Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.     

Molecular Mechanisms Involved in Antibody‐Dependent Enhancement of Dengue Virus Infection in Humans

Dengue is the most common arthropod‐borne viral infection in humans with ～50 million cases annually worldwide. In recent decades, a steady increase in the number of severe dengue cases has been seen. Severe dengue disease is most often observed in individuals that have pre‐existing immunity against heterotypic dengue subtypes and in infants with low levels of maternal dengue antibodies. The generally accepted hypothesis explaining the immunopathogenesis of severe dengue is called antibody‐dependent enhancement of dengue infection. Here, circulating antibodies bind to the newly infecting virus but do not neutralize infection. Rather, these antibodies increase the infected cell mass and virus production. Additionally, antiviral responses are diminished allowing massive virus particle production early in infection. The large infected cell mass and the high viral load are prelude for severe disease development. In this review, we discuss what is known about the trafficking of dengue virus in its human host cells, and the signalling pathways activated after virus detection, both in the absence and presence of antibodies against the virus. This review summarizes work that aims to better understand the complex immunopathogenesis of severe dengue disease.      

Long-range RNA-RNA interactions circularize the dengue virus genome

Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5' and 3' ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy revealed that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5' and 3' CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5' and 3' untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5' and 3' UAR (upstream AUG region). In order to investigate the functional role of 5'-3' UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5' or 3' UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.     

Norovirus Genome Circularization and Efficient Replication Are Facilitated by Binding of PCBP2 and hnRNP A1

Sequences and structures within the terminal genomic regions of plus-strand RNA viruses are targets for the binding of host proteins that modulate functions such as translation, RNA replication, and encapsidation. Using murine norovirus 1 (MNV-1), we describe the presence of long-range RNA-RNA interactions that were stabilized by cellular proteins. The proteins potentially responsible for the stabilization were selected based on their ability to bind the MNV-1 genome and/or having been reported to be involved in the stabilization of RNA-RNA interactions. Cell extracts were preincubated with antibodies against the selected proteins and used for coprecipitation reactions. Extracts treated with antibodies to poly(C) binding protein 2 (PCBP2) and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 significantly reduced the 5′-3′ interaction. Both PCBP2 and hnRNP A1 recombinant proteins stabilized the 5′-3′ interactions and formed ribonucleoprotein complexes with the 5′ and 3′ ends of the MNV-1 genomic RNA. Mutations within the 3′ complementary sequences (CS) that disrupt the 5′-3′-end interactions resulted in a significant reduction of the viral titer, suggesting that the integrity of the 3′-end sequence and/or the lack of complementarity with the 5′ end is important for efficient virus replication. Small interfering RNA-mediated knockdown of PCBP2 or hnRNP A1 resulted in a reduction in virus yield, confirming a role for the observed interactions in efficient viral replication. PCBP2 and hnRNP A1 induced the circularization of MNV-1 RNA, as revealed by electron microscopy. This study provides evidence that PCBP2 and hnRNP A1 bind to the 5′ and 3′ ends of the MNV-1 viral RNA and contribute to RNA circularization, playing a role in the virus life cycle.     

Differential RNA Sequence Requirement for Dengue Virus Replication in Mosquito and Mammalian Cells

Dengue virus cycles between mosquitoes and humans. Each host provides a different environment for viral replication, imposing different selective pressures. We identified a sequence in the dengue virus genome that is essential for viral replication in mosquito cells but not in mammalian cells. This sequence is located at the viral 3′ untranslated region and folds into a small hairpin structure. A systematic mutational analysis using dengue virus infectious clones and reporter viruses allowed the determination of two putative functions in this cis-acting RNA motif, one linked to the structure and the other linked to the nucleotide sequence. We found that single substitutions that did not alter the hairpin structure did not affect dengue virus replication in mammalian cells but abolished replication in mosquito cells. This is the first sequence identified in a flavivirus genome that is exclusively required for viral replication in insect cells.     

Members of the NF90/NFAR protein group are involved in the life cycle of a positive-strand RNA virus

A major issue of current virology concerns the characterization of cellular proteins that operate as functional components of the viral multiplication process. Here we describe a group of host factors designated as 'NFAR proteins' that are recruited by the replication machinery of bovine viral diarrhea virus, a close relative of the human pathogen hepatitis C virus. The NFAR proteins associate specifically with both the termini of the viral RNA genome involving regulatory elements in the 5' and 3' non-translated regions. Modification of the protein interaction sites in the 3' non-translated region yielded viral RNAs that were replication deficient. Viral replication was also inhibited by RNAi approaches that reduced the concentration of RNA helicase A, a member of the NFAR group, in the host cell's cytoplasm. Further experimental data suggest that NFAR proteins mediate a circular conformation of the viral genome that may be important for the coordination of translation and replication. Because NFAR proteins are presumed components of the antiviral response, we suspect that viral recruitment may also serve to weaken cellular defense mechanisms.     

