The Contribution of Viral Proteins to the Synergy of Influenza and Bacterial Co-Infection

A severe course of acute respiratory disease caused by influenza A virus (IAV) infection is often linked with subsequent bacterial superinfection, which is difficult to cure. Thus, synergistic influenza–bacterial co-infection represents a serious medical problem. The pathogenic changes in the infected host are accelerated as a consequence of IAV infection, reflecting its impact on the host immune response. IAV infection triggers a complex process linked with the blocking of innate and adaptive immune mechanisms required for effective antiviral defense. Such disbalance of the immune system allows for easier initiation of bacterial superinfection. Therefore, many new studies have emerged that aim to explain why viral–bacterial co-infection can lead to severe respiratory disease with possible fatal outcomes. In this review, we discuss the key role of several IAV proteins—namely, PB1-F2, hemagglutinin (HA), neuraminidase (NA), and NS1—known to play a role in modulating the immune defense of the host, which consequently escalates the development of secondary bacterial infection, most often caused by Streptococcus pneumoniae. Understanding the mechanisms leading to pathological disorders caused by bacterial superinfection after the previous viral infection is important for the development of more effective means of prevention; for example, by vaccination or through therapy using antiviral drugs targeted at critical viral proteins

The ubiquitination of the influenza A virus PB1-F2 protein is crucial for its biological function.

The aim of the present study was to identify what influences the short half-life of the influenza A virus PB1-F2 protein and whether a prolonged half-life affects the properties of this molecule. We hypothesized that the short half-life of PB1-F2 could conceal the phenotype of the protein. Because proteasome degradation might be involved in PB1-F2 degradation, we focused on ubiquitination, a common label for proteasome targeting. A cluster of lysine residues was demonstrated as an ubiquitination acceptor site in evolutionary and functionally distinct proteins. The PB1-F2 sequence alignment revealed a cluster of lysines on the carboxy terminal end of PB1-F2 in almost all of the GenBank sequences available to date. Using a proximity ligation assay, we identified ubiquitination as a novel posttranslational modification of PB1-F2. Changing the lysines at positions 73, 78, and 85 to arginines suppressed the ubiquitination of A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. The mutation of the C-terminal lysine residue cluster positively affected the overall expression levels of avian A/Honk Kong/156/1997 (H5N1)- and mammalian A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. Moreover, increased PB1-F2 copy numbers strengthened the functions of this virus in the infected cells. The results of a minigenome luciferase reporter assay revealed an enhancement of viral RNA-dependent RNA polymerase activity in the presence of stabilized PB1-F2, regardless of viral origin. IFNβ antagonism was enhanced in 293T cells transfected with a plasmid expressing stabilized K→R mutant variants of PB1-F2. Compared with PB1-F2 wt, the loss of ubiquitination enhanced the antibody response after DNA vaccination. In summary, we revealed that PB1-F2 is an ubiquitinated IAV protein, and this posttranslational modification plays a central role in the regulation of the biological functions of this protein

