RNA interactions in the 5' region of the HIV-1 genome

The untranslated leader of the dimeric HIV-1 RNA genome is folded into a complex structure that plays multiple and essential roles in the viral replication cycle. Here, we have investigated secondary and tertiary structural elements within the 5' 744 nucleotides of the HIV-1 genome using a combination of bioinformatics, enzymatic probing, native gel electrophoresis, and UV-crosslinking experiments. We used a recently developed RNA folding algorithm (Pfold) to predict the common secondary structure of an alignment of 20 divergent HIV-1 sequences. Combining this analysis with biochemical data, we present a secondary structure model for the entire 744 nucleotide fragment, which incorporates previously recognized and novel structural elements. In particular, our data provided strong evidence for a long-distance interaction between the region encompassing the AUG Gag initiation codon and an upstream region and we demonstrate that this feature is highly conserved in distantly related human and animal retroviruses. To obtain information about tertiary interactions we applied an intramolecular UV-crosslinking strategy and identified a novel tertiary interaction within the PBS hairpin structure.     

HIV-1辅助蛋白Vif的结构与功能

HIV-1 Vif(viral infectivity factor)蛋白是由保守的vif基因编码的碱性蛋白质,是HIV-1病毒的辅助调节蛋白之一.研究表明Vif蛋白具有调节病毒侵入、组装、出芽和成熟等功能.此外,Vif蛋白能够特异性地与体内抗病毒因子APOBEC3G相互作用,增强病毒的感染性.因此,针对HIV-1Vif蛋白进行抑制剂设计已经成为抗HIV药物研究的热点之一.本文对HIV-1Vif蛋白的结构与功能研究的最新进展进行了综述.     

Characterization of a novel Cullin5 binding domain in HIV-1 Vif

Human immunodeficiency virus tyoe 1 (HIV-1) Vif counteracts host restriction cytidine deaminase (APOBEC3G) A3G by co-opting the cellular ubiquitin-proteasome machinery. Vif utilizes a viral-specific BC-box to recruit ElonginB-ElonginC and a novel zinc-binding HCCH motif to recruit Cullin5 (Cul5) to form an E3 ubiquitin ligase targeting A3G for polyubiquitination and subsequently proteasomal degradation. To determine the structural requirements in HIV-1 Vif HCCH motif for Cul5 binding and Vif function, we investigated the arrangement of the His and Cys residues, the role of the spacing between them, and the requirement for the conserved residues. Our data demonstrate that exchanging Cys for His and vice versa in the highly conserved Zn-coordinating HCCH motif disrupted Vif function and interaction with Cul5. Moreover, the maintenance of both conserved residues and spacing within the HCCH motif is critical for Vif function. We have identified a "viral Cul5 box" with consensus Hx2YFxCFx4Phix2APhix7-8Cx5H that is required for Cul5 selection and subsequent A3G degradation. This novel motif may represent a potential new target for anti-viral drug development.     

YB-1 stabilizes HIV-1 genomic RNA and enhances viral production

HIV-1 utilizes cellular factors for efficient replication. The viral RNA is different from cellular mRNAs in many aspects, and is prone to attacks by cellular RNA quality control systems. To establish effective infection, the virus has evolved multiple mechanisms to protect its RNA. Here, we show that expression of the Y-box binding protein 1 (YB-1) enhanced the production of HIV-1. Downregulation of endogenous YB-1 in producer cells decreased viral production. YB-1 increased viral protein expression by stabilizing HIV-1 RNAs. The stem loop 2 in the HIV-1 RNA packaging signal was mapped to be the YB-1-responsive element. Taken together, these results indicate that YB-1 stabilizes HIV-1 genomic RNA and thereby enhances HIV-1 gene expression and viral production.     

A structure-based approach for targeting the HIV-1 genomic RNA dimerization initiation site

Dimerization of the genomic RNA is an important step of the HIV-1 replication cycle. The Dimerization Initiation Site (DIS) promotes dimerization of the viral genome by forming a loop-loop complex between two DIS hairpins. Crystal structures of the DIS loop-loop complex revealed an unexpected and strong similitude with the bacterial 16S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. As a consequence of these structural and sequence similarities, the HIV-1 DIS also binds some aminoglycosides, not only in vitro, but also ex vivo, in lymphoid cells and in viral particles. Crystal structures of the DIS loop-loop in complex with several aminoglycoside antibiotics provide a detailed-view of the DIS/drug interaction and reveal some hints about possible modifications to increase the drug affinity and/or specificity.     

