Heterogenous nuclear ribonucleoprotein Q increases protein expression from HIV-1 Rev-dependent transcripts
- Michelle Vincendeau†1,
- Daniel Nagel†1,
- Jara K Brenke1, 2,
- Ruth Brack-Werner1 and
- Kamyar Hadian1, 2Email author
© Vincendeau et al.; licensee BioMed Central Ltd. 2013
Received: 5 January 2012
Accepted: 19 March 2013
Published: 16 May 2013
Heterogenous nuclear ribonucleoproteins (hnRNPs) control many processes of the gene expression machinery including mRNA transcription, splicing, export, stability and translation. Recent data show interaction of the HIV-1 Rev regulatory protein with a subset of hnRNP proteins, that includes hnRNP Q, suggesting that hnRNPs can contribute to regulation of HIV-1 gene expression by Rev.
In this work we address the effect of hnRNP Q on Rev-dependent gene expression. We show that hnRNP Q overexpression increased levels of proteins produced from a Rev-dependent reporter gene in the presence of Rev. Increased protein levels did not correlate with changes in either the levels or the nucleocytoplasmic distribution of Rev-dependent reporter mRNAs. Similar observations were made in persistently HIV-1 infected HeLa cells. In these cells, hnRNP Q overexpression increased levels of the HIV-1 Gag-p24 protein, while levels of viral Rev-dependent mRNAs were not affected.
Our data indicate that hnRNP Q can stimulate the protein production of Rev-dependent mRNAs without changing mRNA levels and mRNA export, respectively. This suggests that hnRNP Q can boost HIV gene expression at the level of protein production.
HIV-1 Rev is critical for HIV-1 replication. Rev recognizes the Rev response element (RRE) in partly and unspliced viral RNAs. Binding of Rev to the RRE counteracts negative effects of instability elements (INS) in viral RNA molecules and promotes their transport to the cytoplasm [1–3]. Additionally, the Rev protein is involved in many other processes, e.g. RNA splicing , translation of viral RNAs [5, 6] and packaging of viral particles . Rev comprises different functional domains responsible for RNA binding/nuclear localization (nuclear localization signal, NLS) , nuclear export/transactivation (nuclear export signal, NES)  and multimerization of the Rev-protein (oligomerization signals) . Rev interacts with many cellular partners, including Exportin 1 (CRM1) and host cell RNA binding factors like DEAD/H box proteins . In addition, we recently showed that the N-terminus of Rev is able to interact with several hnRNPs , a set of RNA binding proteins involved in multiple cellular processes . These interactions seem to play an important role in connecting the diverse functions of Rev during viral gene expression with cellular processes.
The human hnRNP Q protein (synonym: SYNCRIP, Gry-rbp, NSAP1) exists in three isoforms (Q1, Q2 and Q3), which are generated from the same gene by alternative splicing . The full-length protein (Q3) consists of 623 amino acids and contains three RNA-binding domains (RBD), two NLS and a C-terminal Arginine-rich motif (RGG-domain) . HnRNP Q is important for efficient pre-mRNA splicing  and can impact mRNA stability [14, 15]. Furthermore, hnRNP Q is capable of influencing IRES-dependent mRNA translation [16–18]. In the HIV system, hnRNP Q was recently shown to be part of a group of cellular proteins that binds to a segment of HIV-1 RNA containing splice acceptor site A7  located near the RRE. Interestingly, the group of proteins binding to this region also included translational factors. In a recent study we could demonstrate that hnRNP Q interacts with HIV-1 Rev and is able to positively affect HIV replication, with knockdown of hnRNP Q decreasing viral production . In this Short Report we now present additional research data that strengthen the previously published positive effects of hnRNP Q on HIV-1 replication.
As expected, expression of Rev substantially increased reporter protein production (Figure 1D). Co-expression of RevGFP with hnRNP Q-CYN significantly boosted reporter protein levels even further (approx. 2-fold) compared to the CYN-control (Figure 1D). Similar results were obtained with a plasmid expressing hnRNP Q without the CYN-tag (data not shown), confirming the specific influence of hnRNP Q on reporter protein production. In comparison, hnRNP A1 was not as potent as hnRNP Q to induce reporter protein production (Additional file 1A). Interestingly, hnRNP Q did not increase reporter protein production in the absence of Rev (Figure 1D). In addition, a hnRNP Q deletion mutant lacking the RGG domain from amino acids 444–623 (hnRNP Q-(Δ444-623)-CYN) was not able to boost reporter expression and did not bind Rev in exBIFC assays (Figure 1E). As a control, we explored the effect of hnRNP Q on a GagCTE reporter . This reporter drives Gag production under the control of a LTR promoter and its expression is independent of Rev, but rather uses the TAP/CTE pathway. We could detect a small increase in Gag production through the GagCTE reporter, but notably less pronounced when compared to our Rev-dependent reporter construct (compare Figure 1D and Additional file 2). Further, a Rev mutant lacking the N-terminal 14 amino acids (Δ2-14-Rev), a region involved in the binding to different hnRNPs , showed reduced ability to induce Rev-dependent reporter protein production. Surprisingly, hnRNP Q was able to further increase levels of reporter proteins driven by the Δ2-14-Rev mutant indicating that hnRNP Q may also affect reporter protein production by mechanisms independent of Rev-hnRNP Q interaction (Figure 1F). Taken together, our data demonstrate that hnRNP Q is capable of enhancing Rev-mediated reporter protein production.
