Biomedical subjects
B R Cullen
Publications and source records attributed to B R Cullen.
An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein.
The human endogenous retrovirus K (HERV-K) family of endogenous retroviruses consists of approximately 50 proviral copies per haploid human genome. Herein, the HERV-Ks are shown to encode a sequence-specific nuclear RNA export factor, termed K-Rev, that is functionally analogous to the HIV-1 Rev protein. Like HIV-1 Rev, K-Rev binds to both the Crm1 nuclear export factor and to a cis-acting viral RNA target to activate nuclear export of unspliced RNAs. Surprisingly, this HERV-K RNA sequence, which is encoded within the HERV-K long terminal repeat, is also recognized by HIV-1 Rev. These data provide surprising evidence for an evolutionary link between HIV-1 and a group of endogenous retroviruses that first entered the human genome approximately 30 million years ago.
The human tap nuclear RNA export factor contains a novel transportin-dependent nuclear localization signal that lacks nuclear export signal function.
The human Tap protein mediates the sequence-specific nuclear export of RNAs containing the constitutive transport element and is likely also critical for general mRNA export. Here, we demonstrate that a previously defined arginine-rich nuclear localization signal (NLS) present in Tap acts exclusively via the transportin import factor. Previously, transportin has been shown to mediate the nuclear import of several heterogeneous nuclear ribonucleoproteins, including heterogeneous nuclear ribonucleoprotein (hnRNP) A1, by binding to a sequence element termed M9. Although the Tap NLS and the hnRNP A1 M9 element are shown to compete for transportin binding, they show no sequence homology, and the Tap NLS does not conform to the recently defined M9 consensus. The Tap NLS also differs from M9 in that only the latter is able to act as a nuclear export signal. The Tap NLS is therefore the first member of a novel class of transportin-specific NLSs that lack nuclear export signal function.
Analysis of the RNA binding specificity of the human tap protein, a constitutive transport element-specific nuclear RNA export factor.
The human Tap protein has been proposed to mediate Mason Pfizer monkey virus constitutive transport element (CTE)-dependent nuclear RNA export and may also play a role in global mRNA export. Here, we have used in vivo assays, in both yeast and human cells, together with in vitro assays, to further characterize the RNA binding properties of Tap, which has been proposed to contain a novel leucine-rich RNA binding motif. Using the yeast three hybrid assay, we selected RNA molecules that retain Tap binding activity from a pool of randomized CTE sequences. The recovered RNA sequences differed only minimally from the wild-type CTE yet all displayed lower affinity for Tap both in vivo and in vitro. Analysis of the RNA export activity of the recovered CTE variants revealed that Tap affinity was highly predictive of CTE biological activity. Together, these observations provide additional evidence supporting the identification of Tap as the direct cofactor for CTE function and demonstrate that RNA binding by Tap is highly sequence specific.
Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription.
Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR) promoter element by the viral Tat protein is an essential step in the HIV-1 life cycle. Tat function is mediated by the TAR RNA target element encoded within the LTR and is known to require the recruitment of a complex consisting of Tat and the cyclin T1 (CycT1) component of positive transcription elongation factor b (P-TEFb) to TAR. Here, we demonstrate that both TAR and Tat become entirely dispensable for activation of the HIV-1 LTR promoter when CycT1/P-TEFb is artificially recruited to a heterologous promoter proximal RNA target. The level of activation observed was indistinguishable from the level induced by Tat and was neither inhibited nor increased when Tat was expressed in trans. Activation by artificially recruited CycT1 depended on the ability to bind the CDK9 component of P-TEFb. In contrast, although binding to both Tat and TAR was essential for the ability of CycT1 to act as a Tat cofactor, these interactions became dispensable when CycT1 was directly recruited to the LTR. Importantly, activation of the LTR both by Tat and by directly recruited CycT1 was found to be at the level of transcription elongation. Together, these data demonstrate that recruitment of CycT1/P-TEFb to the HIV-1 LTR is fully sufficient to activate this promoter element and imply that the sole role of the Tat/TAR axis in viral transcription is to permit the recruitment of CycT1/P-TEFb.
