genome packaging
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Author(s):  
Robert-William Welke ◽  
Hannah Sabeth Sperber ◽  
Amit Koikkarah ◽  
Laura Menke ◽  
Christian Sieben ◽  
...  

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation and several other crucial processes during hantavirus infection. In this study we have generated fluorescently tagged N protein constructs derived from Puumalavirus, the dominant hantavirus species in Central, Northern and Eastern Europe. We have comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We found a significant spatial correlation of N with vimentin, actin and P-bodies, but not with microtubules. N constructs also co-localized with Gn and Gc, albeit not as strong as the glycoproteins associated with each other. Moreover, we as-sessed oligomerization of N constructs, observing efficient and concentration-dependent multi-merization, with complexes comprising more than 10 individual proteins.


2021 ◽  
Author(s):  
Christen E Tai ◽  
Einav Tayeb-Fligelman ◽  
Sarah Griner ◽  
Lukasz Salwinski ◽  
Jeannette T Bowler ◽  
...  

The SARS-CoV-2 nucleocapsid protein (NCAP) functions in viral RNA genome packaging, virion assembly, RNA synthesis and translation, and regulation of host immune response. RNA-binding is central to these processes. Little is known how NCAP selects its binding partners in the myriad of host and viral RNAs. To address this fundamental question, we employed electrophoresis mobility shift and competition assays to compare NCAP binding to RNAs that are of SARS-CoV-2 vs. non-SARS-CoV-2, long vs. short, and structured vs. unstructured. We found that although NCAP can bind all RNAs tested, it primarily binds structured RNAs, and their association suppresses strong interaction with single-stranded RNAs. NCAP prefers long RNAs, especially those containing multiple structures separated by single-stranded linkers that presumably offer conformational flexibility. Additionally, all three major regions of NCAP bind RNA, including the low complexity domain and dimerization domain that promote formation of NCAP oligomers, amyloid fibrils and liquid-liquid phase separation. Combining these observations, we propose that NCAP-NCAP interactions that mediate higher-order structures during packaging also drive recognition of the genomic RNA and call this mechanism recognition-by-packaging. This study provides a biochemical basis for understanding the complex NCAP-RNA interactions in the viral life cycle and a broad range of similar biological processes.


2021 ◽  
Author(s):  
Kristina V Tugaeva ◽  
Andrey A. Sysoev ◽  
Jake L. R. Smith ◽  
Richard B Cooley ◽  
Alfred A. Antson ◽  
...  

The SARS-CoV-2 nucleocapsid protein (N) is responsible for viral genome packaging and virion assembly. Being highly abundant in the host cell, N interacts with numerous human proteins and undergoes multisite phosphorylation in vivo. When phosphorylated within its Ser/Arg-rich region, a tract highly prone to mutations as exemplified in the Omicron and Delta variants, N recruits human 14-3-3 proteins, potentially hijacking their functions. Here, we show that in addition to phosphorylated Ser197, an alternative, less conserved phosphosite at Thr205 not found in SARS-CoV N binds 14-3-3 with micromolar affinity and is in fact preferred over pS197. Fluorescence anisotropy reveals a distinctive pT205/pS197 binding selectivity towards the seven human 14-3-3 isoforms. Crystal structures explain the structural basis for the discovered selectivity towards SARS-CoV-2 N phosphopeptides, and also enable prediction for how N variants interact with 14-3-3, suggesting a link between the strength of this interaction and replicative fitness of emerging coronavirus variants.


mBio ◽  
2021 ◽  
Author(s):  
Alice Duchon ◽  
Steven Santos ◽  
Jianbo Chen ◽  
Matthew Brown ◽  
Olga A. Nikolaitchik ◽  
...  

To generate infectious virions, HIV-1 must package its full-length RNA as the genome during particle assembly. HIV-1 Gag:RNA interactions mediate genome packaging, but the mechanism remains unclear.


