Mouse oligoadenylate synthetase 1b inhibits West Nile virus replication by acting through the stem-loop 2 in the 3′-untranslated region

Dengue virus, a member of the family Flaviviridae, poses a serious public health threat worldwide. Dengue virus is a positive-sense RNA virus that harbors a genome of approximately 10.7 kb. Replication of dengue virus is mediated coordinately by cis-acting genomic sequences, viral proteins, and host cell factors. We have isolated and identified several host cell factors from baby hamster kidney cell extracts that bind with high specificity and high affinity to sequences within the untranslated regions of the dengue virus genome. Among the factors identified, Y box-binding protein-1 (YB-1) and the heterogeneous nuclear ribonucleoproteins (hnRNPs), hnRNP A1, hnRNP A2/B1, and hnRNP Q, bind to the dengue virus 3'-untranslated region. Further analysis indicated that YB-1 binds to the dengue virus 3' stem loop, a conserved structural feature located at the 3' terminus of the 3'-untranslated region of many flaviviruses. Analysis of the impact of YB-1 on replication of dengue virus in YB-1+/+ and YB-1-/- mouse embryo fibroblasts indicated that host YB-1 mediates an antiviral effect. Further studies demonstrated that this antiviral impact is due, at least in part, to a repressive role of YB-1 on dengue virus translation via a mechanism that requires viral genomic sequences. These results suggest a novel role for YB-1 as an antiviral host cell factor.

Effective siRNA targeting of the 3′ untranslated region of the West Nile virus genome

Functional non-coding RNAs derived from the flavivirus 3′ untranslated region

Over the past 7 years, West Nile zoonosis has been an emerging concern for public health in Europe, Middle East and more recently in North America. West Nile virus causes epidemic outbreaks in humans and infected patients may exhibit severe neurological symptoms. Because susceptibility and sensitivity to West Nile virus infections may depend on host genetic factors, a mouse model has been established to investigate the genetic determinism of host susceptibility to West Nile virus. A nonsense mutation in gene encoding the 1b isoform of the 2'-5'oligoadenylate synthetase (OAS1b) was constantly associated with the susceptibility of mouse strains to experimental West Nile virus infection. Oligoadenylate synthetase are interferon-inducible proteins playing a role in the endogeneous antiviral pathway. It was of interest to establish whether interferon-alpha and OAS 1B were sufficient to mediate resistance to West Nile virus infection. In the present study, we showed that interferon-alpha had the ability to modulate West Nile virus infection in mouse. In vitro, interferon-alpha protected mouse neuroblastoma cells against West Nile virus infection if cells have been pretreated with the cytokine for several hours. As a consequence of the presence of a stop codon, the Oas1b gene of the susceptible mice encodes a truncated and presumably inactive form, while resistant mice have a normal copy of the gene. Stable mouse neuroblastoma cell clones overexpressing mutant or wild-type OAS 1B were established. Replication of West Nile virus was less efficient in cells that produce the normal copy of OAS 1B as compared to those expressing the truncated form. Our data illustrate the notion that interferon-alpha and Oas genes may be critical for West Nile virus pathogenesis.

