Profiles

Robert Daniels

Robert Daniels

Docent

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Works at Department of Biochemistry and Biophysics
Telephone 08-16 24 60
Email robert.daniels@dbb.su.se
Visiting address Svante Arrhenius väg 16
Room A 423
Postal address Institutionen för biokemi och biofysik 106 91 Stockholm

Research

The maturation and assembly of viral membrane glycoproteins.

Millions of people died world-wide as a result of the type A influenza pandemics of 1918 (H1N1), 1957 (H2N2) and 1968 (H3N2). The large number of avian influenza subtypes and the ability of influenza to undergo genetic reassortment and rapidly evolve raise the possibility that other pandemic strains can develop such as the pandemic H1N1 strain of swine origin in 2009 and the H7N9 strain in 2013. Currently, we cannot predict if all of the hemagglutinin (H) and neuraminidase (N) subtypes are capable of surfacing in the human population, but several factors (e.g. cell surface receptors, optimal replication temperatures, and mutations in the viral RNA polymerase) are known to act as barriers to genetic reassortment between human and avian influenza. Our lab is focused on investigating how the different subtypes of the influenza surface antigen neuraminidase assemble, mature and function under varying cellular and environmental conditions. This knowledge is then applied to the ever expanding influenza database to identify conserved and variable regions within and across subtypes to pinpoint features that are evolving or must evolve to cross the species barrier. Through cellular and biochemical analysis of how these alterations change neuraminidase and viral assembly and impact viral propagation, we can begin to illustrate a picture of the potential threats each subtype poses for genetic reassortment and the magnitude of the complimentary changes that are necessary for them to successfully enter the human population.

 

Group members

Dan Dou, PhD Student

Johan Nordholm, PhD Student

Rebecca Revol, PhD Student

Hao Wang, PhD Student

Henrik Östbye, PhD Student

 

Selected Publications

  • Nordholm J, Petitou J, Östbye H, da Silva DV, Dou D, Wang H, Daniels R (2017). “Translational regulation of viral secretory proteins by the 5’ coding regions and a viral RNA-binding protein.” Journal of Cell Biology, Jul 2017, jcb.201702102; DOI: 10.1083/jcb.201702102
  • Dou D, Hernandez-Neuta I, Wang H, Östbye H, Qian X, Thiele S, Resa-Infante P, Kouassi N, Sender V, Hentrich K, Mellroth P, Henriques-Normark B, Gabriel G, Nilsson M, Daniels R (2017). “Analysis of IAV replication and co-infection dynamics by a versatile RNA viral genome labeling method.” Cell Reports, Jul 5;20(1):251-263
  • Dai M, Guo H, Dortmans J, Dekkers J, Nordholm J, Daniels R, van Kuppeveld FJM , de Vries E, de Haan CAM. (2016). “Identification of residues that affect oligomerization and/or enzymatic activity of influenza virus H5N1 neuraminidase proteins.” Journal of Virology, 90(20):9457-70.
  • da Silva DV, Nordholm J, Dou D, Rossman JS, Daniels R. (2015). “The influenza NA protein transmembrane and head domain have co-evolved.” Journal of Virology, 89(2):1094-104.
  • Dou D, da Silva DV, Nordholm J, Wang H, Daniels R. (2014). “Type II transmembrane domain hydrophobicity dictates the co-translational dependence for inversion.” Molecular Biology of the Cell, 25(21):3363-74.
  • Nordholm J, da Silva DV, Damjanovic J, Dou D, Daniels R. (2013). “Polar residues and their positional context dictate the transmembrane domain interactions of influenza A neuraminidases. ” Journal of Biological Chemistry, 288(15):10652-60.
  • da Silva DV, Nordholm J, Madjo U, Pfeiffer A, Daniels R. (2013). “Assembly of subtype 1 influenza neuraminidase is driven by both the transmembrane and head domains.” Journal of Biological Chemistry, 288(1):644-53.
  • Nordholm J, da Silva DV, Damjanovic J, Dou D, Daniels R. (2013).
    “Polar residues and their positional context dictate the transmembrane domain interactions of influenza A neuraminidases.”
    Journal of Biological Chemistry, 288(15):10652-60.
  • da Silva DV, Nordholm J, Madjo U, Pfeiffer A, Daniels R. (2012).
    “Assembly of subtype 1 influenza neuraminidase is driven by both the transmembrane and head domains.”
    Journal of Biological Chemistry, 288(1):644-53.
  • Mellroth P, Daniels R, Eberhardt A, Ronnlund D, Blom H, Widengren J, Normark S, Henriques-Normark B. (2012).
    “LytA, the major autolysin of Streptococcus pneumoniae, requires access to the nascent peptidoglycan.”
    Journal of Biological Chemistry, 287(14):11018-29

