The movement of proteins between the cytoplasm and nucleus mediated by the importin superfamily of proteins is essential to many cellular processes, including differentiation and development, and is critical to disease states such as viral disease and oncogenesis. We recently developed a high-throughput screen to identify specific and general inhibitors of protein nuclear import, from which ivermectin was identified as a potential inhibitor of importin α/β-mediated transport. In the present study, we characterized in detail the nuclear transport inhibitory properties of ivermectin, demonstrating that it is a broad-spectrum inhibitor of importin α/β nuclear import, with no effect on a range of other nuclear import pathways, including that mediated by importin β1 alone. Importantly, we establish for the first time that ivermectin has potent antiviral activity towards both HIV-1 and dengue virus, both of which are strongly reliant on importin α/β nuclear import, with respect to the HIV-1 integrase and NS5 (non-structural protein 5) polymerase proteins respectively. Ivermectin would appear to be an invaluable tool for the study of protein nuclear import, as well as the basis for future development of antiviral agents.

SUMMARY The prokaryotic translation elongation factors were identified as essential cofactors for RNA-dependent RNA polymerase activity of the bacteriophage Qβ more than 40 years ago. A growing body of evidence now shows that eukaryotic translation elongation factors (eEFs), predominantly eEF1A, acting in partially characterized complexes sometimes involving additional eEFs, facilitate virus replication. The functions of eEF1A as a protein chaperone and an RNA- and actin-binding protein enable its “moonlighting” roles as a virus replication cofactor. A diverse group of viruses, from human immunodeficiency type 1 and West Nile virus to tomato bushy stunt virus, have adapted to use eEFs as cofactors for viral transcription, translation, assembly, and pathogenesis. Here we review the mechanisms used by viral pathogens to usurp these abundant cellular proteins for their replication.

The ability of human immunodeficiency virus‐1 (HIV‐1) to undergo efficient reverse transcription is dependent on a number of parameters. These include the binding of the tRNA3Lys to the HIV‐1 primer binding site and the subsequent interaction with the heterodimeric reverse transcriptase. Recently, we demonstrated that TAR RNA was also necessary for efficient HIV‐1 reverse transcription. Given the fact that the Tat protein is involved in the activation of HIV‐1 gene expression in conjunction with TAR, we wished to determine whether Tat might also be involved in the control of HIV‐1 reverse transcription. HIV‐1 virions deleted in the tat gene were unable to initiate reverse transcription efficiently upon infection of peripheral blood mononuclear cells (PBMCs). This defect was not due to decreased amounts of genomic RNA, reverse transcriptase or other HIV‐1 proteins which were incorporated into the virion. Following transfection of wild‐type but not mutant tat genes into cell lines producing HIV‐1 lacking tat , the virions produced could be complemented for defects in reverse transcription upon subsequent infection of PBMCs. In contrast, the defect in reverse transcription seen with HIV‐1 lacking the tat gene could not be complemented when the target cells rather than the producer cells contained tat . Viruses lacking tat were also defective in endogenous assays of reverse transcription, although these viruses contained similar levels of reverse transcriptase. These results indicate that the Tat protein, in addition to regulating the level of gene expression, is also important for efficient HIV‐1 reverse transcription.

The ability of self-replicating RNA (replicon) vaccine vectors derived from the Australian flavivirus Kunjin (KUN) to induce protective αβ CD8⁺T-cell responses was examined. KUN replicons encoding a model immunogen were delivered by three different vaccine modalities: (i) as naked RNA transcribed in vitro, (ii) as plasmid DNA constructed to allow in vivo transcription of replicon RNA by cellular RNA polymerase II (DNA based), and (iii) as replicon RNA encapsidated into virus-like particles. A single immunization with any of these KUN replicon vaccines induced CD8⁺T-cell responses at levels comparable to those induced by recombinant vaccinia virus encoding the same immunogen. Immunization with only 0.1 μg of DNA-based KUN replicons elicited CD8⁺T-cell responses similar to those seen after immunization with 100 μg of a conventional DNA vaccine. Naked RNA immunization with KUN replicons also protected mice against challenges with recombinant vaccinia virus and B16 tumor cells. These results demonstrate the value of KUN replicon vectors for inducing protective antiviral and anticancer CD8⁺T-cell responses.

Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus.

Background: The interferon (IFN)-induced, dsRNA-dependent serine/threonine protein kinase, PKR, plays a key regulatory role in the IFN-mediated anti-viral response by blocking translation in the infected cell by phosphorylating the alpha subunit of elongation factor 2 (eIF2). The human immunodeficiency virus type 1 (HIV-1) evades the anti-viral IFN response through the binding of one of its major transcriptional regulatory proteins, Tat, to PKR. HIV-1 Tat acts as a substrate homologue for the enzyme, competing with eIF2α, and inhibiting the translational block. It has been shown that during the interaction with PKR, Tat becomes phosphorylated at three residues: serine 62, threonine 64 and serine 68. We have investigated the effect of this phosphorylation on the function of Tat in viral transcription. HIV-1 Tat activates transcription elongation by first binding to TAR RNA, a stem-loop structure found at the 5' end of all viral transcripts. Our results showed faster, greater and stronger binding of Tat to TAR RNA after phosphorylation by PKR. Results: We have investigated the effect of phosphorylation on Tat-mediated transactivation. Our results showed faster, greater and stronger binding of Tat to TAR RNA after phosphorylation by PKR. In vitro phosphorylation experiments with a series of bacterial expression constructs carrying the wild-type tat gene or mutants of the gene with alanine substitutions at one, two, or all three of the serine/threonine PKR phosphorylation sites, showed that these were subject to different levels of phosphorylation by PKR and displayed distinct kinetic behaviour. These results also suggested a cooperative role for the phosphorylation of S68 in conjunction with S62 and T64. We examined the effect of phosphorylation on Tat-mediated transactivation of the HIV-1 LTR in vivo with a series of analogous mammalian expression constructs. Co-transfection experiments showed a gradual reduction in transactivation as the number of mutated phosphorylation sites increased, and a 4-fold decrease in LTR transactivation with the Tat triple mutant that could not be phosphorylated by PKR. Furthermore, the transfection data also suggested that the presence of S68 is necessary for optimal Tat-mediated transactivation. Conclusion: These results support the hypothesis that phosphorylation of Tat may be important for its function in HIV-1 LTR transactivation.