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Newly synthesized nuclear NP, M and NS1 proteins migrated from the nucleus over a period of several hours. Rates of transport were higher for NP than for M or NS1 and suggest that nuclear NP may play a role in multiplication.
In a coupled cell-free system for the transcription and translation of the influenza mRNA's, containing detergent-disrupted purified NWS influenza virion and a micrococcal nuclease-preincubated rabbit reticulocyte lysate, five unglycosylated viral proteins (NS1, M, NP, P1, and P3) were easily produced and isolated. Their identification was based on the electrophoretic separation of peptide fragments resulting from their partial digestion with proteases of restricted specificity (D.W. Cleveland, S. G. Fisher, N. W. Kirschner, and U. K. Laemmli, J. Biol. Chem. 252:1102-1106, 1977).
Dengue infection remains a major global public health challenge, with no specific antiviral therapy currently available. The dengue virus non-structural protein 1 (NS1) exists in both intracellular and secreted forms playing a pivotal role in viral replication, immune evasion, and pathogenesis, particularly by contributing to endothelial disruption and vascular leakage during severe disease, thereby making it a promising therapeutic target. In silico screening identified berberine, betulinic acid, and ursolic acid as top candidates, exhibiting high binding affinities and stable interactions within the NS1 binding pocket. These computational predictions were further validated by biophysical assays, which demonstrated strong and specific binding interactions between the purified NS1 protein and the selected compounds. All three compounds significantly reduced viral genome levels, with the highest inhibition observed for berberine (60%), and followed by betulinic acid (40%) and ursolic acid (28%). Consistently, berberine showed the most potent inhibition of both intracellular and extracellular NS1. Overall, these findings highlight the inhibitory potential of natural compounds against DENV NS1 and provide a strong foundation for the development of NS1-targeted antivirals as a novel therapeutic strategy against dengue infection.
The native 30-S ribosomal subunits from Escherichia coli are shown to be associated with two proteins which are different from the known ribosome-associated and ribosomal proteins. Neither protein is foune on native 50-S subunits or on intact ribosomes in the cell extract. The purified proteins re-bind in vitro to free 30-S subunits, but do not bind to either free 50-S subunits or intact ribosomes. The proteins, denoted NS1 and NS2, have been purified and characterized. Both proteins showed the same molecular weight of 9500 by sodium dodecyl sulfate gel electrophoresis but 34 000 by gel filtration. Upon treatment with cross-linking reagents the purified proteins gave higher molecular weight species up to the tetrameric ones showing that they exist in solution as tetramers. The amino acid compositions, tryptic fingerprint patterns and N-terminal sequences of the two proteins have been determined. These data show that NS1 and NS2 possess distinct primary structures but with extensive sequence homology. Antibodies raised against the purified proteins cross-reacted in double immuno-diffusion tests confirming further the homology. Because of the similarity in properties a sample of the DNA-binding protein HD (Berthold, V. and Geider, K. (1976) Eur. J. Biochem. 71, 443--449) was compared to NS1 and NS2. In terms of several criteria, the protein HD is found to be a mixture of two proteins, namely NS1 and NS2. The present report is the first instance of an association of DNA-binding proteins to the ribosome.
The proteins of vesicular stomatitis virus (VSV) were analyzed on the basis of charge as well as size in polyacrylamide gels containing urea and acetic acid. The phosphorprotein NS was resolved into two major species. The less phosphorylated NS1 species contained about 10% fewer phosphate residues than the second species, NS2. These two phosphorylated forms were compartmentalized both in the virus and in the infected cell cytoplasm. Cores from virions and the core-containing fraction of the infected cell cytoplasm contained only the NS1 form. All of the more highly phosphorylated NS2 form and some of the NS1 form were found to be free of cores, whether they were derived from virions or from the infected cell. Therefore, the degree of phosphorylation appeared to determine whether or not the NS protein became bound to VSV cores. Moreover, the amount of bound NS1 protein relative to nucleocapsids increased as the pH of the culture medium was raised from 6.6 to 7.4. Because an increased in pH increases VSV replication (Fiszman et al., J. Virol. 13:801-808, 1974; Palma and Huang, in W.S. Robinson and C.F. Fox, ed., Mechanisms of Virus Disease, ICN-UCLA Symposia, p. 87-100, 1974), the NS1 protein may either regulate overall VSV RNA synthesis or regulate the switch between transcription and replication.
In previous studies we showed that a ninth polypeptide with a molecular weight of approximately 11,000 (NS2) found in influenza virus-infected cells was unique, that it could be synthesized in vitro, and that its expression in vivo required early protein synthesis. On the basis of these results we suggested that one of the eight genome RNA segments of influenza virus codes for two polypeptides [Lamb, R.A., Etkind, P.R. & Choppin, P.W. (1978) Virology 91, 60-78]. We describe here differences in the electrophoretic mobility of the NS2 polypeptides of different strains of influenza A virus. These results provided further evidence that NS2 is virus coded and also made possible genetic studies using recombinants between two virus strains (HK and PR8) whose NS2 polypeptides differ. These studies showed that the gene for NS2 reassorts with that of the nonstructural polypeptide NS1, which is coded by genome segment 8. A mRNA for NS2 has been separated from that of NS1 and the other viral polypeptides by centrifugation and has been translated in vitro. Hybridization of genome segment 8 to the total mRNAs from infected cells specifically prevented the synthesis of NS2 and NS1. These results indicate that influenza virus genome segment 8 is transcribed into two separate mRNAs that code for two polypeptides, NS1 and NS2. Possible mechanisms for the transcription of the two mRNAs from either contiguous or overlapping genes are discussed.
