Translational control of papillomavirus mRNAs in the spotlight.

High-risk human papillomaviruses (HPVs) cause most cases of cervical cancer, a disease with an increasing impact worldwide. Recent studies have shown that the synthesis of viral oncoproteins is strongly subject to translational control. Thus, targeting the protein synthesis machinery might open novel avenues to develop innovative therapies aiming to improve patients' survival.

Molecular basis of RNA recombination in the 3'UTR of chikungunya virus genome.

Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication.

Zika virus non-coding RNAs antagonize antiviral responses by PKR-mediated translational arrest.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that causes severe outbreaks in human populations. ZIKV infection leads to the accumulation of small non-coding viral RNAs (known as sfRNAs) that are crucial for evasion of antiviral responses and for viral pathogenesis. However, the mechanistic understanding of how sfRNAs function remains incomplete. Here, we use recombinant ZIKVs and ribosome profiling of infected human cells to show that sfRNAs block translation of antiviral genes. Mechanistically, we demonstrate that specific RNA structures present in sfRNAs trigger PKR activation, which instead of limiting viral replication, enhances viral particle production. Although ZIKV infection induces mRNA expression of antiviral genes, translation efficiency of type I interferon and interferon stimulated genes were significantly downregulated by PKR activation. Our results reveal a novel viral adaptation mechanism mediated by sfRNAs, where ZIKV increases its fitness by repurposing the antiviral role of PKR into a proviral factor.

Toward a Categorization of Virus-ncRNA Interactions in the World of RNA to Disentangle the Tiny Secrets of Dengue Virus.

In recent years, the function of noncoding RNAs (ncRNAs) as regulatory molecules of cell physiology has begun to be better understood. Advances in viral molecular biology have shown that host ncRNAs, cellular factors, and virus-derived ncRNAs and their interplay are strongly disturbed during viral infections. Nevertheless, the folding of RNA virus genomes has also been identified as a critical factor in regulating canonical and non-canonical functions. Due to the influence of host ncRNAs and the structure of RNA viral genomes, complex molecular and cellular processes in infections are modulated. We propose three main categories to organize the current information about RNA-RNA interactions in some well-known human viruses. The first category shows examples of host ncRNAs associated with the immune response triggered in viral infections. Even though miRNAs introduce a standpoint, they are briefly presented to keep researchers moving forward in uncovering other RNAs. The second category outlines interactions between virus-host ncRNAs, while the third describes how the structure of the RNA viral genome serves as a scaffold for processing virus-derived RNAs. Our grouping may provide a comprehensive framework to classify ncRNA-host-cell interactions for emerging viruses and diseases. In this sense, we introduced them to organize DENV-host-cell interactions.

Assembly of the Tripartite and RNA Condensates of the Respiratory Syncytial Virus Factory Proteins In Vitro: Role of the Transcription Antiterminator M2-1.

A wide variety of viruses replicate in liquid-like viral factories. Non-segmented negative stranded RNA viruses share a nucleoprotein (N) and a phosphoprotein (P) that together emerge as the main drivers of liquid-liquid phase separation. The respiratory syncytial virus includes the transcription antiterminator M2-1, which binds RNA and maximizes RNA transcriptase processivity. We recapitulate the assembly mechanism of condensates of the three proteins and the role played by RNA. M2-1 displays a strong propensity for condensation by itself and with RNA through the formation of electrostatically driven protein-RNA coacervates based on the amphiphilic behavior of M2-1 and finely tuned by stoichiometry. M2-1 incorporates into tripartite condensates with N and P, modulating their size through an interplay with P, where M2-1 is both client and modulator. RNA is incorporated into the tripartite condensates adopting a heterogeneous distribution, reminiscent of the M2-1-RNA IBAG granules within the viral factories. Ionic strength dependence indicates that M2-1 behaves differently in the protein phase as opposed to the protein-RNA phase, in line with the subcompartmentalization observed in viral factories. This work dissects the biochemical grounds for the formation and fate of the RSV condensates in vitro and provides clues to interrogate the mechanism under the highly complex infection context.

Metabolism of NK cells during viral infections.