Inhibition of HIV-1 progeny virion release by cell-surface CD4 is relieved by expression of the viral Nef protein.
BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) Nef protein is required for efficient virus replication in vivo and displays a number of distinct and apparently unrelated biological activities in vitro. Of these, one of the most readily demonstrated is the efficient internalization and degradation of cell-surface CD4, the receptor for the HIV-1 envelope protein. The biological purpose of this internalization has, however, remained unclear. RESULTS: Using human 293T cells expressing high levels of cell-surface CD4 or CD8, we demonstrate that CD4, but not CD8, can dramatically reduce the release of infectious virions bearing the HIV-1 envelope protein and induce a concomitant increase in the accumulation of cell-associated HIV-1 structural proteins. In contrast, CD4 had no effect on the release of HIV-1 bearing a heterologous envelope protein unable to bind CD4. Nef expression totally reversed CD4-mediated inhibition but only if the CD4 used remained susceptible to Nef-induced internalization. CONCLUSIONS: These results support the hypothesis that cell-surface CD4 can interact with the envelope protein present on budding HIV-1 virions to inhibit their release. The internalization and degradation of cell-surface CD4 induced by the viral Nef protein can fully reverse this inhibition and is, therefore, likely to facilitate the spread of virus in vivo.
The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences.
The constitutive transport element (CTE) encoded by simian type D retroviruses directs unspliced viral RNAs into a nuclear export pathway that is congruent with the pathway used by cellular mRNAs. Here, we show that quail cells are refractory to CTE function but become highly permissive upon expression of the human Tap protein, a candidate CTE cofactor. Tap contains a novel sequence-specific RNA binding domain that is sufficient for CTE binding but inadequate to support CTE function. Using microinjection assays, we have defined two NLSs and one NES in Tap. Mutational inactivation of the Tap NES, which lies outside the RNA-binding domain, not only blocks Tap function but also generates dominant-negative forms of Tap. Whereas replacement of the Tap NES with the well-defined Rev NES rescues the ability of Tap to support CTE function, this substitution also confers sensitivity to agents that block the activity of Crm1, the Rev NES cofactor. Together, these data validate Tap as the first human sequence-specific nuclear mRNA export factor and identify a novel type of NES that can support nuclear mRNA export but does not act via Crm1.
Definition of a consensus transportin-specific nucleocytoplasmic transport signal.
The low cytoplasmic and high nuclear concentration of the GTP-bound form of Ran provides directionality for both nuclear protein import and export. Both import and export factors bind RanGTP directly, yet this interaction produces opposite effects; in the former case, RanGTP binding induces nuclear cargo release, whereas in the latter, RanGTP binding induces nuclear cargo assembly. Therefore, nuclear import and export receptors and their protein recognition sites are predicted to be distinct. Nevertheless, the approximately 38-amino acid M9 sequence present in heterogeneous nuclear ribonucleoprotein A1 has been reported to serve as both a nuclear localization signal and a nuclear export signal, even though only one protein, the nuclear import factor transportin, has been shown to bind M9 directly. We have used a combination of mutational randomization followed by selection for transportin binding to exhaustively define amino acids in M9 that are critical for transportin binding in vivo. As expected, the resultant approximately 12-amino acid transportin-binding consensus sequence is also predictive of nuclear localization signal activity. Surprisingly, however, this extensive mutational analysis failed to dissect M9 nuclear localization signal and nuclear export signal function. Nevertheless, transportin appears unlikely to be the M9 export receptor, as RanGTP can be shown to block M9 binding by transportin not only in vitro, but also in the nucleus in vivo. This analysis therefore predicts the existence of a nuclear export receptor distinct from transportin that nevertheless shares a common protein-binding site on heterogeneous nuclear ribonucleoprotein A1.
Role of chemokine receptors in HIV-1 infection and pathogenesis.
