Recently, a novel 87-amino acid influenza A virus protein with proapoptotic properties, PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most known human influenza A virus isolates. Here we characterize the molecular structure of a biologically active synthetic version of the protein (sPB1-F2). Western blot analysis, chemical cross-linking, and NMR spectroscopy afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization mediated by two distinct domains located in the N and C termini, respectively. CD and 1 H NMR spectroscopic analyses indicate that the stability of structured regions in the molecule clearly depends upon the hydrophobicity of the solvent. In aqueous solutions, the behavior of sPB1-F2 is typical of a largely random coil peptide that, however, adopts ␣-helical structure upon the addition of membrane mimetics.1 H NMR analysis of three overlapping peptides afforded, for the first time, direct experimental evidence of the presence of a C-terminal region with strong ␣-helical propensity comprising amino acid residues Ile 55 -Lys 85 connected via an essentially random coil structure to a much weaker helix-like region, located in the N terminus between residues Trp 9 and Lys 20 . The C-terminal helix is not a true amphipathic helix and is more compact than previously predicted. It corresponds to a positively charged region previously shown to include the mitochondrial targeting sequence of PB1-F2. The consequences of the strong oligomerization and helical propensities of the molecule are discussed and used to formulate a hypothetical model of its interaction with the mitochondrial membrane.Influenza A virus (IAV) 3 is one of the most common pathogens threatening humans and animals, with the potential to cause disastrous pandemics. In the last century, it was the origin of at least three pandemics, the most serious outbreak being the "Spanish flu" (1918 -1919) that claimed 20 -50 million casualties worldwide (for a review, see Ref. 1). Apart from various mammals, IAV also infects avian hosts, and particularly aquatic birds have been shown to be the primary reservoir. Sporadically, some of these avian strains acquire the capability to infect other mammals or humans either as a whole or more likely upon genetic reassortment with prevailing human IAV strains. This process termed antigenic (viral) shift appears to occur via the pig as an intermediate host and "mixing vessel" and can lead to new IAV subtypes of mixed surface antigens (2, 3).The genome of IAV, a representative of the orthomyxoviruses, consists of eight separate linear segments of negative sense RNA and was thought to encode 10 gene products. Only very recently, while screening for major histocompatibility complex class I epitopes derived from out-of-frame viral polypeptides, an 11th IAV gene product, named PB1-F2, was incidentally discovered (4). Like the two proteins M1 (matrix protein) and M2 (ion channel) encoded on the M gene segment and t...

BackgroundThe HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6.ResultsConsistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site.ConclusionsOverall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and virus core formation, and consequently the infectivity of released virions.

SummaryThe 11 th influenza A virus (IAV) protein PB1-F2 is encoded by an alternative reading frame of the PB1 polymerase gene and found in the nucleus, cytosol and at the mitochondria of infected cells, the latter is consistent with experimental evidence for its pro-apoptotic function. Here, the function of PB1-F2 as a phosphoprotein was characterized. PB1-F2 derived from isolate IAVPR8 and synthetic fragments thereof were phosphorylated in vitro by purified protein kinase C (PKC) and cellular extract. Constitutively active PKCa interacts with PB1-F2 in yeast two-hybrid assays.

Mutational analysis of the four conserved proline residues in human immunodeficiency virus type 1 (HIV-1) Vpr reveals that only Pro-35 is required for efficient replication of R5-tropic, but not of X4-tropic, viruses in human lymphoid tissue (HLT) cultivated ex vivo. While Vpr-mediated apoptosis and G 2 cell cycle arrest, as well as the expression and subcellular localization of Vpr, were independent, the capacity for encapsidation of Vpr into budding virions was dependent on Pro-35. 1 H nuclear magnetic resonance data suggest that mutation of Pro-35 causes a conformational change in the hydrophobic core of the molecule, whose integrity is required for the encapsidation of Vpr, and thus, Pro-35 supports the replication of R5-tropic HIV-1 in HLT.