Translational regulation of APOBEC3G mRNA by Vif requires its 5'UTR and contributes to restoring HIV-1 infectivity.

The essential HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells expressing cytidine deaminases APOBEC3G (A3G) and A3F by decreasing their cellular level, and preventing their incorporation into virions. Unlike the Vif-induced degradation of A3G, the functional role of the inhibition of A3G translation by Vif remained unclear. Here, we show that two stem-loop structures within the 5'-untranslated region of A3G mRNA are crucial for translation inhibition by Vif in cells, and most Vif alleles neutralize A3G translation efficiently. Interestingly, K26R mutation in Vif abolishes degradation of A3G by the proteasome but has no effect at the translational level, indicating these two pathways are independent. These two mechanisms, proteasomal degradation and translational inhibition, similarly contribute to decrease the cellular level of A3G by Vif and to prevent its incorporation into virions. Importantly, inhibition of A3G translation is sufficient to partially restore viral infectivity in the absence of proteosomal degradation. These findings demonstrate that HIV-1 has evolved redundant mechanisms to specifically inhibit the potent antiviral activity of A3G.

Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system.

Among Candida species, the opportunistic fungal pathogen Candida glabrata has become the second most common causative agent of candidiasis in the world and a major public health concern. Yet, few molecular tools and resources are available to explore the biology of C. glabrata and to better understand its virulence during infection. In this study, we describe a robust experimental strategy to generate loss-of-function mutants in C. glabrata. The procedure is based on the development of three main tools: (i) a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system, (ii) an online program facilitating the selection of the most efficient guide RNAs for a given C. glabrata gene, and (iii) the identification of mutant strains by the Surveyor technique and sequencing. As a proof-of-concept, we have tested the virulence of some mutants in vivo in a Drosophila melanogaster infection model. Our results suggest that yps11 and a previously uncharacterized serine/threonine kinase are involved, directly or indirectly, in the ability of the pathogenic yeast to infect this model host organism.

Structural basis of genomic RNA (gRNA) dimerization and packaging determinants of mouse mammary tumor virus (MMTV).

BACKGROUND: One of the hallmarks of retroviral life cycle is the efficient and specific packaging of two copies of retroviral gRNA in the form of a non-covalent RNA dimer by the assembling virions. It is becoming increasingly clear that the process of dimerization is closely linked with gRNA packaging, and in some retroviruses, the latter depends on the former. Earlier mutational analysis of the 5' end of the MMTV genome indicated that MMTV gRNA packaging determinants comprise sequences both within the 5' untranslated region (5' UTR) and the beginning of gag. RESULTS: The RNA secondary structure of MMTV gRNA packaging sequences was elucidated employing selective 2'hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE analyses revealed the presence of a U5/Gag long-range interaction (U5/Gag LRI), not predicted by minimum free-energy structure predictions that potentially stabilizes the global structure of this region. Structure conservation along with base-pair covariations between different strains of MMTV further supported the SHAPE-validated model. The 5' region of the MMTV gRNA contains multiple palindromic (pal) sequences that could initiate intermolecular interaction during RNA dimerization. In vitro RNA dimerization, SHAPE analysis, and structure prediction approaches on a series of pal mutants revealed that MMTV RNA utilizes a palindromic point of contact to initiate intermolecular interactions between two gRNAs, leading to dimerization. This contact point resides within pal II (5' CGGCCG 3') at the 5' UTR and contains a canonical "GC" dyad and therefore likely constitutes the MMTV RNA dimerization initiation site (DIS). Further analyses of these pal mutants employing in vivo genetic approaches indicate that pal II, as well as pal sequences located in the primer binding site (PBS) are both required for efficient MMTV gRNA packaging. CONCLUSIONS: Employing structural prediction, biochemical, and genetic approaches, we show that pal II functions as a primary point of contact between two MMTV RNAs, leading to gRNA dimerization and its subsequent encapsidation into the assembling virus particles. The results presented here enhance our understanding of the MMTV gRNA dimerization and packaging processes and the role of structural motifs with respect to RNA-RNA and possibly RNA-protein interactions that might be taking place during MMTV life cycle.

Importance of the RNA secondary structure for the relative accumulation of clustered viral microRNAs.

Micro (mi)RNAs are small non-coding RNAs with key regulatory functions. Recent advances in the field allowed researchers to identify their targets. However, much less is known regarding the regulation of miRNAs themselves. The accumulation of these tiny regulators can be modulated at various levels during their biogenesis from the transcription of the primary transcript (pri-miRNA) to the stability of the mature miRNA. Here, we studied the importance of the pri-miRNA secondary structure for the regulation of mature miRNA accumulation. To this end, we used the Kaposi's sarcoma herpesvirus, which encodes a cluster of 12 pre-miRNAs. Using small RNA profiling and quantitative northern blot analysis, we measured the absolute amount of each mature miRNAs in different cellular context. We found that the difference in expression between the least and most expressed viral miRNAs could be as high as 60-fold. Using high-throughput selective 2'-hydroxyl acylation analyzed by primer extension, we then determined the secondary structure of the long primary transcript. We found that highly expressed miRNAs derived from optimally structured regions within the pri-miRNA. Finally, we confirmed the importance of the local structure by swapping stem-loops or by targeted mutagenesis of selected miRNAs, which resulted in a perturbed accumulation of the mature miRNA.

HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation.

The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3'UTR than for the 5'UTR, even though this region contained at least one high affinity Vif binding site (apparent K(d) = 27 +/- 6 nM). Several Vif binding sites were identified in 5' and 3'UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5'UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.

In vitro dimerization of human immunodeficiency virus type 1 (HIV-1) spliced RNAs.

The human immunodeficiency virus type 1 (HIV-1) packages its genomic RNA as a dimer of homologous RNA molecules that has to be selected among a multitude of cellular and viral RNAs. Interestingly, spliced viral mRNAs are packaged into viral particles with a relatively low efficiency despite the fact that they contain most of the extended packaging signal found in the 5' untranslated region of the genomic RNA, including the dimerization initiation site (DIS). As a consequence, HIV-1 spliced viral RNAs can theoretically homodimerize and heterodimerize with the genomic RNA, and thus they should directly compete with genomic RNA for packaging. To shed light on this issue, we investigated for the first time the in vitro dimerization properties of spliced HIV-1 RNAs. We found that singly spliced (env, vpr) and multispliced (tat, rev, and nef) RNA fragments are able to dimerize in vitro, and to efficiently form heterodimers with genomic RNA. Chemical probing experiments and inhibition of RNA dimerization by an antisense oligonucleotide directed against the DIS indicated that the DIS is structurally functional in spliced HIV-1 RNA, and that RNA dimerization occurs through a loop-loop interaction. In addition, by combining in vitro transcription and dimerization assays, we show that heterodimers can be efficiently formed only when the two RNA fragments are synthesized simultaneously, in the same environment. Together, our results support a model in which RNA dimerization would occur during transcription in the nucleus and could thus play a major role in splicing, transport, and localization of HIV-1 RNA.

Cooperative and specific binding of Vif to the 5' region of HIV-1 genomic RNA.

The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F.