1 vaccines. Development of such inhibitor/vaccines would have to take into consideration the variation in amino acid sequence analyzed in this study as this could determine epitope presentation on MHC class I antigen for afferent immune response.     

Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups

A soluble form of the HIV-1 envelope glycoprotein gp160 devoid of the transmembrane anchor domain was found to bind to cholesteryl-hemisuccinate agarose. The external subunit gp120 failed to bind to the resin, suggesting that the site responsible for the binding to cholesterol was located in the transmembrane protein gp41. We constructed a series of maltose binding protein (MBP) fusion proteins representing overlapping fragments of the gp41 molecule and we studied their capacity to bind to cholesteryl beads. The domain responsible for binding to cholesterol was localised within the residues 668 to 684 immediately adjacent to the membrane spanning domain. We identified a short sequence (LWYIK, aa 678-683) comparable to the cholesterol interaction amino acid consensus pattern published by Li and Papadopoulos [Endocrinology 139 (1998) 4991]. We demonstrated that the sequence LWYIK synthesized fused to the MBP was able to bind to cholesteryl groups. A synthetic peptide containing the sequence LWYIK was found to inhibit the interaction between cholesteryl beads and MBP44, an MBP fusion HIV-1 envelope protein that contains the putative cholesterol binding domain. Human sera obtained from HIV-1 seropositive patients did not react in ELISA to the LWYIK sequence, suggesting that this region is not exposed to the immune system. The biological significance of the interaction between gp41 and cholesterol is discussed.     

Function of the human immunodeficiency virus types 1 and 2 Rev proteins is dependent on their ability to interact with a structured region present in env gene mRNA.

The interaction of the human immunodeficiency virus type 1 (HIV-1) Rev protein with a structured region in env mRNA (the Rev-responsive element [RRE]) mediates the export of structural mRNAs from the nucleus to the cytoplasm. We demonstrated that unlike HIV-1 Rev, which functions with both the HIV-1 and HIV-2 RREs, HIV-2 Rev functions only with the HIV-2 RRE. Rev-RRE binding studies suggested that the lack of nonreciprocal complementation stems from the inability of HIV-2 Rev to interact with HIV-1 RRE RNA. Maintenance of RNA secondary structure, rather than the primary nucleotide sequence, appeared to be the major determinant for interaction of both HIV-1 and HIV-2 Rev with the HIV-2 RRE. Moreover, the binding domain of the HIV-2 RRE recognized by HIV-1 Rev was dissimilar to the binding domain of the HIV-1 RRE, in terms of both secondary structure and primary nucleotide sequence. Our results support the hypothesis that function of HIV Rev proteins and possibly the functionally similar Rex proteins encoded by the human T-cell leukemia viruses (HTLVs) HTLV-I and HTLV-II is controlled by the presence of RNA secondary structure generated within the RRE RNA.     

Gag localization and virus-like particle release mediated by the matrix domain of human T-lymphotropic virus type 1 Gag are less dependent on phosphatidylinositol-(4,5)-bisphosphate than those mediated by the matrix domain of HIV-1 Gag

The human immunodeficiency virus type 1 (HIV-1) Gag matrix (MA) domain facilitates Gag targeting and binding to the plasma membrane (PM) during virus assembly. Interaction with a PM phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)], plays a key role in these MA functions. Previous studies showed that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P(2), mislocalizes HIV-1 Gag to the cytosol and greatly reduces HIV-1 release efficiency. In this study, we sought to determine the role of the MA-PI(4,5)P(2) interaction in Gag localization and membrane binding of a deltaretrovirus, human T-lymphotropic virus type 1 (HTLV-1). We compared the chimeric HIV-1 Gag (HTMA), in which MA was replaced with HTLV-1 MA, with wild-type HIV-1 and HTLV-1 Gag for PI(4,5)P(2) dependence. Our results demonstrate that, unlike HIV-1 Gag, subcellular localization of and VLP release by HTLV-1 and HTMA Gag were minimally sensitive to 5ptaseIV overexpression. These results suggest that the interaction of HTLV-1 MA with PI(4,5)P(2) is not essential for HTLV-1 particle assembly. Furthermore, liposome-binding analyses showed that both HTLV-1 and HTMA Gag can bind membrane efficiently even in the absence of PI(4,5)P(2). Efficient HTLV-1 Gag binding to liposomes was largely driven by electrostatic interaction, unlike that of HIV-1 Gag, which required specific interaction with PI(4,5)P(2). Furthermore, membrane binding of HTLV-1 Gag in vitro was not suppressed by RNA, in contrast to HIV-1 Gag. Altogether, our data suggest that Gag targeting and membrane binding mediated by HTLV-1 MA does not require PI(4,5)P(2) and that distinct mechanisms regulate HIV-1 and HTLV-1 Gag membrane binding.     

