Lemay et al identified HuR as a binding partner for HIV-1 RT in a yeast two-hybrid screen of a random primed cDNA library derived from CEMC7 lymphocytes, and a physical interaction in vitro was proposed based on time-resolved fluorescence data . In the reported fluorescence experiment, serial dilutions of GST-HuR (or GST alone) were incubated with a constant amount of C-terminal hexahistidine tagged HIV-1 RT for 24 hours at 4°C. Subsequently, the interaction was probed by fluorescence energy transfer using anti-GST antibodies conjugated with the donor TBPEu3+ and anti-hexahistidine antibodies conjugated with the acceptor XL665. In our experiments, we discovered that the GST-HuR fusion protein is not stable in solution for extended periods and is subject to degradation, casting doubt on the validity of the above interpretation. Indeed, Lemay et al. may have looked at an interaction, most-likely a non-specific one, between HIV-1 RT and a degraded/unfolded form of GST-HuR. In this regard it is well known that marginally stable proteins are prone to aggregation, a non-specific protein-protein interaction. It should be noted, however, that Lemay et al cloned HuR into the pGEX-4T-1 vector whereas we cloned it into the pGEX-2T vector. The resultant fusion proteins are identical in amino acid sequence except that the Lemay et al construct contains an additional proline residue located between the thrombin cleavage site and N-terminus of HuR. Although unlikely, this minor difference could contribute to differences in the relative solution stabilities of the GST-HuR constructs used in the two different studies.
Lemay et al. also reported that knockdown of HuR expression in HeLa P4.2 cells by RNA interference inhibited both the early and late steps of HIV-1 reverse transcription. While this could be caused by a direct effect of HuR on reverse transcription, it also could arise via indirect effects on other host cell factors that are important in HIV-1 replication. For example, it is well documented that tumor-necrosis factor-alpha (TNF-α) levels in cells significantly impact HIV-1 replication [23–26] and that the expression of many inflammatory cytokines, including TNF-α, is tightly regulated at the post-transcriptional level by HuR . Interestingly, several studies have demonstrated that HuR may undergo post-translational phosphorylation at S202 and/or S242 [28, 29]. Therefore, one cannot rule out the possibility that Lemay et al identified an interaction between a post-translationally modified HuR protein and HIV-1 RT in their yeast-two hybrid screen, and that this interaction may be of biological relevance.
In summary, the NMR chemical shift titration experiments with purified proteins, presented in this report, demonstrate unambiguously that no direct protein-protein interactions between HIV-1 RT and HuR are present in vitro up to concentrations of ~200 μM. It should be noted that the RT used in our study is derived from an LAI isolate (group M, subtype B), whereas the RT used in the Lemay et al study was derived from a BH10 isolate (group M, subtype B). There are amino acid differences between these two isolates in their RNase H domains at codons 447 [N (BH10) → S (LAI)], 461 (K → R), 468 (P → T), 471 (N → D), 482 (Y → H) and 559 (V → I). However, all of these substitutions exist as polymorphisms in the RT subtype B sequences deposited in the Stanford HIV database. Furthermore, although we found no evidence for a direct protein-protein interaction between HuR and HIV-1 RT in this study, an indirect interaction may be mediated by RNA. However, we could not detect any influence of HuR on HIV-1 RT DNA synthesis, even on T/P substrates that contain AREs and bound both HIV-1 RT and HuR (Fig. 4B). Therefore, our results suggest that HuR does not interfere with HIV-1 replication through a direct interaction with the viral reverse transcription complex, but through indirect effects possibly mediated via unidentified host factors and/or RNA.
In the search for host-pathogen interactions, a burgeoning field in modern virology, many potential interactions have been identified for HIV-1 in the last 2 or 3 years through high through-put screens [8–10, 12, 30, 31]. Our present follow up study using purified proteins illuminates some of the potential pitfalls associated with such approaches, and highlights the urgent need to carry out stringent biophysical validation of any putative interaction.