Influenza Polymerase Activity Correlates with the Strength of Interaction between Nucleoprotein and PB2 through the Host-Specific Residue K/E627

The ribonucleoprotein (RNP) complex is the essential transcription-replication machinery of the influenza virus. It is composed of the trimeric polymerase (PA, PB1 and PB2), nucleoprotein (NP) and RNA. Elucidating the molecular mechanisms of RNP assembly is central to our understanding of the control of viral transcription and replication and the dependence of these processes on the host cell. In this report, we show, by RNP reconstitution assays and co-immunoprecipitation, that the interaction between NP and polymerase is crucial for the function of the RNP. The functional association of NP and polymerase involves the C-terminal ‘627’ domain of PB2 and it requires NP arginine-150 and either lysine-627 or arginine-630 of PB2. Using surface plasmon resonance, we demonstrate that the interaction between NP and PB2 takes place without the involvement of RNA. At 33, 37 and 41°C in mammalian cells, more positive charges at aa. 627 and 630 of PB2 lead to stronger NP-polymerase interaction, which directly correlates with the higher RNP activity. In conclusion, our study provides new information on the NP-PB2 interaction and shows that the strength of NP-polymerase interaction and the resulting RNP activity are promoted by the positive charges at aa. 627 and 630 of PB2

Trends in template/fragment-free protein structure prediction

Predicting the structure of a protein from its amino acid sequence is a long-standing unsolved problem in computational biology. Its solution would be of both fundamental and practical importance as the gap between the number of known sequences and the number of experimentally solved structures widens rapidly. Currently, the most successful approaches are based on fragment/template reassembly. Lacking progress in template-free structure prediction calls for novel ideas and approaches. This article reviews trends in the development of physical and specific knowledge-based energy functions as well as sampling techniques for fragment-free structure prediction. Recent physical- and knowledge-based studies demonstrated that it is possible to sample and predict highly accurate protein structures without borrowing native fragments from known protein structures. These emerging approaches with fully flexible sampling have the potential to move the field forward

The influence of subminimal inhibitory concentrations of netilmicin and ceftriaxone on the interaction of Escherichia coli with host defences.

The effect of sub-MICs of netilmicin and ceftriaxone on the interaction between encapsulated and unencapsulated strains of Escherichia coli and certain host defence mechanisms, i.e. complement activation, opsonization, phagocytosis by human polymorphonuclear leucocytes (PMN), and serum bactericidal activity have been studied. Experiments were carried out testing antibiotics either alone or in combination. Non-capsulated strains of E. coli activated complement rapidly and were easily phagocytosed and killed after opsonization in human pooled serum. Pretreatment of these strains with sub-MICs of antibiotics did not change the rate of opsonization or the degree of uptake by PMN, but did enhance serum sensitivity. Capsulated strains of E. coli were both poorly opsonized and resistant to serum bactericidal activity. Treatment of these strains with sub-MICs of antibiotics enhanced complement consumption as well as phagocytosis by PMN, but did not affect serum-resistance

The role of the influenza virus RNA polymerase in host shut-off

Viruses induce an antiviral host response by activating the expression of antiviral host genes. However, viruses have evolved a wide range of strategies to counteract antiviral host responses. One of the strategies used by many viruses is the general inhibition of host gene expression, also referred to as a host shut-off mechanism. Here we discuss our recent findings that influenza virus infection results in the inhibition and degradation of host RNA polymerase II (Pol II) and that the viral RNA polymerase plays a critical role in this process. In particular, we found that Pol II is ubiquitylated in influenza virus infected cells and ubiquitylation can be induced by the expression of the RNA polymerase. Moreover, the expression of an antiviral host gene could be inhibited by the overexpression of the RNA polymerase. Both ubiquitylation and the inhibition of the host gene were dependent on the ability of the RNA polymerase to bind to Pol II. Further studies will be required to understand the interplay between the host and viral transcriptional machineries and to elucidate the exact molecular mechanisms that lead to the inhibition and degradation of Pol II as a result of viral RNA polymerase binding. These findings extend our understanding of how influenza virus counteracts antiviral host responses and underpin studies into the mechanisms by which the RNA polymerase determines virulence