核酸定量检测试验应用于HIV-1感染实验室诊断的探讨

目的:探讨核酸定量检测在HIV-1感染实验室诊断中的应用。方法:选取145例第四代抗原/抗体联合诊断筛查试验为阳性反应的血浆样本,分别用Western印迹和HIV-1核酸定量方法进行检测,综合对比分析2种方法检测结果。结果:Western印迹检出阳性样本120例,不确定样本17例,阴性样本8例;HIV-1核酸定量试验检出结果大于检测限样本131例,其中包括12例Western印迹不确定样本、2例Western印迹阴性样本;有3例Western印迹阳性样本用HIV-1核酸定量检测试验未能检出。结论:核酸定量检测试验对于HIV-1感染阳性样本是一种有效的实验室诊断方法;对HIV-1核酸定量检测结果为"TND"的样本,建议加做Western印迹或结合其他补充试验结果进行综合诊断。     

HIV-1 Replication Benefits from the RNA Epitranscriptomic Code

The effects of RNA methylation on HIV-1 replication remain largely unknown. Recent studies have discovered new insights into the effect of 2′-O-methylation and 5-methylcytidine marks on the HIV-1 RNA genome. As so far, HIV-1 benefits from diverse RNA methylations through distinct mechanisms. In this review, we summarize the recent advances in this emerging field and discuss the role of RNA methylation writers and readers in HIV-1 infection, which may help to find alternative strategies to control HIV-1 infection.     

Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation

The HIV-1 ribonucleoprotein (RNP) contains the major structural protein, pr55(Gag), viral genomic RNA, as well as the host protein, Staufen1. In this report, we show that the nonsense-mediated decay (NMD) factor UPF1 is also a component of the HIV-1 RNP. We investigated the role of UPF1 in HIV-1-expressing cells. Depletion of UPF1 by siRNA resulted in a dramatic reduction in steady-state HIV-1 RNA and pr55(Gag). Pr55(Gag) synthesis, but not the cognate genomic RNA, was efficiently rescued by expression of an siRNA-insensitive UPF1, demonstrating that UPF1 positively influences HIV-1 RNA translatability. Conversely, overexpression of UPF1 led to a dramatic up-regulation of HIV-1 expression at the RNA and protein synthesis levels. The effects of UPF1 on HIV-1 RNA stability were observed in the nucleus and cytoplasm and required ongoing translation. We also demonstrate that the effects exerted by UPF1 on HIV-1 expression were dependent on its ATPase activity, but were separable from its role in NMD and did not require interaction with UPF2.     

人免疫缺陷病毒I型Vif蛋白的原核表达、纯化及单克隆抗体制备

目的：克隆、表达、纯化人免疫缺陷病毒I型（HIV-1）Vif蛋白,制备其单克隆抗体。方法：提取感染了HIV的细胞基因组DNA,PCR扩增vif基因,插入表达载体pET32a,转化大肠杆菌BL21（DE3）获得工程菌株,IPTG诱导蛋白表达,Western印迹鉴定目的蛋白,亲和层析纯化目的蛋白;免疫BALB／c小鼠,制备单克隆抗体。结果：构建了Vif蛋白的原核表达载体vif-pET32a,并在大肠杆菌中获得高表达,目的蛋白以包涵体形式存在;纯化获得高纯度的重组Vif蛋白,蛋白浓度可达0．56mg／mL;建立了抗Vif蛋白单克隆抗体细胞株,制备了腹水,滴度可达1：16×10^6,抗体纯化后保持了活性和特异性。结论：在原核表达系统中表达、纯化了重组Vif蛋白,制备了针对Vif蛋白的单克隆抗体,为研究Vif蛋白的功能和抗原性奠定了基础。     

Identification of Two APOBEC3F Splice Variants Displaying HIV-1 Antiviral Activity and Contrasting Sensitivity to Vif