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Structural and functional analysis of the avian leukemia virus constitutive transport element.
The observation that cells restrict the nuclear export of incompletely spliced transcripts via the canonical nuclear mRNA export pathway implies that all retroviruses should have evolved a way to direct the unspliced form of their genomic RNA into an alternate export pathway. While the Crm1-dependent pathway used by complex retroviruses to export incompletely spliced viral transcripts is now fairly well understood, less is known about how simple retroviruses accomplish this task. However, the Mason-Pfizer monkey virus (MPMV) has been shown to encode a structured RNA sequence, termed the constitutive transport element (CTE), that recruits a cellular RNA export factor termed Tap. Here we demonstrate that a CTE previously proposed to be present in the avian sarcoma/leukemia (ASV/ALV) family of retroviruses indeed functions as a potent RNA export signal. We have mapped single- and double-stranded regions present in the ASV/ALV CTE in vitro and report that this CTE is predicted to fold into a structure bearing three distinct RNA stem-loops. However, only the central stem-loop is critical for CTE function and this 69-nt structure is, in fact, sufficient when present as a dimer. While the ASV/ALV CTE is shown to function independently of Crm1, as also previously reported for the MPMV CTE, it lacks any evident sequence homology to the highly conserved MPMV CTE sequence. Together, these data define the secondary structure and biological activity of an avian CTE sequence that may access a novel nuclear RNA export pathway.
HIV-1 Nef protein: An invitation to a kill.
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Analysis of the effect of natural sequence variation in Tat and in cyclin T on the formation and RNA binding properties of Tat-cyclin T complexes.
The biological activity of the human immunodeficiency virus type 1 (HIV-1) Tat (Tat1) transcriptional activator requires the recruitment of a Tat1-CyclinT1 (CycT1) complex to the TAR RNA target encoded within the viral long terminal repeat (LTR). While other primate immunodeficiency viruses, such as HIV-2 and mandrill simian immunodeficiency virus (SIVmnd), also encode Tat proteins that activate transcription via RNA targets, these proteins differ significantly, both from each other and from Tat1, in terms of their ability to activate transcription directed by LTR promoter elements found in different HIV and SIV isolates. Here, we show that CycT1 also serves as an essential cofactor for HIV-2 Tat (Tat2) and SIVmnd Tat (Tat-M) function. Moreover, the CycT1 complex formed by each Tat protein displays a distinct RNA target specificity that accurately predicts the level of activation observed with a particular LTR. While Tat2 and Tat-M share the ability of Tat1 to bind to CycT1, they differ from Tat1 in that they are also able to bind to the related but distinct CycT2. However, the resultant Tat-CycT2 complexes fail to bind TAR and are therefore abortive. Surprisingly, mutation of a single residue in CycT2 (asparagine 260 to cysteine) rescues the ability of CycT2 to bind Tat1 and also activates not only TAR binding by all three Tat-CycT2 complexes but also Tat function. Therefore, the RNA target specificity of different Tat-CycT1 complexes is modulated by natural sequence variation in both the viral Tat transcriptional activator and in the host cell CycT molecule recruited by Tat. Further, the RNA target specificity of the resultant Tat-CycT1 complex accurately predicts the ability of that complex to activate transcription from a given LTR promoter element.
The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin beta-dependent nuclear localization signals.
Protein nuclear import is generally mediated by basic nuclear localization signals (NLSs) that serve as targets for the importin alpha (Imp alpha) NLS receptor. Imp alpha is in turn bound by importin beta (Imp beta), which targets the resultant protein complex to the nucleus. Here, we report that the arginine-rich NLS sequences present in the human immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to interact with Imp alpha and instead bind directly to Imp beta. Using in vitro nuclear import assays, we demonstrate that Imp alpha is entirely dispensable for Tat and Rev nuclear import. In contrast, Imp beta proved both sufficient and necessary, in that other beta-like import factors, such as transportin, were unable to support Tat or Rev nuclear import. Using in vitro competition assays, it was demonstrated that the target sites on Imp beta for Imp alpha, Tat, and Rev binding either are identical or at least overlap. The interaction of Tat and Rev with Imp beta is also similar to Imp alpha binding in that it is inhibited by RanGTP but not RanGDP, a finding that may in part explain why the interaction of the Rev nuclear RNA export factor with target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these studies define a novel class of arginine-rich NLS sequences that are direct targets for Imp beta and that therefore function independently of Imp alpha.