Translation Elongation Factor 1-Alpha Interacts Specifically with the Human Immunodeficiency Virus Type 1 Gag Polyprotein

Human immunodeficiency virus type 1 (HIV-1) gag-encoded proteins play key functions at almost all stages of the viral life cycle. Since these functions may require association with cellular factors, the HIV-1 matrix protein (MA) was used as bait in a yeast two-hybrid screen to identify MA-interacting proteins. MA was found to interact with elongation factor 1-alpha (EF1alpha), an essential component of the translation machinery that delivers aminoacyl-tRNA to ribosomes. EF1alpha was then shown to bind the entire HIV-1 Gag polyprotein. This interaction is mediated not only by MA, but also by the nucleocapsid domain, which provides a second, independent EF1alpha-binding site on the Gag polyprotein. EF1alpha is incorporated within HIV-1 virion membranes, where it is cleaved by the viral protease and protected from digestion by exogenously added subtilisin. The specificity of the interaction is demonstrated by the fact that EF1alpha does not bind to nonlentiviral MAs and does not associate with Moloney murine leukemia virus virions. The Gag-EF1alpha interaction appears to be mediated by RNA, in that basic residues in MA and NC are required for binding to EF1alpha, RNase disrupts the interaction, and a Gag mutant with undetectable EF1alpha-binding activity is impaired in its ability to associate with tRNA in cells. Finally, the interaction between MA and EF1alpha impairs translation in vitro, a result consistent with a previously proposed model in which inhibition of translation by the accumulation of Gag serves to release viral RNA from polysomes, permitting the RNA to be packaged into nascent virions.     

Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy

Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short alpha-helical peptide derived from Rev (Rev 34-50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2'-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K(D) = 20 +/- 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K(D) = 0.24 +/- 0.040 microM), the second site inhibited Rev binding in a competitive fashion (K(D) = 1. 8 +/- 0.8 microM), and the third much weaker site (or sites) is attributed to nonspecific binding (K(D) >/= 40 microM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE.     

HIV-1 Matrix Protein Binding to RNA

The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein plays multiple roles in the viral replication cycle. One essential role is to target PrGag proteins to their lipid raft-associated phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] assembly sites at the plasma membranes of infected cells. In addition to this role, several reports have implicated nucleic acid binding properties to retroviral MAs. Evidence indicates that RNA binding enhances the binding specificity of MA to PI(4,5)P₂-containing membranes and supports a hypothesis in which RNA binding to MA acts as a chaperone that protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to plasma membrane assembly sites. To gain a better understanding of HIV-1 MA-RNA interactions, we have analyzed the interaction of HIV MA with RNA ligands that were selected previously for their high affinities to MA. Binding interactions were characterized via bead binding, fluorescence anisotropy, gel shift, and analytical ultracentrifugation methods. Moreover, MA residues that are involved in RNA binding were identified from NMR chemical shift data. Our results indicate that the MA RNA and PI(4,5)P₂ binding sites overlap and suggest models for Gag-membrane and Gag-RNA interactions and for the HIV assembly pathway.     

RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors.     

Calmodulin Disrupts the Structure of the HIV-1 MA Protein

The MA protein from HIV-1 is a small, multifunctional protein responsible for regulating various stages of the viral replication cycle. To achieve its diverse tasks, MA interacts with host cell proteins and it has been reported that one of these is the ubiquitous calcium-sensing calmodulin (CaM), which is up-regulated upon HIV-1 infection. The nature of the CaM-MA interaction has been the subject of structural studies, using peptides based on the MA sequence, that have led to conflicting conclusions. The results presented here show that CaM binds intact MA with 1:1 stoichiometry in a Ca²⁺-dependent manner and that the complex adopts a highly extended conformation in solution as revealed by small-angle X-ray scattering. Alterations in tryptophan fluorescence suggest that the two buried tryptophans (W16 and W36) located in the first two alpha-helices of MA mediate the CaM interaction. Major chemical shift changes occur in the NMR spectrum of MA upon complex formation, whereas chemical shift changes in the CaM spectrum are quite modest and are assigned to residues within the normal target protein-binding hydrophobic clefts of CaM. The NMR data indicate that CaM binds MA via its N- and C-terminal lobes and induces a dramatic conformational change involving a significant loss of secondary and tertiary structure within MA. Circular dichroism experiments suggest that MA loses ∼ 20% of its α-helical content upon CaM binding. Thus, CaM binding is expected to impact upon the accessibility of interaction sites within MA that are involved in its various functions.     