RNA sequences and structures required for the recruitment and activity of the dengue virus polymerase

Dengue virus RNA-dependent RNA polymerase specifically binds to the viral genome by interacting with a promoter element known as stem-loop A (SLA). Although a great deal has been learned in recent years about the function of this promoter in dengue virus-infected cells, the molecular details that explain how the SLA interacts with the polymerase to promote viral RNA synthesis remain poorly understood. Using RNA binding and polymerase activity assays, we defined two elements of the SLA that are involved in polymerase interaction and RNA synthesis. Mutations at the top of the SLA resulted in RNAs that retained the ability to bind the polymerase but impaired promoter-dependent RNA synthesis. These results indicate that protein binding to the SLA is not sufficient to induce polymerase activity and that specific nucleotides of the SLA are necessary to render an active polymerase-promoter complex for RNA synthesis. We also report that protein binding to the viral RNA induces conformational changes downstream of the promoter element. Furthermore, we found that structured RNA elements at the 3' end of the template repress dengue virus polymerase activity in the context of a fully active SLA promoter. Using assays to evaluate initiation of RNA synthesis at the viral 3'-UTR, we found that the RNA-RNA interaction mediated by 5'-3'-hybridization was able to release the silencing effect of the 3'-stem-loop structure. We propose that the long range RNA-RNA interactions in the viral genome play multiple roles during RNA synthesis. Together, we provide new molecular details about the promoter-dependent dengue virus RNA polymerase activity.     

A Host YB-1 Ribonucleoprotein Complex Is Hijacked by Hepatitis C Virus for the Control of NS3-Dependent Particle Production

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.     

RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors.     

The 5' and 3' downstream AUG region elements are required for mosquito-borne flavivirus RNA replication

Flaviviruses require complementarity between the 5' and 3' ends of the genome for RNA replication. For mosquito-borne flaviviruses, the cyclization sequences (CS) and upstream of AUG region (UAR) elements at the genomic termini are necessary for viral RNA replication, and a third motif, the downstream of AUG region (DAR), was recently designated for dengue virus. The 3' DAR sequence is also part of a hairpin (HP-3'SL), and both complementarity between 5' and 3' DAR motifs and formation of the HP-3'SL in the absence of the 5' end are conserved among mosquito-borne flaviviruses. Using West Nile virus as a model, we demonstrate that 5'-3' DAR complementarity and HP-3'SL formation are essential for viral RNA replication.     

Molecular Docking Based Screening of Plant Flavonoids as Dengue NS1 Inhibitors

Dengue infection has turned into a serious health concern globally due to its high morbidity rate and a high possibility of increase in its mortality rate on the account of unavailability of any proper treatment for severe dengue infection. The situation demands an urgent development of efficient and practicable treatment to deal with Dengue virus (DENV). Flavonoids, a class of phytochemicals present in medicinal plants, possess anti-viral activity and can be strong drug candidates against viruses. NS1 glycoprotein of Dengue virus is involved in its RNA replication and can be a strong target for screening of drugs against this virus. Current study focuses on the identification of flavonoids which can block Asn-130 glycosylation site of Dengue virus NS1 to inhibit viral replication as glycosylation of NS1 is required for its biological functioning. Molecular docking approach was used in this study and the results revealed that flavonoids have strong potential interactions with active site of NS1. Six flavonoids (Deoxycalyxin A; 3,5,7,3'',4''-pentahydroxyflavonol-3-O-beta-D-galactopyranoside; (3R)-3'',8-Dihydroxyvestitol; Sanggenon O; Epigallocatechin gallate; Chamaejasmin) blocked the Asn-130 glycosylation site of NS1 and could be able to inhibit the viral replication. It can be concluded from this study that these flavonoids could serve as antiviral drugs for dengue infections. Further in-vitro analyses are required to confirm their efficacy and to evaluate their drug potency.     

Derivation and characterization of a dengue type 1 host range-restricted mutant virus that is attenuated and highly immunogenic in monkeys

We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3' stem-loop structure (3'-SL) that is conserved among all flavivirus species. The 3'-SL constitutes the downstream terminal similar95 nucleotides of the 3' noncoding region in flavivirus RNA. Here we report the introduction of these same mutational changes into the analogous region of an infectious DNA derived from the genome of a human-virulent dengue serotype 1 virus (DEN1), strain Western Pacific (DEN1WP). The resulting DEN1 mutant (DEN1mutF) exhibited a host range-restricted phenotype similar to that of DEN2mutF virus. DEN1mutF virus was attenuated in a monkey model for dengue infection in which viremia is taken as a correlate of human virulence. In spite of the markedly reduced levels of viremia that it induced in monkeys compared to DEN1WP, DEN1mutF was highly immunogenic. In addition, DEN1mutF-immunized monkeys retained high levels of neutralizing antibodies in serum and were protected from challenge with high doses of the DEN1WP parent for as long as 17 months after the single immunizing dose. Phenotypic revertants of DEN1mutF and DEN2mutF were each detected after a total of 24 days in C6/36 cell cultures. Complete nucleotide sequence analysis of DEN1mutF RNA and that of a revertant virus, DEN1mutFRev, revealed that (i) the DEN1mutF genome contained no additional mutations upstream from the 3'-SL compared to the DEN1WP parent genome and (ii) the DEN1mutFRev genome contained de novo mutations, consistent with our previous hypothesis that the defect in DEN2mutF replication in C6/36 cells was at the level of RNA replication. A strategy for the development of a tetravalent dengue vaccine is discussed.     

Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.     

A brief review on dengue molecular virology, diagnosis, treatment and prevalence in Pakistan

ABSTRACT: Dengue virus infection is a serious health problem infecting 2.5 billion people worldwide. Dengue is now endemic in more than 100 countries, including Pakistan. Each year hundreds of people get infected with dengue in Pakistan. Currently, there is no vaccine available for the prevention of Dengue virus infection due to four viral serotypes. Dengue infection can cause death of patients in its most severity, meanwhile many antiviral compounds are being tested against dengue virus infection to eradicate this disease but still there is a need to develop an efficient, low-cost and safe vaccine that can target all the four serotypes of dengue virus. This review summarizes dengue molecular virology, important drug targets, prevalence in Pakistan, diagnosis, treatment and medicinal plant inhibitors against dengue.