Cellular metabolism is essential for the correct function of immune system cells, including Natural Killer cells (NK). These cells depend on energy to carry out their effector functions, especially in the early stages of viral infection. NK cells participate in the innate immune response against viruses and tumors. Their main functions are cytotoxicity and cytokine production. Metabolic changes can impact intracellular signals, molecule production, secretion, and cell activation which is essential as the first line of immune defense. Metabolic variations in different immune cells in response to a tumor or pathogen infection have been described; however, little is known about NK cell metabolism in the context of viral infection. This review summarizes the activation-specific metabolic changes in NK cells, the immunometabolism of NK cells during early, late, and chronic antiviral responses, and the metabolic alterations in NK cells in SARS-CoV2 infection. The modulation points of these metabolic routes are also discussed to explore potential new immunotherapies against viral infections.

Detection of high antibodies titers against rat leukemia virus in an outbreak of reproductive disorders and lymphomas in Wistar rats.

Young female Wistar rats from a specific pathogen free breeding colony presented an outbreak of infertility along with neurological symptoms and malignant lymphomas. We evaluated the presence and the potential role of the rat leukemia virus (RaLV) in the disease because these clinical signs could be compatible with a retrovirus. RaLV is a mammalian type C endogenous retrovirus initially isolated from in vitro Sprague-Dawley rat embryo cultures. There are no reports of clinical disease in rats associated with this virus, and little is known about its interaction with the host. Using reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, we studied the synthesis of the viral particles and the development of an immune response against the virus in this rat colony. The results showed that healthy and diseased Wistar rats synthetized viral RNA but only diseased animals developed a detectable immune response against RaLV envelop protein. Furthermore, rats with lymphomas tended to have higher titers of antibodies against RaLV epitopes than those with infertility or neurological symptoms. The results suggest that increases in the RaLV infectious particle loads could be involved in the development of lymphomas in young rats. The potential causes of RaLV reactivation are discussed.

The RNA-dependent RNA polymerase of the infectious pancreatic necrosis virus is linked to viral mRNA acting as a cap substitute.

The infectious pancreatic necrosis virus (IPNV) is responsible for significant economic losses in the aquaculture industry. It is an unenveloped virus with an icosahedral capsid. Its viral genome comprises two dsRNA segments, A and B. Segment A contains a small ORF, which encodes VP5, and a large ORF, which encodes a polyprotein that generates the structural proteins and the viral protease. Segment B encodes the RNA-dependent RNA polymerase (RdRp), called VP1 in this free form, or Vpg when it covalently attaches to the viral RNA. The viral genome does not have cap or poly(A). Instead, each 5' end is linked to the Vpg. Recently, we demonstrated that mRNA-A contains an internal ribosome entry site (IRES) to command polyprotein synthesis. However, the presence of Vpg on IPNV mRNAs and its impact on cellular translation has not been investigated. This research demonstrates that IPNV mRNAs are linked to Vpg and that this protein inhibits cap-dependent translation on infected cells. Also, it is demonstrated that Vpg interacts with eIF4E and that rapamycin treatment partially diminishes the viral protein synthesis. In addition, we determined that an IRES does not command translation of IPNV mRNA-B. We show that VPg serves as a cap substitute during the initiation of IPNV translation, contributing to understanding the replicative cycle of Birnaviruses. Our results indicate that the viral protein VP1/Vpg is multifunctional, having a significant role during IPNV RNA synthesis as the RdRp and the primer for IPNV RNA synthesis and translation as the viral protein genome, acting as a cap substitute.

In Vivo Visualization of Mobile mRNA Particles in Plants Using BglG.

Cells have developed mechanisms for cytoplasmic RNA transport and localization that participate in the regulation and subcellular localization of protein synthesis. In addition, plants can exchange RNA molecules between cells through plasmodesmata and to distant tissues in the phloem. These mechanisms are hijacked by RNA viruses to establish their replication complexes and to disseminate their genomes throughout the plant organism with the help of virus-encoded movement proteins (MP). Live imaging of RNA molecules is a fundamental approach to understand the regulation and molecular basis of these processes. The most widely used experimental systems for the in vivo visualization of genetically encoded RNA molecules are based on fluorescently tagged RNA binding proteins that bind to specific motifs inserted into the RNA, thus allowing the tracking of the specific RNA molecule by fluorescent microscopy. Recently, we developed the use of the E. coli RNA binding protein BglG for the imaging of RNAs tagged with BglG-binding sites in planta. We describe here the detailed method by which we use this in vivo RNA tagging system for the real-time imaging of Tobacco mosaic virus (TMV) MP mRNA.

RNA-Binding Proteins as Regulators of Internal Initiation of Viral mRNA Translation.

