scholarly journals Icosahedral viruses defined by their positively charged domains: a signature for viral identity and capsid assembly strategy

2019 ◽  
Author(s):  
Rodrigo D. Requião ◽  
Rodolfo L. Carneiro ◽  
Mariana Hoyer Moreira ◽  
Marcelo Ribeiro-Alves ◽  
Silvana Rossetto ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Level ◽  
Protein Domain ◽  
Capsid Proteins ◽  
Capsid Assembly ◽  
Genome Packaging ◽  
Structure Comparison ◽  
Net Charge ◽  
Icosahedral Viruses ◽  
Positively Charged

AbstractCapsid proteins often present a positively charged arginine-rich region at the N and/or C-termini that for some icosahedral viruses has a fundamental role in genome packaging and particle stability. These sequences show little to no conservation at the amino-acid level and are structurally dynamic so that they cannot be easily detected by common sequence or structure comparison. As a result, the occurrence and distribution of positively charged protein domain across the viral and the overall protein universe are unknown. We developed a methodology based on the net charge calculation of discrete segments of the protein sequence that allows us to identify proteins containing amino-acid stretches with an extremely high net charge. We observed that among all organisms, icosahedral viruses are especially enriched in extremely positively charged segments (Q ≥ +17), with a distinctive bias towards arginine instead of lysine. We used viral particle structural data to calculate the total electrostatic charge derived from the most positively charged protein segment of capsid proteins and correlated these values with genome charge arising from the phosphates of each nucleotide. We obtained a positive correlation (r = 0.91, p-value < 0001) for a group of 17 viral families, corresponding to 40% of all families with icosahedral structures described so far. These data indicated that unrelated viruses with diverse genome types adopt a common underlying mechanism for capsid assembly and genome stabilization based on R-arms. Outliers from a linear fit pointed to families with alternative strategies of capsid assembly and genome packaging.Significance StatementViruses can be characterized by the existence of a capsid, an intricate proteinaceous container that encases the viral genome. Therefore, capsid assembly and function are essential to viral replication. Here we specify virus families with diverse capsid structure and sequence, for each capsid packing capacity depends on a distinctive structural feature: a highly positively charged segment of amino acids residues, preferentially made of arginine. We also show that proteins with the same characteristics are rarely found in cellular proteins. Therefore, we identified a conserved viral functional element that can be used to infer capsid assembly mechanisms and inspire the design of protein nanoparticles and broad-spectrum antiviral treatments.

scholarly journals Amino Acid Contacts between Sigma 70 Domain 4 and the Transcription Activators RhaS and RhaR

2004 ◽  
Vol 186 (18) ◽  
pp. 6277-6285 ◽  
Author(s):  
Jason R. Wickstrum ◽  
Susan M. Egan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Level ◽  
Transcription Activation ◽  
Charged Amino Acids ◽  
Specific Contact ◽  
Loss Of Contact ◽  
Transcription Activators ◽  
Sigma 70 ◽  
Positively Charged

ABSTRACT The RhaS and RhaR proteins are transcription activators that respond to the availability of l-rhamnose and activate transcription of the operons in the Escherichia coli l-rhamnose catabolic regulon. RhaR activates transcription of rhaSR, and RhaS activates transcription of the operon that encodes the l-rhamnose catabolic enzymes, rhaBAD, as well as the operon that encodes the l-rhamnose transport protein, rhaT. RhaS is 30% identical to RhaR at the amino acid level, and both are members of the AraC/XylS family of transcription activators. The RhaS and RhaR binding sites overlap the −35 hexamers of the promoters they regulate, suggesting they may contact the σ70 subunit of RNA polymerase as part of their mechanisms of transcription activation. In support of this hypothesis, our lab previously identified an interaction between RhaS residue D241 and σ70 residue R599. In the present study, we first identified two positively charged amino acids in σ70, K593 and R599, and three negatively charged amino acids in RhaR, D276, E284, and D285, that were important for RhaR-mediated transcription activation of the rhaSR operon. Using a genetic loss-of-contact approach we have obtained evidence for a specific contact between RhaR D276 and σ70 R599. Finally, previous results from our lab separately showed that RhaS D250A and σ70 K593A were defective at the rhaBAD promoter. Our genetic loss-of-contact analysis of these residues indicates that they identify a second site of contact between RhaS and σ70.