Until recently, West Nile (WN) virus and Kunjin (KUN) virus were classified as distinct virus types within the Flavivirus genus. However genetic and antigenic studies on isolates of these two viruses indicate that the relationship between them is more complex. To properly define the relationship between KUN and WN viruses, sequence analyses were performed on 32 isolates of KUN virus and 28 isolates of WN virus from different geographic areas, including a WN virus isolate from the recent outbreak in New York. Two independent 500 - 550 nucleotide regions of the genome were sequenced: (i) a region coding for a portion of the envelope protein surrounding the potential glycosylation site at amino acid 154; and (ii) a region encompassing the 3' end of the non-structural (NS) 5 gene and 5' end of the 3' untranslated region (UTR). Sequence comparisons from both regions revealed that the KUN virus isolates from Australia were tightly grouped whereas the WN virus isolates exhibited significant divergence and could be differentiated into three distinct groups. Some WN virus isolates shared greater sequence identity with Australian KUN virus isolates than with other WN virus isolates, while a KUN virus isolate from Sarawak, Malaysia, showed greatest sequence identity with WN virus strains. Virus isolates were also tested for antigenic variation using a panel of seven monoclonal antibodies (Mab) produced to either KUN or WN viruses. The binding patterns of these antibodies in ELISA demonstrated that KUN virus isolates from Australia were antigenically homologous and distinct from the WN virus isolates and the Malaysian KUN virus isolate. Similarly, all WN virus isolates except one displayed a distinct Mab binding profile. The remaining WN virus isolate (Sarafend), the Malaysian KUN virus isolate and Koutango virus also displayed distinct Mab binding patterns. The results in this thesis suggest that KUN and WN viruses comprise a group of closely related viruses that can be differentiated into a number of subgroups on the basis of genetic and antigenic analyses.During the course of this study, viral cultures were identified that contained both a KUN-like virus and a WN-like virus. The observation that the KUN virus population grew more efficiently in a mosquito cell line (C6/36) while the WN virus population replicated more effectively in mammalian cells (Vero) allowed enrichment for either virus by culturing the mixture in the appropriate cell line. A novel strategy was then designed to separate the two virus populations. Limit dilution of the enriched virus preparations was then performed in the appropriate cell line by infecting microtitre cultures with serial ten fold dilutions. Culture wells that contained a pure population of virus were then identified by removing and retaining the culture fluid from each well and immunostaining fixed cell monolayers with virus-specific Mabs. Subsequent passage of the 'cloned' viruses in either C6/36 or Vero cells and analysis of the infected cultures by specific Mab staining, PCR and nucleotide sequencing confirmed the identity of the virus and that in each case an homogenous virus population had been obtained. This procedure is particularly useful for isolating virus populations from heterogeneous mixtures that fail to develop discrete plaques in infected cell monolayers.

Dengue virus serotypes 1-4 are members of mosquito-borne flavivirus genus of Flaviviridae family that encode one long open reading frame (ORF) that is translated to a polyprotein. Both host and virally encoded proteases function in the processing of the polyprotein by co-translational and posttranslational mechanisms to yield 10 mature proteins prior to viral RNA replication. To study cis- and trans-acting factors involved in viral RNA replication, many groups [1-8] have constructed cDNAs encoding West Nile virus (WNV), DENV, or yellow fever virus reporter replicon RNAs. The replicon plasmids constructed in our laboratory for WNV [9] and the DENV4 replicon described here are arranged in the order of 5'-untranslated region (UTR), the N-terminal coding sequence of capsid (C), Renilla luciferase (Rluc) reporter gene with a translation termination codon, and an internal ribosome entry site (IRES) element from encephalomyocarditis virus (EMCV) for cap-independent translation of the downstream ORF that codes for a polyprotein precursor, CterE-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, followed by the 3'-UTR. In the second DENV4 replicon, the Rluc gene is fused sequentially downstream to the 20 amino acid (aa) FMDV 2A protease coding sequence, neomycin resistance gene (Neo(r)), a termination codon, and the EMCV leader followed by the same polyprotein coding sequence and 3'-UTR as in the first replicon. The first replicon is useful to study by transient transfection experiments the cis-acting elements and trans-acting factors involved in viral RNA replication. The second DENV4 replicon is used to establish a stable monkey kidney (Vero) cell line by transfection of replicon RNA and selection in the presence of the G418, an analog of neomycin. This replicon is useful for screening and identifying antiviral compounds that are potential inhibitors of viral replication.