Viruses are obligate intracellular parasites that depend on the host's protein synthesis machinery for translating their mRNAs. The viral mRNA (vRNA) competes with the host mRNA to recruit the translational machinery, including ribosomes, tRNAs, and the limited eukaryotic translation initiation factor (eIFs) pool. Many viruses utilize non-canonical strategies such as targeting host eIFs and RNA elements known as internal ribosome entry sites (IRESs) to reprogram cellular gene expression, ensuring preferential translation of vRNAs. In this review, we discuss vRNA IRES-mediated translation initiation, highlighting the role of RNA-binding proteins (RBPs), other than the canonical translation initiation factors, in regulating their activity.

Effectiveness of face masks in blocking the transmission of SARS-CoV-2: A preliminary evaluation of masks used by SARS-CoV-2-infected individuals.

In 2019, a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is transmitted via the airborne route, caused a new pandemic namely, "coronavirus disease 2019" (COVID-19). Although the effectiveness of face masks to prevent the transmission of SARS-CoV-2 is debated, no study has evaluated the virus-blocking efficacy of masks used by patients. We aimed to evaluate this efficacy of masks used by SARS-CoV-2-infected individuals. Data, masks used, and nasopharyngeal swab samples were obtained from these patients. Forty-five paired samples of nasopharyngeal swabs and masks were obtained and processed; the majority of masks were woven. Viral RNAs were amplified using quantitative reverse-transcription polymerase chain reaction and detected only on the inner parts of masks. Median viral load (VL) values of swabs and masks were 1.954x106 and 2,51x103, respectively. Statistically, there was a difference of approximately 1000 RNA copies/mL between swabs and masks and no significant difference in VL values among different types of masks. There were statistically significant differences in VL values between men and women and between symptomatic and asymptomatic patients. Our findings suggest the blocking of virus transmission by different types of masks and reinforce the use of masks by both infected and non-infected individuals.

Epitranscriptomic regulation of HIV-1 full-length RNA packaging.

During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that deposition of N6-methyladenosine (m6A) regulates full-length RNA packaging. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promoted the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and contributes to full-length RNA demethylation. We further identified two highly conserved adenosines within the 5'-UTR that have a crucial functional role in m6A methylation and packaging of the full-length RNA. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the HIV-1 full-length RNA molecules that will be used as viral genomes.

Polypyrimidine-Tract-Binding Protein Isoforms Differentially Regulate the Hepatitis C Virus Internal Ribosome Entry Site.

Translation initiation of the hepatitis C virus (HCV) mRNA depends on an internal ribosome entry site (IRES) that encompasses most of the 5'UTR and includes nucleotides of the core coding region. This study shows that the polypyrimidine-tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the HCV 5'UTR, stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Our results show that PTB1 and PTB4, but not PTB2, stimulate HCV IRES activity in HuH-7 and HEK293T cells. In HuH-7 cells, PTB1 promotes HCV IRES-mediated initiation more strongly than PTB4. Mutations in PTB1, PTB4, RRM1/RRM2, or RRM3/RRM4, which disrupt the RRM's ability to bind RNA, abrogated the protein's capacity to stimulate HCV IRES activity in HuH-7 cells. In HEK293T cells, PTB1 and PTB4 stimulate HCV IRES activity to similar levels. In HEK293T cells, mutations in RRM1/RRM2 did not impact PTB1's ability to promote HCV IRES activity; and mutations in PTB1 RRM3/RRM4 domains reduced, but did not abolish, the protein's capacity to stimulate HCV IRES activity. In HEK293T cells, mutations in PTB4 RRM1/RRM2 abrogated the protein's ability to promote HCV IRES activity, and mutations in RRM3/RRM4 have no impact on PTB4 ability to enhance HCV IRES activity. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity in a cell type-specific manner. We conclude that PTB1 and PTB4, but not PTB2, act as IRES transacting factors of the HCV IRES.

The double-stranded RNA-binding protein, Staufen1, is an IRES-transacting factor regulating HIV-1 cap-independent translation initiation.