scholarly journals Surface Loop Dynamics in Adeno-Associated Virus Capsid Assembly

2008 ◽  
Vol 82 (11) ◽  
pp. 5178-5189 ◽  
Author(s):  
Nina DiPrimio ◽  
Aravind Asokan ◽  
Lakshmanan Govindasamy ◽  
Mavis Agbandje-McKenna ◽  
R. Jude Samulski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Virus Titer ◽  
Amino Acid Position ◽  
Capsid Assembly ◽  
Genome Packaging ◽  
Particle Assembly ◽  
Adeno Associated Virus ◽  
Viral Genomes ◽  
Amino Acid Peptide

ABSTRACT The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.

scholarly journals The structure of a highly conserved picocyanobacterial protein reveals a Tudor domain with a novel tRNA binding function

2019 ◽  
Author(s):  
Katherine M Bauer ◽  
Rose Dicovitsky ◽  
Maria Pellegrini ◽  
Olga Zhaxybayeva ◽  
Michael J Ragusa
Keyword(s):  
Amino Acid ◽  
Amino Acid Level ◽  
Computational Docking ◽  
Binding Function ◽  
Acid Protein ◽  
Tudor Domain ◽  
And Function ◽  
Positively Charged ◽  
Amino Acid Protein

ABSTRACTCyanobacteria of the Prochlorococcus and marine Synechococcus genera are the most abundant photosynthetic microbes in the ocean. Intriguingly, the genomes of these bacteria are very divergent even within each genus, both in gene content and at amino acid level of the encoded proteins. One striking exception to this is a 62 amino acid protein, termed Prochlorococcus/SynechococcusHyper Conserved Protein (PSHCP). PSHCP is not only found in all sequenced Prochlorococcus and marine Synechococcus genomes but it is also nearly 100% identical in its amino acid sequence across all sampled genomes. Such universal distribution and sequence conservation suggests an essential cellular role of the encoded protein in these bacteria. However, the function of PSHCP is unknown. We used Nuclear Magnetic Resonance (NMR) spectroscopy to determine its structure. We found that 52 of the 62 amino acids in PSHCP form a Tudor domain, while the remainder of the protein is disordered. NMR titration experiments revealed that PSHCP has only a weak affinity for DNA, but an 18.5 fold higher affinity for tRNA, hinting at an involvement of PSHCP in translation. Computational docking and mutagenesis studies identified a positively charged patch surrounding residue K30 that serves as the primary docking site for tRNA on PSHCP. These results provide the first insight into the structure and function of PSHCP and suggest a new function for Tudor domains in recognizing tRNA.

Salamander rods and cones contain distinct transducin alpha subunits

2000 ◽  
Vol 17 (6) ◽  
pp. 847-854 ◽  
Author(s):  
JAMES C. RYAN ◽  
SERGEY ZNOIKO ◽  
LIN XU ◽  
ROSALIE K. CROUCH ◽  
JIAN-XING MA
Keyword(s):  
Amino Acid ◽  
Blot Analysis ◽  
Cellular Localization ◽  
Amino Acid Level ◽  
Single Copy ◽  
Lower Vertebrate ◽  
Rods And Cones ◽  
Sequence Identity ◽  
Outer Segments ◽  
Cone Pigments

The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin α subunits (Gαt) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of Gαt. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander Gαt-1 has 91.2–93.7% amino acid sequence identity to mammalian rod Gαt subunits and 79.7–80.9% to mammalian cone Gαts. The salamander Gαt-2 has 86.2–87.9% sequence identity to mammalian cone Gαts and 78.9–80.9% to mammalian rod Gαts at the amino acid level. The Gαt-1 cDNA encodes 350 amino acids while the Gαt-2 cDNA encodes 354 residues, which is typical for rod and cone Gαts, respectively, and we thus identified the Gαt-1 as rod and Gαt-2 as cone Gαt. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone Gαt subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod Gαt and 2.6 kb for cone Gαt. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod Gαt is localized to rods, predominantly in the outer segments; similarly, cone Gαt is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.

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