The 3′untranslated region (UTR) in alphavirus genomes functions in virus replication and plays a role in determining virus host range. However, the molecular evolution of virus UTRs is understudied compared to the evolution of protein-coding regions. Chikungunya virus (CHIKV) has the longest 3′UTR among the alphaviruses (500–700 nt), and 3′UTR length and sequence structure vary substantially among different CHIKV lineages. Previous studies showed that genomic deletions and insertions are key drivers of CHIKV 3′UTR evolution. Inspired by hypothesized deletion events in the evolutionary history of CHIKV, we used experimental evolution to examine CHIKV adaptation in response to a large 3′UTR deletion. We engineered a CHIKV mutant with a 258 nt deletion in the 3′UTR (ΔDR1/2). This deletion reduced viral replication on mosquito cells, but did not reduce replication on mammalian cells. To examine how selective pressures from vertebrate and invertebrate hosts shape CHIKV evolution after a deletion in the 3′UTR, we passaged ΔDR1/2 virus populations strictly on primate cells, strictly on mosquito cells, or with alternating primate/mosquito cell passages. We found that virus populations passaged on a single host cell line increased in fitness relative to the ancestral deletion mutant on their selected host, and viruses that were alternately passaged improved on both hosts. Surprisingly, whole genome sequencing revealed few changes in the 3′UTR of passaged populations. Rather, virus populations evolved improved fitness through mutations in protein coding regions that were associated with specific hosts.

T cells from patients with systemic lupus erythematosus express decreased levels of the T cell receptor-associated CD3 zeta chain, a feature directly linked to their aberrant function. The decrease in CD3zeta protein expression is in part due to decreased levels of functional wild type isoform of the 3'-untranslated region (UTR) of CD3zeta mRNA with concomitant increased levels of an unstable alternatively spliced isoform. In order to identify factors involved in the post-transcriptional regulation of CD3zeta, we performed mass spectrometric analysis of Jurkat T cell nuclear proteins "pulled down" by a CD3zeta 3'-UTR oligonucleotide, which identified the splicing protein alternative splicing factor/splicing factor 2 (ASF/SF2). We show for the first time that ASF/SF2 binds specifically to the 3'-UTR of CD3zeta and regulates expression of CD3zeta protein by limiting the production of the alternatively spliced isoform. During activation of human T cells, an increase in the wild type CD3zeta mRNA is associated with increased expression of ASF/SF2. Finally, we show a significant correlation between ASF/SF2 and CD3zeta protein levels in T cells from systemic lupus erythematosus patients. Thus, our results identify ASF/SF2 as a novel factor in the regulation of alternative splicing of the 3'-UTR of CD3zeta and protein expression in human T cells.

OASL1 is a member of the 2’-5’-oligoadenylate synthetase (OAS) family and promotes viral clearance by activating RNase L. OASL1 interacts with the 5’-untranslated region (UTR) of interferon regulatory factor 7 (Irf7) and inhibits its translation. To identify the secondary structure required for OASL1 binding, we examined the 5’-UTR of the Irf7 transcript using “selective 2’-hydroxyl acylation analyzed by primer extension” (SHAPE). SHAPE takes advantage of the selective acylation of residues in single-stranded regions by 1-methyl-7-nitroisatoic anhydride (1M7). We found five major acylation sites located in, or next to, predicted single-stranded regions of the Irf7 5’-UTR. These results demonstrate the involvement of the stem structure of the Irf7 5’-UTR in the regulation of Irf7 translation, mediated by OASL1. [BMB Reports 2014; 47(10): 558-562]