Funding Sources

Swedish Foundation for Strategic Research through the Center for Biomembrane - Research
Swedish Research Council
Carl Trygger Foundation
Harald Jeanssons Stiftelse

 

 

Publications

A selection from Stockholm University publication database
  • Johan Nordholm (et al.). Journal of Cell Biology

    A primary function of 5’ regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. Here we show the regions coding for the ER-targeting sequences of the influenza proteins NA and HA also function as translational regulatory elements, which are controlled by the viral RNA-binding protein NS1. The translational increase depends on the nucleotide composition of the NA and HA ER-targeting sequences, their 5’ positioning, and is facilitated by the NS1 RNA-binding domain, which can associate with ER membranes. Inserting the ER-targeting sequence coding region of NA into different 5’UTRs confirmed that NS1 can promote the translation of secretory protein mRNAs based on the nucleotides within this region rather than the resulting amino acids. By analysing human protein mRNA sequences we found evidence that this mechanism of using 5’ coding regions and particular RNA-binding proteins to achieve gene-specific regulation may extend to human secreted proteins.

  • 2016. Meiling Dai (et al.). Journal of Virology 90 (20), 9457-9470

    Influenza A virus (IAV) attachment to and release from sialoside receptors is determined by the balance between hemagglutinin (HA) and neuraminidase (NA). The molecular determinants that mediate the specificity and activity of NA are still poorly understood. In this study, we aimed to design the optimal recombinant soluble NA protein to identify residues that affect NA enzymatic activity. To this end, recombinant soluble versions of four different NA proteins from H5N1 viruses were compared with their full-length counterparts. The soluble NA ectodomains were fused to three commonly used tetramerization domains. Our results indicate that the particular oligomerization domain used does not affect the K-m value but may affect the specific enzymatic activity. This particularly holds true when the stalk domain is included and for NA ectodomains that display a low intrinsic ability to oligomerize. NA ectodomains extended with a Tetrabrachion domain, which forms a nearly parallel four-helix bundle, better mimicked the enzymatic properties of full-length proteins than when other coiled-coil tetramerization domains were used, which probably distort the stalk domain. Comparison of different NA proteins and mutagenic analysis of recombinant soluble versions thereof resulted in the identification of several residues that affected oligomerization of the NA head domain (position 95) and therefore the specific activity or sialic acid binding affinity (K-m value; positions 252 and 347). This study demonstrates the potential of using recombinant soluble NA proteins to reveal determinants of NA assembly and enzymatic activity. IMPORTANCE The IAV HA and NA glycoproteins are important determinants of host tropism and pathogenicity. However, NA is relatively understudied compared to HA. Analysis of soluble versions of these glycoproteins is an attractive way to study their activities, as they are easily purified from cell culture media and applied in downstream assays. In the present study, we analyzed the enzymatic activity of different NA ectodomains with three commonly used tetramerization domains and compared them with fulllength NA proteins. By performing a mutagenic analysis, we identified several residues that affected NA assembly, activity, and/or substrate binding. In addition, our results indicate that the design of the recombinant soluble NA protein, including the particular tetramerization domain, is an important determinant for maintaining the enzymatic properties within the head domain. NA ectodomains extended with a Tetrabrachion domain better mimicked the full-length proteins than when the other tetramerization domains were used.

  • 2015. Diogo V. da Silva (et al.). Journal of Virology 89 (2), 1094-1104

    Transmembrane domains (TMDs) from single-spanning membrane proteins are commonly viewed as membrane anchors for functional domains. Influenza virus neuraminidase (NA) exemplifies this concept, as it retains enzymatic function upon proteolytic release from the membrane. However, the subtype 1 NA TMDs have become increasingly more polar in human strains since 1918, which suggests that selection pressure exists on this domain. Here, we investigated the N1 TMD-head domain relationship by exchanging a prototypical old TMD (1933) with a recent (2009), more polar TMD and an engineered hydrophobic TMD. Each exchange altered the TMD association, decreased the NA folding efficiency, and significantly reduced viral budding and replication at 37 degrees C compared to at 33 degrees C, at which NA folds more efficiently. Passaging the chimera viruses at 37 degrees C restored the NA folding efficiency, viral budding, and infectivity by selecting for NA TMD mutations that correspond with their polar or hydrophobic assembly properties. These results demonstrate that single-spanning membrane protein TMDs can influence distal domain folding, as well as membrane-related processes, and suggest the NA TMD in H1N1 viruses has become more polar to maintain compatibility with the evolving enzymatic head domain. IMPORTANCE The neuranainidase (NA) protein from influenza A viruses (IAVs) functions to promote viral release and is one of the major surface antigens. The receptor-destroying activity in NA resides in the distal head domain that is linked to the viral membrane by an N-terminal hydrophobic transmembrane domain (TMD). Over the last century, the subtype 1 NA TMDs (N1) in human H1N1 viruses have become increasingly more polar, and the head domains have changed to alter their antigenicity. Here, we provide the first evidence that an old N1 head domain from 1933 is incompatible with a recent (2009), more polar N1 TMD sequence and that, during viral replication, the head domain drives the selection of TMD mutations. These mutations modify the intrinsic TMD assembly to restore the head domain folding compatibility and the resultant budding deficiency. This likely explains why the N1 TMDs have become more polar and suggests the N1 TMD and head domain have coevolved.

