The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA.

The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3'UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3'UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A-eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5'-3' end interaction, mediated by both viral and cellular proteins.

The internal ribosome entry site of the Dengue virus mRNA is active when cap-dependent translation initiation is inhibited.

Dengue virus (DENV) is an enveloped, positive-sense, single-stranded RNA virus belonging to the Flaviviridae family. Translation initiation of the DENV mRNA can occur following a cap-dependent or a cap-independent mechanism. Two non-mutually exclusive cap-independent mechanisms of translation initiation have been described for the DENV mRNA. The first corresponds to a 5'end-dependent internal ribosome entry site (IRES)-independent mechanism, while the second relies on IRES-dependent initiation. In this report, we study the recently discovered DENV IRES. Results show that the DENV IRES is functional in the rabbit reticulocyte (RRL) in vitro translation system. In accordance, the activity of DENV IRES was resistant to the cleavage of eIF4G by the Foot-and-mouth disease virus leader protease in RRL. In cells, the DENV IRES exhibited only a marginal activity under standard culture conditions. The DENV IRES showed weak activity in HEK 293T cells; however, the DENV IRES activity was significantly enhanced in HEK 293T cells expressing the Human rhinovirus 2A protease. These findings suggest that the DENV IRES enables viral protein synthesis under conditions that suppress canonical translation initiation.IMPORTANCE Dengue virus (DENV), the etiological agent of Dengue, a febrile and hemorrhagic disease, infects millions of people per year in tropical and subtropical countries. When infecting cells, DENV induces stress conditions known to inhibit canonical protein synthesis. Under these conditions, DENV mRNA thrives using non-canonical modes of translation initiation. In this study, we characterize the mechanism dependent upon an internal ribosome entry site (IRES). Herein, we describe the activity of the DENV IRES in vitro and cells. We show that in cells, DENV IRES enables the viral mRNA to translate under conditions that suppress canonical translation initiation.

Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation.

The full-length mRNAs of the human immunodeficiency virus type-1 (HIV-1), the human T-cell lymphotropic virus type-1 (HTLV-1), and the mouse mammary tumor virus (MMTV) harbor IRESs. The activity of the retroviral-IRESs requires IRES-transacting factors (ITAFs), being hnRNP A1, a known ITAF for the HIV-1 IRES. In this study, we show that hnRNP A1 is also an ITAF for the HTLV-1 and MMTV IRESs. The MMTV IRES proved to be more responsive to hnRNP A1 than either the HTLV-1 or the HIV-1 IRESs. The impact of post-translational modifications of hnRNP A1 on HIV-1, HTLV-1 and MMTV IRES activity was also assessed. Results show that the HIV-1 and HTLV-1 IRESs were equally responsive to hnRNP A1 and its phosphorylation mutants S4A/S6A, S4D/S6D and S199A/D. However, the S4D/S6D mutant stimulated the activity from the MMTV-IRES to levels significantly higher than the wild type hnRNP A1. PRMT5-induced symmetrical di-methylation of arginine residues of hnRNP A1 enabled the ITAF to stimulate the HIV-1 and HTLV-1 IRESs while reducing the stimulatory ability of the ITAF over the MMTV IRES. We conclude that retroviral IRES activity is not only dependent on the recruited ITAFs but also relies on how these proteins are modified at the post-translational level.

Mother-to-Child Transmission of Andes Virus through Breast Milk, Chile1.

Andes virus (ANDV) is the only hantavirus transmitted between humans through close contact. We detected the genome and proteins of ANDV in breast milk cells from an infected mother in Chile who transmitted the virus to her child, suggesting gastrointestinal infection through breast milk as a route of ANDV person-to-person transmission.

Cap-independent translation initiation of the unspliced RNA of retroviruses.

Retroviruses are a unique family of RNA viruses that utilize a virally encoded reverse transcriptase (RT) to replicate their genomic RNA (gRNA) through a proviral DNA intermediate. The provirus is permanently integrated into the host cell chromosome and is expressed by the host cell transcription, RNA processing, and translation machinery. Retroviral messenger RNAs (mRNAs) entirely resemble a cellular mRNA as they have a 5'cap structure, 5'untranslated region (UTR), an open reading frame (ORF), 3'UTR, and a 3'poly(A) tail. The primary transcription product interacts with the cellular RNA processing machinery and is spliced, exported to the cytoplasm, and translated. However, a proportion of the pre-mRNA subverts typical RNA processing giving rise to the full-length RNA. In the cytoplasm, the full-length retroviral RNA fulfills a dual role acting as mRNA and as the gRNA. Simple retroviruses generate two pools of full-length RNA, one for each purpose. However, complex retroviruses have a single pool of full-length RNA, which is destined for translation or encapsidation. As for eukaryotic mRNAs, translational control of retroviral protein synthesis is mostly exerted at the step of initiation. Interestingly, some retroviral mRNAs, both simple and complex, use a dual mechanism to initiate protein synthesis, a cap-dependent initiation mechanism, or via internal initiation using an internal ribosome entry site (IRES). In this review, we describe and discuss data regarding the molecular mechanism driving the canonical cap-dependent and IRES-mediated translation initiation for retroviral mRNA, focusing the discussion mainly on the most studied retroviral mRNA, the HIV-1 mRNA.