The 5' and 3' untranslated regions (UTRs) of coxsackievirus B3 (CVB3) RNA form highly ordered secondary structures that have been confirmed to play important regulatory roles in viral cap-independent internal translation initiation and RNA replication. We previously demonstrated that deletions in different regions of the 5' UTR significantly reduced viral RNA translation and infectivity. Such observations suggested strongly that viral RNA translation and replication could be blocked if highly specific antisense oligodeoxynucleotides (AS-ODNs) were applied to target crucial sites within the 5' and 3' UTRs. In this study, seven phosphorothioate AS-ODNs were synthesized, and the antiviral activity was evaluated by Lipofectin transfection of HeLa cells with AS-ODNs followed by infection of CVB3. Analysis by Western blotting, reverse transcription-PCR, and viral plaque assay demonstrated that viral protein synthesis, genome replication, and infectivity of CVB3 were strongly inhibited by the AS-ODNs complementary to different regions of the 5' and 3' UTRs. The most effective sites are located at the proximate terminus of the 5' UTR (AS-1), the proximate terminus of the 3' UTR (AS-7), the core sequence of the internal ribosome entry site (AS-2), and the translation initiation codon region (AS-4). These AS-ODNs showed highly sequence-specific and dose-dependent inhibitory effects on both viral protein synthesis and RNA replication. It is noteworthy that the highest inhibitory activities were obtained with AS-1 and AS-7 targeting the termini of the 5' and 3' UTRs. The percent inhibition values of AS-1 and AS-7 for CVB3 protein VP1 synthesis and RNA replication were 70.6 and 79.6 for AS-1 and 73.7 and 79.7 for AS-7, respectively. These data suggest that CVB3 infectivity can be inhibited effectively by AS-ODNs.

HuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3' untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3' UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3' UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3' UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR. Hepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3' untranslated region (UTR) of HCV RNA. At the 3' UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unraveled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions.

Retroviruses have evolved multiple strategies to direct the synthesis of a complex proteome from a single primary transcript. Their mechanisms are modulated by a breadth of virus-host interactions, which are of significant fundamental interest because they ultimately affect the efficiency of virus replication and disease pathogenesis. Motifs located within the untranslated region (UTR) of the retroviral RNA have established roles in transcriptional trans-activation, RNA packaging, and genome reverse transcription; and a growing literature has revealed a necessary role of the UTR in modulating the efficiency of viral protein synthesis. Examples include a 5' UTR post-transcriptional control element (PCE), present in at least eight retroviruses, that interacts with cellular RNA helicase A to facilitate cap-dependent polyribosome association; and 3' UTR constitutive transport element (CTE) of Mason-Pfizer monkey virus that interacts with Tap/NXF1 and SR protein 9G8 to facilitate RNA export and translational utilization. By contrast, nuclear protein hnRNP E1 negatively modulates HIV-1 Gag, Env, and Rev protein synthesis. Alternative initiation strategies by ribosomal frameshifting and leaky scanning enable polycistronic translation of the cap-dependent viral transcript. Other studies posit cap-independent translation initiation by internal ribosome entry at structural features of the 5' UTR of selected retroviruses. The retroviral armamentarium also commands mechanisms to counter cellular post-transcriptional innate defenses, including protein kinase R, 2',5'-oligoadenylate synthetase and the small RNA pathway. This review will discuss recent and historically-recognized insights into retrovirus translational control. The expanding knowledge of retroviral post-transcriptional control is vital to understanding the biology of the retroviral proteome. In a broad perspective, each new insight offers a prospective target for antiviral therapy and strategic improvement of gene transfer vectors.

Usutu virus (USUV) and West Nile virus (WNV) are two closely related emerging mosquito-borne flaviviruses. Their natural hosts are wild birds, but they can also cause severe neurological disorders in humans. Both viruses are efficiently suppressed by type I interferon (IFN), which interferes with viral replication, dissemination, pathogenesis and transmission. Here, we show that the replication of USUV and WNV are inhibited through a common set of IFN–induced genes (ISGs), with the notable exception of ISG20, which USUV is resistant to. Strikingly, USUV was the only virus among all the other tested mosquito-borne flaviviruses that demonstrated resistance to the 3′–5′ exonuclease activity of ISG20. Our findings highlight that the intrinsic resistance of the USUV genome, irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a unique sequence present in its 3′ untranslated region. Importantly, this genomic region alone can confer ISG20 resistance to a susceptible flavivirus, without compromising its infectivity, suggesting that it could be acquired by other flaviviruses. This study provides new insights into the strategy employed by emerging flaviviruses to overcome host defense mechanisms.