  • 2014. Dan Dou (et al.). Molecular Biology of the Cell 25 (21), 3363-3374

    Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity. This places stringent hydrophobicity requirements on transmembrane domains (TMDs) from single-spanning membrane proteins. On examining the single-spanning influenza A membrane proteins, we found that the strict hydrophobicity requirement applies to the N-out-C-in HA and M2 TMDs but not the N-in-C-out TMDs from the type II membrane protein neuraminidase (NA). To investigate this discrepancy, we analyzed NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, in mammalian cells and ER microsomes. Our results show that the marginally hydrophobic NA TMDs (Delta G(app) > 0 kcal/mol) require the cotranslational insertion process for facilitating their inversion during translocation and a positively charged N-terminal flanking residue and that NA inversion enhances its plasma membrane localization. Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once similar to 70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by similar to 100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins. Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.

  • 2013. Diogo V. da Silva (et al.). Journal of Biological Chemistry 288 (1), 644-653

    Neuraminidase (NA) is one of the two major influenza surface antigens and the main influenza drug target. Although NA has been well characterized and thought to function as a tetramer, the role of the transmembrane domain (TMD) in promoting proper NA assembly has not been systematically studied. Here, we demonstrate that in the absence of the TMD, NA is synthesized and transported in a predominantly inactive state. Substantial activity was rescued by progressive truncations of the stalk domain, suggesting the TMD contributes to NA maturation by tethering the stalk to the membrane. To analyze how the TMD supports NA assembly, the TMD was examined by itself. The NA TMD formed a homotetramer and efficiently trafficked to the plasma membrane, indicating the TMD and enzymatic head domain drive assembly together through matching oligomeric states. In support of this, an unrelated strong oligomeric TMD rescued almost full NA activity, whereas the weak oligomeric mutant of this TMD restored only half of wild type activity. These data illustrate that a large soluble domain can force assembly with a poorly compatible TMD; however, optimal assembly requires coordinated oligomerization between the TMD and the soluble domain.

  • 2013. Johan Nordholm (et al.). Journal of Biological Chemistry 288 (15), 10652-10660

    Interactions that facilitate transmembrane domain (TMD) dimerization have been identified mainly using synthetic TMDs. Here, we investigated how inherent properties within natural TMDs modulate their interaction strength by exploiting the sequence variation in the nine neuraminidase subtypes (N1-N9) and the prior knowledge that a N1 TMD oligomerizes. Initially, consensus TMDs were created from the influenza A virus database, and their interaction strengths were measured in a biological membrane system. The TMD interactions increased with respect to decreasing hydrophobicity across the subtypes (N1-N9) and within the human N1 subtype where the N1 TMDs from the pandemic H1N1 strain of swine origin were found to be significantly less hydrophobic. The hydrophobicity correlation was attributed to the conserved amphipathicity within the TMDs as the interactions were abolished by mutating residues on the polar faces that are unfavorably positioned in the membrane. Similarly, local changes enhanced the interactions only when a larger polar residue existed on the appropriate face in an unfavorable membrane position. Together, the analysis of this unique natural TMD data set demonstrates how polar-mediated TMD interactions from bitopic proteins depend on which polar residues are involved and their positioning with respect to the helix and the membrane bilayer.

  • 2012. Peter Mellroth (et al.). Journal of Biological Chemistry 287 (14), 11018-11029

    Background: The regulation of cell wall hydrolysis by the pneumococcal autolysin LytA is poorly understood. Results: The cell wall is susceptible to extracellular LytA only during the stationary phase or after cell wall synthesis inhibition. Conclusion: LytA is regulated on the substrate level, where peptidoglycan modifications likely prevent LytA hydrolysis. Significance: The control of amidases is essential for bacterial survival, cell-wall synthesis, and division.

Show all publications by Robert Daniels at Stockholm University

Last updated: July 26, 2017

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