The Andes Orthohantavirus NSs Protein Antagonizes the Type I Interferon Response by Inhibiting MAVS Signaling.

The small messenger RNA (SmRNA) of the Andes orthohantavirus (ANDV), a rodent-borne member of the Hantaviridae family of viruses of the Bunyavirales order, encodes a multifunctional nucleocapsid (N) protein and for a nonstructural (NSs) protein of unknown function. We have previously shown the expression of the ANDV-NSs, but only in infected cell cultures. In this study, we extend our early findings by confirming the expression of the ANDV-NSs protein in the lungs of experimentally infected golden Syrian hamsters. Next, we show, using a virus-free system, that the ANDV-NSs protein antagonizes the type I interferon (IFN) induction pathway by suppressing signals downstream of the melanoma differentiation-associated protein 5 (MDA5) and the retinoic acid-inducible gene 1 (RIG-I) and upstream of TBK1. Consistent with this observation, the ANDV-NSs protein antagonized mitochondrial antiviral-signaling protein (MAVS)-induced IFN-ß, NF-κB, IFN-regulatory factor 3 (IRF3), and IFN-sensitive response element (ISRE) promoter activity. Results demonstrate that ANDV-NSs binds to MAVS in cells without disrupting the MAVS-TBK-1 interaction. However, in the presence of the ANDV-NSs ubiquitination of MAVS is reduced. In summary, this study provides evidence showing that the ANDV-NSs protein acts as an antagonist of the cellular innate immune system by suppressing MAVS downstream signaling by a yet not fully understand mechanism. Our findings reveal new insights into the molecular regulation of the hosts' innate immune response by the Andes orthohantavirus.IMPORTANCEAndes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is distinguished from other hantaviruses by its unique ability to spread from person to person. In a previous report, we identified a novel ANDV protein, ANDV-NSs. Until now, ANDV-NSs had no known function. In this new study, we established that ANDV-NSs acts as an antagonist of cellular innate immunity, the first line of defense against invading pathogens, hindering the cellular antiviral response during infection. This study provides novel insights into the mechanisms used by ANDV to establish its infection.

Targeting deoxyhypusine hydroxylase activity impairs cap-independent translation initiation driven by the 5'untranslated region of the HIV-1, HTLV-1, and MMTV mRNAs.

Replication of the human immunodeficiency virus type 1 (HIV-1) is dependent on eIF5A hypusination. Hypusine is formed post-translationally on the eIF5A precursor by two consecutive enzymatic steps; a reversible reaction involving the enzyme deoxyhypusine synthase (DHS) and an irreversible step involving the enzyme deoxyhypusine hydroxylase (DOHH). In this study we explored the effect of inhibiting DOHH activity and therefore eIF5A hypusination, on HIV-1 gene expression. Results show that the expression of proteins from an HIV-1 molecular clone is reduced when DOHH activity is inhibited by Deferiprone (DFP) or Ciclopirox (CPX). Next we evaluated the requirement of DOHH activity for internal ribosome entry site (IRES)-mediated translation initiation driven by the 5'untranslated region (5'UTR) of the full length HIV-1 mRNA. Results show that HIV-1 IRES activity relies on DOHH protein concentration and enzymatic activity. Similar results were obtained for IRES-dependent translation initiation mediated by 5'UTR of the human T-cell lymphotropic virus type 1 (HTLV-1) and the mouse mammary tumor virus (MMTV) mRNAs. Interestingly, activity of the poliovirus IRES, was less sensitive to the targeting of DOHH suggesting that not all viral IRESs are equally dependent on the cellular concentration or the activity of DOHH. In summary we present evidence indicating that the cellular concentration of DOHH and its enzymatic activity play a role in HIV-1, HTLV-1 and MMTV IRES-mediated translation initiation.

Who Regulates Whom? An Overview of RNA Granules and Viral Infections.

After viral infection, host cells respond by mounting an anti-viral stress response in order to create a hostile atmosphere for viral replication, leading to the shut-off of mRNA translation (protein synthesis) and the assembly of RNA granules. Two of these RNA granules have been well characterized in yeast and mammalian cells, stress granules (SGs), which are translationally silent sites of RNA triage and processing bodies (PBs), which are involved in mRNA degradation. This review discusses the role of these RNA granules in the evasion of anti-viral stress responses through virus-induced remodeling of cellular ribonucleoproteins (RNPs).

Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci.

LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants.

Polypyrimidine tract-binding protein binds to the 5' untranslated region of the mouse mammary tumor virus mRNA and stimulates cap-independent translation initiation.