The genome of influenza virus consists of eight segments of single-stranded RNA, each of which encodes a different polypeptide. In addition to the eight recognized gene products, the virus specifies a distinct smaller nonstructural polypeptide (NS2), which is translated from a separate species of virus-specific mRNA. The location on the virus genome of the gene encoding this polypeptide was investigated by hybridization of the NS2 mRNA with isolated subgenomic RNA species, and by correlation of the inheritance of a strain-specific NS2 with inheritance of particular genome RNA segments during recombination between two different virus strains. The genetic information for NS2 was found to reside in the smallest genome RNA segment of the virion, which also encodes the NS1 polypeptide. Considering the sizes of the molecules involved, it is likely that the coding sequences for the two polypeptides overlap.
INTRODUCTION: Dengue virus (DENV) neurological complications are increasingly reported, yet the viral genetic determinants of neurotropism remain poorly characterized. METHODS: We screened 25 DENV1 clinical isolates from the 2014 outbreak in Guangdong, China, for neurotropism in suckling mice, and integrated comparative genomics, pre-expression functional assays, population-scale sequence analysis, and OpenFold3 structural modeling to identify mutations associated with enhanced neuroinvasion. RESULTS: We found that only strain P1253 induced neurological symptoms and mortality via subcutaneous inoculation, producing cortical-selective lesions distinct from the diffuse encephalitic damage observed after intracranial inoculation, and P1253 replicated preferentially in human brain microvascular endothelial cells (HBMEC) compared to contemporaneous strains. Comparative genomics identified three unique mutations in P1253 (NS1 175Y→H, NS2A 89V→F, NS4A 2V→I), and pre-expression assays demonstrated that only NS2A 89V→F significantly enhanced viral replication and cytopathic effect in HBMEC. Analysis of 1,990 complete DENV1 genomes revealed five natural mutant types in the NS2A 89 -96 residue region, with P1253 representing the FIPI quadruple-mutant type, and OpenFold3 structural prediction showed that 89V→F introduced on the VIPI background induced the most significant distal domain reorientation (RMSD 1.605 Å), increasing the centroid-to-centroid distance between residues 89 -96 and 185 -218 from 18.221 Å to 27.462 Å. DISCUSSION: These findings identify NS2A 89V→F as a candidate adaptive mutation associated with enhanced neurotropism in DENV1 and provide a framework for monitoring neurovirulent variants.
The isolation of pure tubules of the outer cortex of the foetal bovine kidney, using a previously described methods, has shown that the tubule suspension obtained is almost entirely constituted of proximal tubule fragments. The proximal cells obtained from this suspension were grown in vitro. The previously called "nephrospecific" antigens NS1-2 were shown to be strictly specific for the cells of the proximal tubule, which is an "histion" of the kidney, that is to say an ultimate unit of differentiation of this organ. The fate of these antigens, henceforth termed "histiospecific antigens" (of the proximal tubule), was followed in primary monolayer cultures of the proximal tubules, using immunodiffusion tests and immunofluorescence. A rapid decrease in this antigen content of the cells was observed during the in vitro culture. An immunoautoradiographic study demonstrated that the synthesis of these antigens was switched off in the confluent cultures. A similar autoradiographic study demonstrated that these same proximal cells at confluence synthesized a bovine alpha1-foetoprotein, which had been detected in earlier studies of kidney cortex cultures. This double antigenic shift is correlated with the in vitro evolution of some morphological markers of the proximal cells, as studied with the transmission and the scanning electron microscope. The factors responsible for these results are discussed.
Globally ≤ 4 billion of the population are at potential risk of contracting dengue virus (DENV) infection. Seasonal outbreaks of dengue are frequently reported causing a high healthcare burden. Undiagnosed DENV can lead to severe morbidity and mortality. Early diagnosis of DENV relies on molecular methods, which are impractical in resource-constrained settings (RCSs). Dengue can be caused by any of the four distinct DENV serotypes. Therefore, a simple method for rapid diagnosis of Pan-DENV serotypes is of utmost importance at RCSs. A fluorescence detection platform for Pan-DENV using RT-RPA and CRISPR/Cas12a was developed targeting nonstructural 1 (NS1) gene for DENV-1, 2, and 3, and envelope (E) gene for DENV-2. Further, crRNA specific to DENV serotypes were designed to facilitate CRISPR/Cas12a detection. Analytical sensitivity was determined using synthetic RNA and DENV serotypes genome. Clinical validation of the assay was performed using RNA extracted from AES/AFI clinical samples. The developed CRISPR/Cas12a-based detection platform can detect all four serotypes of DENV viz 1-4 in a single pot using fluorescence detection. This assay showed the limit of detection ≥ 781 zg reaction- 1, ≥ 1.81 ag reaction-1, ≥ 62.5 fg reaction-1, and ≥ 2.5 pg reaction-1 for synthetic DENV-1, DENV-2, DENV-3, and DENV-4 template, respectively. Our assay demonstrated the analytic sensitivity of ≥ 10 ng reaction-1 for DENV-1 and DENV-4, and ≥ 0.5 ng reaction-1 for DENV-3 and DENV-4 genomes. This assay showed no cross-reactivity with other related etiologies tested causing AFI/AES. With 76 clinical samples (DENV PCR positive = 16, DENV PCR negative = 60), the assay demonstrated 93.7% sensitivity and 100% specificity with an overall accuracy of 98.7% for detection of the Pan-DENV serotypes. Our assay displayed comparable results to that of RT-PCR. The ease of interpretation and rapid detection of the Pan-DENV, represents the potential of the developed assay as an ideal point-of-care test. This assay upon field-deployment could help in reducing healthcare burden, provide differential diagnosis and support initiating early and prompt treatment to patients at RCS.