Identification of a Heterogeneous Nuclear Ribonucleoprotein-recognition Region in the HIV Rev Protein

Abstract

The Rev protein is a key regulator of human immunodeficiency virus type 1 (HIV-1) gene expression. Rev is primarily known as an adaptor protein for nuclear export of HIV RNAs. However, Rev also contributes to numerous other processes by less well known mechanisms. Understanding the functional nature of Rev requires extensive knowledge of its cellular interaction partners. Here we demonstrate that Rev interacts with members of a large family of multifunctional host cell factors called hnRNPs. Rev employs amino acids 9 -14 for specific binding to the heterogeneous nuclear ribonucleoproteins (hnRNP) A1, Q, K, R, and U. In addition, Rev interacts with hnRNP E1 and E2 by a different mechanism. The set of hnRNPs recognized by the N terminus of Rev feature RGG boxes. Exemplary testing of hnRNP A1 revealed a critical role of arginine residues within the RGG box for interaction with Rev. Finally, we demonstrate that expression levels of hnRNP A1, Q, K, R, and U influence HIV-1 production by persistently infected astrocytes, linking these hnRNPs to HIV replication. The novel interaction of HIV-1 Rev with functionally diverse hnRNPs lends further support to the idea that Rev is a multifunctional protein and may be involved in coupling HIV replication to diverse cellular processes and promoting virus-host cell interactions. During human immunodeficiency virus (HIV)3 replication, the transcripts that encode viral structural proteins and the viral RNA genome contain introns and would normally be eliminated by the host cell. The production of these RNAs and their utilization is achieved by strictly controlled alternative splicing mechanisms and the regulatory activities of the HIV trans-activator protein Rev (reviewed in Refs. 1-3).Rev is an RNA-binding protein that specifically binds a recognition element (RRE) within intron-containing HIV RNAs. One of the best studied functions of Rev is the recruitment of cellular factors that mediate nuclear export of Rev-bound RNAs (2, 3). Rev has also been shown to influence splicing (4), stability (3,5,6), and translation (7-9) of the viral RNAs as well as their packaging (10, 11). These regulatory activities of the Rev protein render it a key player in the HIV replication cycle. Detailed and sophisticated studies of the Rev protein identified at least three functional domains in Rev (2): (i) an arginine richmotif that functions both as nuclear localization signal and RNA-binding sequence (AA 35-50), (ii) a bipartite multimerization domain (AA 12-29 and AA 52-60), and (iii) a nuclear export signal (AA 75-83). Host factors shown to bind to these functional domains include B23 and Importin ␤ for the nuclear localization signal and CRM1/Exportin-1 and eIF-5A for the nuclear export signal (2, 12). In addition, Rev has been shown to interact with several RNA helicases (13-15).However, the overall number of cellular interactor proteins identified for Rev is still surprisingly small, compared with, for example, Tat (16). The many activities of Rev (17) and evidence for host-cell regulation of ...

Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

“…Rep proteins themselves are involved in AAV genome transcription and have been shown to affect the ratio of spliced versus unspliced AAV mRNAs (47). An increasing number of studies have suggested that various hnRNPs are involved in the replication of numerous viruses, including herpesviruses (7,23,24,33). Interestingly, some of the hnRNPs recovered were also reported to be able to associate with DNA by recognizing structural motifs and to regulate several aspects of DNA metabolism, such as chromosome maintenance and DNA replication and repair (25).…”

Structural Basis for Homodimerization of the Src-associated during Mitosis, 68-kDa Protein (Sam68) Qua1 Domain

“…Cell Culture and Transfections-U138MG cells were cultured as described previously (46). All overexpression experiments were performed in 6-well plates.…”

Depletion of hnRNP A2/B1 overrides the nuclear retention of the HIV-1 genomic RNA

“…18,24,25 These roles in retroviral RNA splicing and nucleocytoplasmic ReseARch PAPeR trafficking might also be functionally linked 26 a notion that is supported by newer work that shows that several functionally distinct hnRNPs interact with Rev. 27 Although principally nuclear, several hnRNPs indeed have cytoplasmic functions that contribute to the localization, the utilization, and fate of RNA in the cell. [28][29][30][31] Recent work for example emphasizes the importance for hnRNPs that translocate to the cytoplasm in an HIV-1-dependent manner to contribute to vRNA stability and translation in the cytoplasm.…”

“…This could be achieved by a loss of a direct vRNA interaction and/or by the deficiency of a Rev-interacting partner. 27 While we have proposed that the hnRNP A2/B1-A2RE association is important for both nuclear Figure 6. The distribution of hnRNPs and staufen1 in polyribosome gradients.…”