2021 ◽  
Author(s):  
Katharina Kohm ◽  
Valentina A. Floccari ◽  
Veronika T. Lutz ◽  
Birthe Nordmann ◽  
Carolin Mittelstaedt ◽  
...  

The Bacillus phage SPβ has been known for about 50 years, but only a few strains are avalible. We isolated four new wild type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SPβ-like phages. We could also reveal the SPβ-like phage genome replication strategy, the genome packaging mode, and the phage genome opening point. We extracted 55 SPβ-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resembled four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SPβ-like prophages consists of 38 proteins. The integration cassette proved to be not conserved even though present in all strains. It consists of distinct integrases. Analysis of SPβ transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.


Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2300
Author(s):  
Mi Chen ◽  
Lulu Chen ◽  
Jing Wang ◽  
Chunxiao Mou ◽  
Zhenhai Chen

Senecavirus A (SVA) is a member of the genus Senecavirus of the family Picornaviridae. SVA-associated vesicular disease (SAVD) outbreaks have been extensively reported since 2014–2015. Characteristic symptoms include vesicular lesions on the snout and feet as well as lameness in adult pigs and even death in piglets. The capsid protein VP3, a structural protein of SVA, is involved in viral replication and genome packaging. Here, we developed and characterized a mouse monoclonal antibody (mAb) 3E9 against VP3. A motif 192GWFSLHKLTK201 was identified as the linear B-cell epitope recognized by mAb 3E9 by using a panel of GFP-tagged epitope polypeptides. Sequence alignments show that 192GWFSLHKLTK201 was highly conserved in all SVA strains. Subsequently, alanine (A)-scanning mutagenesis indicated that W193, F194, L196, and H197 were the critical residues recognized by mAb 3E9. Further investigation with indirect immunofluorescence assay indicated that the VP3 protein was present in the cytoplasm during SVA replication. In addition, the mAb 3E9 specifically immunoprecipitated the VP3 protein from SVA-infected cells. Taken together, our results indicate that mAb 3E9 could be a powerful tool to work on the function of the VP3 protein during virus infection.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Li Dai ◽  
Digvijay Singh ◽  
Suoang Lu ◽  
Vishal I. Kottadiel ◽  
Reza Vafabakhsh ◽  
...  

AbstractMulti-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, using single-molecule fluorescence, we determine that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise number of active or inactive subunits, we find that the packaging motor can tolerate an inactive subunit. However, motors containing one or more inactive subunits exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a DNA packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, suggesting that strict coordination amongst motor subunits of packaging motors is not crucial for function.


PLoS Biology ◽  
2021 ◽  
Vol 19 (11) ◽  
pp. e3001423
Author(s):  
Peter Naniima ◽  
Eleonora Naimo ◽  
Sandra Koch ◽  
Ute Curth ◽  
Khaled R. Alkharsah ◽  
...  

Herpesviruses cause severe diseases particularly in immunocompromised patients. Both genome packaging and release from the capsid require a unique portal channel occupying one of the 12 capsid vertices. Here, we report the 2.6 Å crystal structure of the pentameric pORF19 of the γ-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV) resembling the portal cap that seals this portal channel. We also present the structure of its β-herpesviral ortholog, revealing a striking structural similarity to its α- and γ-herpesviral counterparts despite apparent differences in capsid association. We demonstrate pORF19 pentamer formation in solution and provide insights into how pentamerization is triggered in infected cells. Mutagenesis in its lateral interfaces blocked pORF19 pentamerization and severely affected KSHV capsid assembly and production of infectious progeny. Our results pave the way to better understand the role of pORF19 in capsid assembly and identify a potential novel drug target for the treatment of herpesvirus-induced diseases.


Author(s):  
Elise K. Mullins ◽  
Thomas W. Powers ◽  
Jim Zobel ◽  
Kory M. Clawson ◽  
Lauren F. Barnes ◽  
...  

We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.


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