A heterologous, high-affinity RNA ligand for human immunodeficiency virus Gag protein has RNA packaging activity

Retroviral RNA encapsidation depends on the specific binding of Gag proteins to packaging (psi) signals in genomic RNA. We investigated whether an in vitro-selected, high-affinity RNA ligand for the nucleocapsid (NC) portion of the Gag protein from human immunodeficiency virus type 1 (HIV-1) could mediate packaging into HIV-1 virions. We find that this ligand can functionally substitute for one of the Gag-binding elements (termed SL3) in the HIV-1 psi locus to support packaging and viral infectivity in cis. By contrast, this ligand, which fails to dimerize spontaneously in vitro, is unable to replace a different psi element (termed SL1) which is required for both Gag binding and dimerization of the HIV-1 genome. A single point mutation within the ligand that eliminates high-affinity in vitro Gag binding also abolishes its packaging activity at the SL3 position. These results demonstrate that specific binding of Gag or NC protein is a critical determinant of genomic RNA packaging.     

Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

The matrix domain (MA) of the HIV-1 precursor Gag (PrGag) protein directs PrGag proteins to assembly sites at the plasma membrane by virtue of its affinity to the phospholipid, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂). MA also binds to RNA at a site that overlaps its PI(4,5)P₂ site, suggesting that RNA binding may protect MA from associating with inappropriate cellular membranes prior to PrGag delivery to the PM. Based on this, we have developed an assay in which small molecule competitors to MA-RNA binding can be characterized, with the assumption that such compounds might interfere with essential MA functions and help elucidate additional features of MA binding. Following this approach, we have identified four compounds, including three thiadiazolanes, that compete with RNA for MA binding. We also have identified MA residues involved in thiadiazolane binding and found that they overlap the MA PI(4,5)P₂ and RNA sites. Cell culture studies demonstrated that thiadiazolanes inhibit HIV-1 replication but are associated with significant levels of toxicity. Nevertheless, these observations provide new insights into MA binding and pave the way for the development of antivirals that target the HIV-1 matrix domain.     

Examination of specific binding activity of aptamer RNAs to the HIV-NC by using a cell-based in vivo assay for protein-RNA interaction

The nucleocapsid (NC) protein of the Human Immunodeficiency Virus-1 plays a key role in viral genomic packaging by specifically recognizing the Psi(Psi) RNA sequence within the HIV-1 genome RNA. Recently, a novel cell-based assay was developed to probe the specific interactions in vivo between the NC and Psi-RNA using E.coli cells (J. Virol. 81: 6151-55, 2007). In order to examine the extendibility of this cell-based assay to RNAs other than Psi-RNA, this study tested the RNA aptamers isolated in vitro using the SELEX method, but whose specific binding ability to NC in a living cellular environment has not been established. The results demonstrate for the first time that each of those aptamer RNAs can bind specifically to NC in a NC zinc finger motif dependent manner within the cell. This confirms that the cell-based assay developed for NC-Psi interaction can be further extended and applied to NC-binding RNAs other than Psi-RNA.     

Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism.

The interaction of the human immunodeficiency virus (HIV) Gag protein with the plasma membrane of a cell is a critical event in the assembly of HIV particles. The matrix protein region (MA) of HIV type 1 (HIV-1) Pr55Gag has previously been demonstrated to confer membrane-binding properties on the precursor polyprotein. Both the myristic acid moiety and additional determinants within MA are essential for plasma membrane binding and subsequent particle formation. In this study, we demonstrated the myristylation-dependent membrane interaction of MA in an in vivo membrane-binding assay. When expressed within mammalian cells, MA was found both in association with cellular membranes and in a membrane-free form. In contrast, the intact precursor Pr55Gag molecule analyzed in an identical manner was found almost exclusively bound to membranes. Both membrane-bound and membrane-free forms of MA were myristylated and phosphorylated. Differential membrane binding was not due to the formation of multimers, as dimeric and trimeric forms of MA were also found in both membrane-bound and membrane-free fractions. To define the requirements for membrane binding of MA, we analyzed the membrane binding of a series of MA deletion mutants. Surprisingly, deletions within alpha-helical regions forming the globular head of MA led to a dramatic increase in overall membrane binding. The stability of the MA-membrane interaction was not affected by these deletions, and no deletion eliminated membrane binding of the molecule. These results establish that myristic acid is a primary determinant of the stability of the Gag protein-membrane interaction and provide support for the hypothesis that a significant proportion of HIV-1 MA molecules may adopt a conformation in which myristic acid is hidden and unavailable for membrane interaction.