Translation initiation of the viral genomic mRNA (vRNA) of human immunodeficiency virus-type 1 (HIV-1) can be mediated by a cap- or an internal ribosome entry site (IRES)-dependent mechanism. A previous report shows that Staufen1, a cellular double-stranded (ds) RNA-binding protein (RBP), binds to the 5'untranslated region (5'UTR) of the HIV-1 vRNA and promotes its cap-dependent translation. In this study, we now evaluate the role of Staufen1 as an HIV-1 IRES-transacting factor (ITAF). We first confirm that Staufen1 associates with both the HIV-1 vRNA and the Gag protein during HIV-1 replication. We found that in HIV-1-expressing cells, siRNA-mediated depletion of Staufen1 reduces HIV-1 vRNA translation. Using dual-luciferase bicistronic mRNAs, we show that the siRNA-mediated depletion and cDNA-mediated overexpression of Staufen1 acutely regulates HIV-1 IRES activity. Furthermore, we show that Staufen1-vRNA interaction is required for the enhancement of HIV-1 IRES activity. Interestingly, we find that only Staufen1 harboring an intact dsRNA-binding domain 3 (dsRBD3) rescues HIV-1 IRES activity in Staufen1 CRISPR-Cas9 gene edited cells. Finally, we show that the expression of Staufen1-dsRBD3 alone enhances HIV-1 IRES activity. This study provides evidence of a novel role for Staufen1 as an ITAF promoting HIV-1 vRNA IRES activity.

Direct RNA Sequencing Reveals SARS-CoV-2 m6A Sites and Possible Differential DRACH Motif Methylation among Variants.

The causative agent of COVID-19 pandemic, SARS-CoV-2, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 150 modified bases that are essential for proper function. Among internal modified bases, the N6-methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced thus far are, in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because the incorporation of dNTPs hides RNA base modifications. Here, we present an initial exploration of Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3'-untranslated region. We identified fifteen m6A methylated positions, of which, six are in ORF N. Additionally, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants, we show that variants Beta and Eta have a fourth position C > U change in DRACH at 28,884b that could affect methylation. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.

Portable and Label-Free Quantitative Loop-Mediated Isothermal Amplification (LF-qLamp) for Reliable COVID-19 Diagnostics in Three Minutes of Reaction Time: Arduino-Based Detection System Assisted by a pH Microelectrode.

Loop-mediated isothermal amplification (LAMP) has been recently studied as an alternative method for cost-effective diagnostics in the context of the current COVID-19 pandemic. Recent reports document that LAMP-based diagnostic methods have a comparable sensitivity and specificity to that of RT-qPCR. We report the use of a portable Arduino-based LAMP-based amplification system assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 min of the amplification reaction. We show that this simple system enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 µL of reaction mix. We also spiked saliva samples with SARS-CoV-2 synthetic material and corroborated that the LAMP reaction can be successfully monitored in real time using microelectrodes in saliva samples as well. These results may have profound implications for the design of real-time and portable quantitative systems for the reliable detection of viral pathogens including SARS-CoV-2.

On the Weak Binding and Spectroscopic Signature of SARS-CoV-2 nsp14 Interaction with RNA.

The SARS-CoV-2 non-structural protein 14 (nsp14), known as exoribonuclease is encoded from the large polyprotein of viral genome and is a major constituent of the transcription replication complex (TRC) machinery of the viral RNA synthesis. This protein is highly conserved among the coronaviruses and is a potential target for the development of a therapeutic drug. Here, we report the SARS-CoV-2 nsp14 expression, show its structural characterization, and ss-RNA exonuclease activity through vibrational and electronic spectroscopies. The deconvolution of amide-I band in the FTIR spectrum of the protein revealed a composition of 35 % α-helix and 25 % ß-sheets. The binding between protein and RNA is evidenced from the spectral changes in the amide-I region of the nsp14, showing protein conformational changes during the binding process. A value of 20.60±3.81 mol L-1 of the binding constant (KD ) is obtained for nsp14/RNA complex. The findings reported here can motivate further studies to develop structural models for better understanding the mechanism of exonuclease enzymes for correcting the viral genome and can help in the development of drugs against SARS-CoV-2.

Risk factor for breast cancer development under exposure to bovine leukemia virus in Colombian women: A case-control study.

Viruses have been implicated in cancer development in both humans and animals. The role of viruses in cancer is typically to initiate cellular transformation through cellular DNA damage, although specific mechanisms remain unknown. Silent and long-term viral infections need to be present, in order to initiate cancer disease. In efforts to establish a causative role of viruses, first is needed to demonstrate the strength and consistency of associations in different populations. The aim of this study was to determine the association of bovine leukemia virus (BLV), a causative agent of leukemia in cattle, with breast cancer and its biomarkers used as prognosis of the severity of the disease (Ki67, HER2, hormonal receptors) in Colombian women. An unmatched, observational case-control study was conducted among women undergoing breast surgery between 2016-2018. Malignant samples (n = 75) were considered as cases and benign samples (n = 83) as controls. Nested-liquid PCR, in-situ PCR and immunohistochemistry were used for viral detection in blood and breast tissues. For the risk assessment, only BLV positive samples from breast tissues were included in the analysis. BLV was higher in cases group (61.3%) compared with controls (48.2%), with a statistically significant association between the virus and breast cancer in the unconditional logistic regression (adjusted-OR = 2.450,95%CI:1.088-5.517, p = 0.031). In this study, BLV was found in both blood and breast tissues of participants and an association between breast cancer and the virus was confirmed in Colombia, as an intermediate risk factor.

