Translational regulation of viral secretory proteins by the 5' coding regions and a viral RNA-binding protein.

A primary function of 5' regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. In this study, we show how the regions coding for the ER-targeting sequences of the influenza glycoproteins NA and HA also function as translational regulatory elements that are controlled by the viral RNA-binding protein (RBP) NS1. The translational increase depends on the nucleotide composition and 5' positioning of the ER-targeting sequence coding regions and is facilitated by the RNA-binding domain of NS1, 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 analyzing human protein mRNA sequences, we found evidence that this mechanism of using 5' coding regions and particular RBPs to achieve gene-specific regulation may extend to human-secreted proteins.

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins.

UNLABELLED: 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 Km 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 (Km 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 full-length 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.

The influenza virus neuraminidase protein transmembrane and head domains have coevolved.

UNLABELLED: 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°C compared to at 33°C, at which NA folds more efficiently. Passaging the chimera viruses at 37°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 neuraminidase (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.

Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion.

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 (ΔG(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 ~70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by ~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.

Polar residues and their positional context dictate the transmembrane domain interactions of influenza A neuraminidases.

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.

Assembly of subtype 1 influenza neuraminidase is driven by both the transmembrane and head domains.

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.

Luminescent lanthanide complexes with analyte-triggered antenna formation.

A new strategy for accessing analyte-responsive luminescent probes is presented. The lanthanide luminescence of Eu and Tb centers is switched on by the analyte-triggered formation of a sensitizing antenna from a nonsensitizing caged precursor. As the cage can be freely varied, an array of probes for different analytes (Pd(0/2+), H(2)O(2), F(-), ß-galactosidase) can be created from the same core structure. The probe design affords nanomolar to micromolar detection limits, provides the capability to detect two analytes in parallel, and can be utilized to monitor enzymatic activity in live cells.