In this Short Report we addressed the effect of hnRNP Q on Rev-dependent reporter production and HIV-1 replication. For overexpression we used the full-length hnRNP Q coding sequence. Overexpression of hnRNP Q increased protein levels produced from Rev-dependent transcripts without affecting amounts or nucleocytoplasmic distribution of Rev-dependent transcripts. This point is remarkable as hnRNP Q is known to influence Hepatitis C virus RNA replication , splicing events  and mRNA stability [14, 15]. The increase in p24 production is congruent with our recent findings that knockdown of hnRNP Q in persistently infected astrocytes leads to diminished p24 production . Of note, the influences by hnRNP Q on the Rev-dependent reporter protein production were visibly higher when compared to a GagCTE reporter indicating a specific effect of hnRNP Q on the Rev/RRE axis. Moreover, hnRNP Q had a bigger effect on Rev-dependent reporter protein production than hnRNP A1 and the effects by these two hnRNPs were distinct of each other. While hnRNP Q did not affect mRNA levels, hnRNP A1 markedly increased reporter mRNA levels in the nucleus. One possibility to explain these hnRNP A1 effects is that hnRNP A1 is able to stabilize INS containing mRNAs . Over time the elevated number of reporter mRNAs lead to an increased protein production. However, hnRNP A1 has also other influences on viral replication (e.g. on the level of splicing) that could lead to the induced reporter protein and p24 production.
Interestingly, the influence of hnRNP Q on protein production from a Rev dependent reporter gene was only visible in the presence of HIV-1 Rev. Rev interaction with hnRNP Q was shown to involve the N-terminus of Rev . Surprisingly, we still see a positive effect of hnRNP Q on the activity of a Rev-deletion mutant lacking the N-terminus. This could be due to secondary binding sites in Rev for hnRNP Q since we previously reported that deletion of the N-terminus of Rev significantly diminished, but did not abrogate binding of Rev to hnRNP Q . However, we cannot exclude that there is also an activity of hnRNP Q on HIV-1 protein production independent of direct binding to Rev. In future studies we will decipher the mechanisms behind the mutual as well as individual impacts of Rev and hnRNP proteins to regulate HIV-1 replication.
Recently the Rev co-factors Sam68, eIF5A, hRIP and DDX3, which are essential for nuclear export of Rev-dependent mRNAs , were shown to be able to regulate HIV-1 replication on the translational level . This expands the impact of multifunctional Rev-interaction partners to processes in the cytoplasm. Our new data suggest that hnRNP Q could also be involved in the translational control of HIV-1 replication. Indeed, hnRNP Q was shown to influence translation of different mRNAs, including HCV mRNA , BiP mRNA  and Rev-erb alpha within circadian oscillation  proving its presence and function in the cytoplasmic compartment. The very diverse set of hnRNP proteins comprise a variety of cellular functions (mRNA transcription, splicing, trafficking, stability, translation, etc.) inside the nucleus as well as the cytoplasm [28, 29] and many hnRNPs are known to regulate HIV-1 replication (discussed in  and [30, 31]). Thus, hnRNP Q may also recruit other hnRNPs to co-activate HIV-1 replication. Future studies will address the role of hnRNP Q in the translation of HIV-1 proteins.
Taken together, we demonstrate supporting data that hnRNP Q increases p24 production in persistently infected cells and Rev-dependent reporter protein production. These effects are not associated with changes in RNA levels or nucleocytoplasmic distribution of Rev-dependent mRNAs, suggesting that hnRNP Q can contribute to positive effects on HIV-1 protein production.
KH was supported by the German research council (DFG), grant 1710/1-3 to RBW. We thank Dr. Felber for providing us with plasmids for GagCTE reporter and Tat expression.
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