Diagnostic performance and clinical implications of rapid SARS-CoV-2 antigen testing in Mexico using real-world nationwide COVID-19 registry data.

BACKGROUND: SARS-CoV-2 testing capacity is important to monitor epidemic dynamics and as a mitigation strategy. Given difficulties of large-scale quantitative reverse transcription polymerase chain reaction (qRT-PCR) implementation, rapid antigen tests (Rapid Ag-T) have been proposed as alternatives in settings like Mexico. Here, we evaluated diagnostic performance of Rapid Ag-T for SARS-CoV-2 infection and its associated clinical implications compared to qRT-PCR testing in Mexico. METHODS: We analyzed data from the COVID-19 registry of the Mexican General Directorate of Epidemiology up to April 30th, 2021 (n = 6,632,938) and cases with both qRT-PCR and Rapid Ag-T (n = 216,388). We evaluated diagnostic performance using accuracy measures and assessed time-dependent changes in the Area Under the Receiver Operating Characteristic curve (AUROC). We also explored test discordances as predictors of hospitalization, intubation, severe COVID-19 and mortality. RESULTS: Rapid Ag-T is primarily used in Mexico City. Rapid Ag-T have low sensitivity 37.6% (95%CI 36.6-38.7), high specificity 95.5% (95%CI 95.1-95.8) and acceptable positive 86.1% (95%CI 85.0-86.6) and negative predictive values 67.2% (95%CI 66.2-69.2). Rapid Ag-T has optimal diagnostic performance up to days 3 after symptom onset, and its performance is modified by testing location, comorbidity, and age. qRT-PCR (-) / Rapid Ag-T (+) cases had higher risk of adverse COVID-19 outcomes (HR 1.54 95% CI 1.41-1.68) and were older, qRT-PCR (+)/ Rapid Ag-T(-) cases had slightly higher risk or adverse outcomes and ≥7 days from symptom onset (HR 1.53 95% CI 1.48-1.59). Cases detected with rapid Ag-T were younger, without comorbidities, and milder COVID-19 course. CONCLUSIONS: Rapid Ag-T could be used as an alternative to qRT-PCR for large scale SARS-CoV-2 testing in Mexico. Interpretation of Rapid Ag-T results should be done with caution to minimize the risk associated with false negative results.

First evidence of enterovirus A71 and echovirus 30 in Uruguay and genetic relationship with strains circulating in the South American region.

Human enteroviruses (EVs) comprise more than 100 types of coxsackievirus, echovirus, poliovirus and numbered enteroviruses, which are mainly transmitted by the faecal-oral route leading to diverse diseases such as aseptic meningitis, encephalitis, and acute flaccid paralysis, among others. Since enteroviruses are excreted in faeces, wastewater-based epidemiology approaches are useful to describe EV diversity in a community. In Uruguay, knowledge about enteroviruses is extremely limited. This study assessed the diversity of enteroviruses through Illumina next-generation sequencing of VP1-amplicons obtained by RT-PCR directly applied to viral concentrates of 84 wastewater samples collected in Uruguay during 2011-2012 and 2017-2018. Fifty out of the 84 samples were positive for enteroviruses. There were detected 27 different types belonging to Enterovirus A species (CVA2-A6, A10, A16, EV-A71, A90), Enterovirus B species (CVA9, B1-B5, E1, E6, E11, E14, E21, E30) and Enterovirus C species (CVA1, A13, A19, A22, A24, EV-C99). Enterovirus A71 (EV-A71) and echovirus 30 (E30) strains were studied more in depth through phylogenetic analysis, together with some strains previously detected by us in Argentina. Results unveiled that EV-A71 sub-genogroup C2 circulates in both countries at least since 2011-2012, and that the C1-like emerging variant recently entered in Argentina. We also confirmed the circulation of echovirus 30 genotypes E and F in Argentina, and reported the detection of genotype E in Uruguay. To the best of our knowledge this is the first report of the EV-A71 C1-like emerging variant in South-America, and the first report of EV-A71 and E30 in Uruguay.