The hepatitis C virus (HCV) 3'-untranslated region (UTR) and negative-strand RNA sequences contribute cis-acting functions essential to viral RNA replication. Although efficient suppression of HCV replicon RNA in cell culture has been demonstrated with small interfering RNAs (siRNAs) directed against various sequences in the 5' UTR and coding regions, data regarding siRNA targeting of the 3' UTR have been lacking. Furthermore, it has not been definitively shown whether the active constructs, identified to date, exert their effect exclusively via suppression of the replicon positive strand, negative strand or some combination of both strands. In the present study, we assayed inhibitory activity of various siRNAs targeting the 3' UTR by transient transfection in a subgenomic replicon cell culture model. A survey of 13 candidate target sites in the 3'-UTR X sequence indicated a uniformly low activity of siRNA constructs against the steady-state level of replicon. In contrast, the majority of these same siRNAs exhibited high activity against HCV X sequences of either polarity when these targets were presented in the context of a mammalian polymerase II mRNA transcript. Transfection of siRNAs directed against other regions of the replicon revealed differences in the magnitude of inhibitory effects against positive-strand and negative-strand target sites. Strand preference of siRNA activity was further demonstrated through the introduction of base-pair-destabilizing mutations that promote strand-specific targeting. The results suggest that the HCV positive-strand 5' UTR and coding region are efficiently and directly targeted by siRNA, whereas the 3' UTR and the entire negative strand are relatively resistant to RNA interference.

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-strand RNA virus. The viral genome is capped at the 5′ end, followed by an untranslated region (UTR). There is a poly(A) tail at the 3′ end, preceded by a UTR. The self-interaction between the RNA regulatory elements present within the 5′ and 3′ UTRs and their interaction with host/virus-encoded proteins mediate the function of the 5′ and 3′ UTRs. Using an RNA-protein interaction detection (RaPID) assay coupled to liquid chromatography with tandem mass spectrometry, we identified host interaction partners of SARS-CoV-2 5′ and 3′ UTRs and generated an RNA-protein interaction network. By combining these data with the previously known protein-protein interaction data proposed to be involved in virus replication, we generated the RNA-protein-protein interaction (RPPI) network, likely to be essential for controlling SARS-CoV-2 replication. Notably, bioinformatics analysis of the RPPI network revealed the enrichment of factors involved in translation initiation and RNA metabolism. Lysosome-associated membrane protein-2a (Lamp2a), the receptor for chaperone-mediated autophagy, is one of the host proteins that interact with the 5′ UTR. Further studies showed that the Lamp2 level is upregulated in SARS-CoV-2-infected cells and that the absence of the Lamp2a isoform enhanced the viral RNA level whereas its overexpression significantly reduced the viral RNA level. Lamp2a and viral RNA colocalize in the infected cells, and there is an increased autophagic flux in infected cells, although there is no change in the formation of autophagolysosomes. In summary, our study provides a useful resource of SARS-CoV-2 5′ and 3′ UTR binding proteins and reveals the role of Lamp2a protein during SARS-CoV-2 infection.IMPORTANCE Replication of a positive-strand RNA virus involves an RNA-protein complex consisting of viral genomic RNA, host RNA(s), virus-encoded proteins, and host proteins. Dissecting out individual components of the replication complex will help decode the mechanism of viral replication. 5′ and 3′ UTRs in positive-strand RNA viruses play essential regulatory roles in virus replication. Here, we identified the host proteins that associate with the UTRs of SARS-CoV-2, combined those data with the previously known protein-protein interaction data (expected to be involved in virus replication), and generated the RNA-protein-protein interaction (RPPI) network. Analysis of the RPPI network revealed the enrichment of factors involved in translation initiation and RNA metabolism, which are important for virus replication. Analysis of one of the interaction partners of the 5′-UTR (Lamp2a) demonstrated its role in reducing the viral RNA level in SARS-CoV-2-infected cells. Collectively, our study provides a resource of SARS-CoV-2 UTR-binding proteins and identifies an important role for host Lamp2a protein during viral infection.