The 5' untranslated region (UTR) of the full-length mRNA of the mouse mammary tumor virus (MMTV) harbors an internal ribosomal entry site (IRES). In this study, we show that the polypyrimidine tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the MMTV 5' UTR stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Results show that PTB1 and PTB4, but not PTB2, stimulate MMTV-IRES activity. PTB1 promotes MMTV-IRES-mediated initiation more strongly than PTB4. When expressed in combination, PTB1 further enhanced PTB4 stimulation of the MMTV-IRES, while PTB2 fully abrogates PTB4-induced stimulation. PTB1-induced stimulation of MMTV-IRES was not altered in the presence of PTB4 or PTB2. Mutational analysis reveals that stimulation of MMTV-IRES activity is abrogated when PTB1 is mutated either in RRM1/RRM2 or RRM3/RRM4. In contrast, a PTB4 RRM1/RRM2 mutant has reduced effect over MMTV-IRES activity, while stimulation of the MMTV-IRES activity is still observed when the PTB4 RRM3/RMM4 mutant is used. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity. In contrast, PTB2 acts as a negative modulator of PTB4-induced stimulation of MMTV-IRES. We conclude that PTB1 and PTB4 act as IRES trans-acting factors of the MMTV-IRES.

RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila.

How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.

The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

The 3' untranslated region of the Andes hantavirus small mRNA functionally replaces the poly(A) tail and stimulates cap-dependent translation initiation from the viral mRNA.

In the process of translation of eukaryotic mRNAs, the 5' cap and the 3' poly(A) tail interact synergistically to stimulate protein synthesis. Unlike its cellular counterparts, the small mRNA (SmRNA) of Andes hantavirus (ANDV), a member of the Bunyaviridae, lacks a 3' poly(A) tail. Here we report that the 3' untranslated region (3'UTR) of the ANDV SmRNA functionally replaces a poly(A) tail and synergistically stimulates cap-dependent translation initiation from the viral mRNA. Stimulation of translation by the 3'UTR of the ANDV SmRNA was found to be independent of viral proteins and of host poly(A)-binding protein.

Translation initiation of viral mRNAs.

Viruses depend on cells for their replication but have evolved mechanisms to achieve this in an efficient and, in some instances, a cell-type-specific manner. The expression of viral proteins is frequently subject to translational control. The dominant target of such control is the initiation step of protein synthesis. Indeed, during the early stages of infection, viral mRNAs must compete with their host counterparts for the protein synthetic machinery, especially for the limited pool of eukaryotic translation initiation factors (eIFs) that mediate the recruitment of ribosomes to both viral and cellular mRNAs. To circumvent this competition viruses use diverse strategies so that ribosomes can be recruited selectively to viral mRNAs. In this review we focus on the initiation of protein synthesis and outline some of the strategies used by viruses to ensure efficient translation initiation of their mRNAs.

Is single-strand conformation polymorphism analysis of the full 5' untranslated region an adequate approach to study hepatitis C virus quasispecies distribution?

Single-strand conformation polymorphism (SSCP) analysis is used by many laboratories to study the quasispecies distribution of the hepatitis C virus (HCV). Here we question the validity of this experimental approach, as conclusions are drawn from the analysis of the migration patterns of two ssDNA molecules and not from RNA. Using previously characterized mutants of the HCV 5' untranslated regions, we show that contrary to what has been predicted, SSCP migration patterns of DNA amplicons with differences in their nucleotide sequences generated from the full 5' UTR of HCV are not necessarily unique.

Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation.

The HCV internal ribosome entry site (IRES) spans a region of approximately 340 nt that encompasses most of the 5' untranslated region (5'UTR) of the viral mRNA and the first 24-40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5'UTR on IRES activity, naturally occurring variants of the 5'UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg(2+) ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation.

Influence of extrahepatic viral infection on the natural history of hepatitis C.

HCV is primarily hepatotropic, but there is mounting evidence pointing to infection and replication of extrahepatic sites. Here we evaluated the occurrence of HCV infection of peripheral blood mononuclear cells (PBMC) and explored the possible association between viral extrahepatic infection and the natural history of the disease. Forty seven Chilean, HCV infected, treatment naïve patients were included in the study. HCV RNA was isolated from plasma and PBMC and subsequently reverse transcribed, amplified and sequenced. Most patients harbored HCV 1b genotype and the most common route of infection showed to be blood transfusion. HCV RNA was readily detected in PBMCs of 34 out of the 47 patients (72%). We report that HCV sequences found in PBMC differ from those in plasma of the same subjects strongly suggesting HCV compartmentalization. In addition, we found that patients with detectable HCV RNA in PBMC had a tendency for being more likely cirrhotic [OR 3.8 (95% CI: 0.98 to 14)]. In conclusion, this study provides further arguments for the existence of HCV infection of extrahepatic sites and suggests that extrahepatic infection could be a factor influencing the natural history of the disease.