Highly divergent lentiviral Tat proteins activate viral gene expression by a common mechanism.
The human immunodeficiency virus type 1 (HIV-1) Tat protein (hTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter by a unique mechanism requiring recruitment of the human cyclin T1 (hCycT1) cofactor to the viral TAR RNA target element. While activation of equine infectious anemia virus (EIAV) gene expression by the EIAV Tat (eTat) protein appears similar in that the target element is a promoter proximal RNA, eTat shows little sequence homology to hTat, does not activate the HIV-1 LTR, and is not active in human cells that effectively support hTat function. To address whether eTat and hTat utilize similar or distinct mechanisms of action, we have cloned the equine homolog of hCycT1 (eCycT1) and examined whether it is required to mediate eTat function. Here, we report that expression of eCycT1 in human cells fully rescues eTat function and that eCycT1 and eTat form a protein complex that specifically binds to the EIAV, but not the HIV-1, TAR element. While hCycT1 is also shown to interact with eTat, the lack of eTat function in human cells is explained by the failure of the resultant protein complex to bind to EIAV TAR. Critical sequences in eCycT1 required to support eTat function are located very close to the amino terminus, i.e., distal to the HIV-1 Tat-TAR interaction motif previously identified in the hCycT1 protein. Together, these data provide a molecular explanation for the species tropism displayed by eTat and demonstrate that highly divergent lentiviral Tat proteins activate transcription from their cognate LTR promoters by essentially identical mechanisms.
Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat.
Human cyclin T1 (hCycT1), a major subunit of the essential elongation factor P-TEFb, has been proposed to act as a cofactor for human immunodeficiency virus type 1 (HIV-1) Tat. Here, we show that murine cyclin T1 (mCycT1) binds the activation domain of HIV-1 Tat but, unlike hCycT1, cannot mediate Tat function because it cannot be recruited efficiently to TAR. In fact, overexpression of mCycT1, but not hCycT1, specifically inhibits Tat-TAR function in human cells. This discordant phenotype results from a single amino acid difference between hCycT1 and mCycT1, a tyrosine in place of a cysteine at residue 261. These data indicate that the ability of Tat to recruit CycT1/P-TEFb to TAR determines the species restriction of HIV-1 Tat function in murine cells and therefore demonstrate that this recruitment is a critical function of the Tat protein.
Determination of the functional domain organization of the importin alpha nuclear import factor.
Although importin alpha (Imp alpha) has been shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to the importin beta nuclear import factor, little is known about the functional domains present in Imp alpha, with the exception that importin beta binding is known to map close to the Imp alpha NH2 terminus. Here, we demonstrate that sequences essential for binding to the CAS nuclear export factor are located near the Imp alpha COOH terminus and include a critical acidic motif. Although point mutations introduced into this acidic motif inactivated both CAS binding and Imp alpha nuclear export, a putative leucine-rich nuclear export signal proved to be neither necessary nor sufficient for Imp alpha nuclear export. Analysis of sequences within Imp alpha that bind to the SV-40 T antigen NLS or to the similar LEF-1 NLS revealed that both NLSs interact with a subset of the eight degenerate armadillo (Arm) repeats that form the central part of Imp alpha. However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region of Imp alpha may serve to facilitate binding to similar but nonidentical basic NLSs. Importantly, the SV-40 T NLS proved able to specifically inhibit the interaction of Imp alpha with CAS in vitro, thus explaining why the SV-40 T NLS is unable to also function as a nuclear export signal.
Retroviruses as model systems for the study of nuclear RNA export pathways.
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The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop.
Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.