Approximately half of all human genes undergo alternative mRNA splicing. This process often yields homologous gene products exhibiting diverse functions. Alternative splicing of APOBEC3G (A3G) and APOBEC3F (A3F), the major host resistance factors targeted by the HIV-1 protein Vif, has not been explored. We investigated the effects of alternative splicing on A3G/A3F gene expression and antiviral activity. Three alternatively spliced A3G mRNAs and two alternatively spliced A3F mRNAs were detected in peripheral blood mononuclear cells in each of 10 uninfected, healthy donors. Expression of these splice variants was altered in different cell subsets and in response to cellular stimulation. Alternatively spliced A3G variants were insensitive to degradation by Vif but displayed no antiviral activity against HIV-1. Conversely, alternative splicing of A3F produced a 37-kDa variant lacking exon 2 (A3FΔ2) that was prominently expressed in macrophages and monocytes and was resistant to Vif-mediated degradation. Alternative splicing also produced a 24-kDa variant of A3F lacking exons 2–4 (A3FΔ2–4) that was highly sensitive to Vif. Both A3FΔ2 and A3FΔ2–4 displayed reduced cytidine deaminase activity and moderate antiviral activity. These alternatively spliced A3F gene products, particularly A3FΔ2, were incorporated into HIV virions, albeit at levels less than wild-type A3F. Thus, alternative splicing of A3F mRNA generates truncated antiviral proteins that differ sharply in their sensitivity to Vif.     

Quantitative analysis of the formation of nucleoprotein complexes between HIV-1 Gag protein and genomic RNA using transmission electron microscopy

In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag–gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5′-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein–protein interactions.     

Specific binding of human immunodeficiency virus type 1 (HIV-1) Gag-derived proteins to a 5' HIV-1 genomic RNA sequence.

We developed an in vitro binding assay to study the specific interaction between human immunodeficiency virus type 1 (HIV-1) RNA and the Gag polyprotein. Binding of the in vitro-expressed protein to in vitro-transcribed RNA was determined by altered migration of the protein in polyacrylamide gels. We found that a Gag precursor lacking the matrix domain bound specifically to HIV-1 RNA, while deletion of both matrix and capsid domains diminished the specificity of binding. Among several regions of HIV-1 RNA tested, strongest binding was seen with the 5'-most 261 nucleotides, while antisense RNA from the same region did not bind.     

Thermodynamic examination of trinucleotide bulged RNA in the context of HIV-1 TAR RNA

RNA structures contain many bulges and loops that are expected to be sites for inter- and intra-molecular interactions. Nucleotides in the bulge are expected to influence the structure and recognition of RNA. The same stability is assigned to all trinucleotide bulged RNA in the current secondary structure prediction models. In this study thermal denaturation experiments were performed on four trinucleotide bulged RNA, in the context of HIV-1 TAR RNA, to determine whether the bulge sequence affects RNA stability and its divalent ion interactions. Cytosine-rich bulged RNA were more stable than uracil-rich bulged RNA in 1 M KCl. Interactions of divalent ions were more favorable with uracil-rich bulged RNA by ~2 kcal/mol over cytosine-rich bulged RNA. The UCU-TAR RNA (wild type) is stabilized by 1.7 kcal/mol in 9.5 mM Ca²⁺ as compared with 1 M KCl, whereas no additional gain in stability is measured for CCC-TAR RNA. These results have implications for base substitution experiments traditionally employed to identify metal ion binding sites. To our knowledge, this is the first systematic study to quantify the effect of small sequence changes on RNA stability upon interactions with divalent ions.     

Cooperative dimerization of a stably folded protein directed by a flexible RNA in the assembly of the HIV Rev dimer–RRE stem II complex

The binding of the HIV‐1 Rev protein as an oligomer to a viral RNA element, the Rev‐response element (RRE), mediates nuclear export of genomic RNA. Assembly of the Rev–RRE ribonucleoprotein (RNP) complex is nucleated by the binding of the first Rev molecule to stem IIB of the RRE. This is followed by stepwise addition of a total of ~six Rev molecules along the RRE through a combination of RNA–protein and protein–protein interactions. RRE stem II, which forms a three‐way junction consisting of stems IIA, IIB and IIC, has been shown to bind to two Rev molecules in a cooperative manner, with the second Rev molecule binding to the junction region of stem II. The results of base substitutions at the stem II junction, and characterization of stem II junction variants selected from a randomized library showed that an “open” flexible structure is preferred for binding of the second Rev molecule, and that binding of the second Rev molecule to the junction region is not sequence‐specific. Alanine substitutions of a number of Rev amino acid residues implicated to be important for Rev folding in previous structural studies were found to result in a dramatic decrease in the binding of the second Rev molecule. These results support the model that proper folding of Rev is critical in ensuring that the flexible RRE is able to correctly position Rev molecules for specific RNP assembly, and suggests that targeting Rev folding may be effective in the inhibition of Rev function. Copyright © 2015 John Wiley & Sons, Ltd.