Perspektiva užívání antivirových peptidů proti chřipkovým virům

The threat of a worldwide influenza pandemic has greatly increased over the past decade with the emergence of highly virulent avian influenza strains. The increased frequency of drug-resistant influenza strains against currently available antiviral drugs requires urgent development of new strategies for antiviral therapy, too. The research in the field of therapeutic peptides began to develop extensively in the second half of the 20th century. Since then, the mechanisms of action for several peptides and their antiviral prospect received large attention due to the global threat posed by viruses. Here, we discussed the therapeutic properties of peptides used in influenza treatment. Peptides with antiviral activity against influenza can be divided into three main groups. First, entry blocker peptides such as a Flupep that interact with influenza hemagglutinin, block its binding to host cells and prevent viral fusion. Second, several peptides display virucidal activity, disrupting viral envelopes, e.g., Melittin. Finally, a third set of peptides interacts with the viral polymerase complex and act as viral replication inhibitors such as PB1 derived peptides. Here, we present a review of the current literature describing the antiviral activity, mechanism and future therapeutic potential of these influenza antiviral peptides.Hrozba celosvětové pandemie chřipky výrazně vzrostla oproti minulosti díky vzniku vysoce nakažlivých chřipkových virů ptáků. Zvýšená frekvence chřipkových kmenů rezistentních vůči současným antivioritikům vyžaduje také rozvoj nových strategií pro antivirové terapie. Výzkum v oblasti terapeutických peptidů se začal značně rozvíjet v druhé polovině 20. století. Od té doby, mechanismy působení účinků na několik peptidů a jejich antivirové vyhlídky získaly velkou pozornost díky globání hrozbě představované viry . V této práci jsme diskutovali terapeutické vlastnosti peptidů používaných v léčbě chřikového onemocnění. Peptidy s antivirovou aktivitou proti chřipce mohou být rozděleny do tří hlavních skupin. Za prvé, blokovací peptidy, jako je například Flupep, které vzájemně působní s chřipkovým hemagglutinin, blokují jeho vazbu na hostitelských buňkách a zabraňují virové fúze. Za druhé, některé peptidy zobrazují viroidní aktivitu, přerušují virové obály, například Melittin. Konečnou třetí sada peptidů vzájemně působí s virovými polymerázy komplexy a půsboní jako inhibitory virové replikace, jako je pb1 odvozených peptidů. Prezentujeme zde přehled o současné literatuře popisující antivirovou aktivitu, mechanismus a budoucnost terapeutického potenciálů těchto chřipkových protivirových peptidů

Effect of PB1-F2 stabilization on the induction of the human IFN-β promoter.

<p>(A) A 293T cells were co-transfected with the human IFN-β promoter reporter plasmid pGLhIFNBpr, pRLTK (transfection efficiency normalization) and the respective PB1-F2 expressing plasmids (pPB1-F2 PR8/pPB1-F2 PR8 3KR/pPB1-F2 HK97/pPB1-F2 HK97 4KR/pPB1-F2 Stop3aa). At 24 hr p.t., the IFN-β promoter was further induced through the addition of pIC. At 15, 24 and 40 hr p.t., the supernatant was removed, and the cells were gently washed, directly lysed using passive lysis buffer (Promega) and the luciferase activity was quantified using the Dual Luciferase Reporter Assay System kit (Promega) on a BIOTEK Synergy HT luminometer. The samples were measured in triplicate and normalized to renilla luciferase activity (transfection efficiency) and firefly luciferase activity (human IFN-β promoter induction) determined for pPB1-F2 Stop3aa co-transfected samples. The data represent 3 independent experiments. The bars indicate the average value of the independent experiments, and the error bars indicate the intra-experiment SD values. The asterisk indicates a significant difference between the groups compared (* <i>P<0</i>.<i>05</i>; ** <i>P<0</i>.<i>01</i>; *** <i>P<0</i>.<i>001</i>). (B) PB1-F2 protein and Beta-actin was detected through western blotting to compare the induction of IFN-β promoter with the relative amount of the PB1-F2 in the same samples.</p

Identification of the cluster of C-terminal K residues as an ubiquitination site of PB1-F2.

<p>MDCK cells were transfected transiently with 1 μg pPB1-F2 PR8, pPB1-F2 PR8 3KR or pPB1-F2 Stop3aa. At 24 hr p.t., the cells were fixed and stained for the presence of the subpopulation of ubiquitinated PB1-F2 (labeled UBQ-PB1-F2, red spots) and PB1-F2 (green). (A) and analyzed with confocal microscopy. (B) Z stack acquisition was applied to confirm the absence of UBQ-PB1-F2 (red spots), regardless of the focal plane displayed. Orthogonal section reconstruction confirmed that the distribution of PB1-F2 PR8 3KR was equal to the wt PB1-F2 protein. (C) No UBQ-PB1-F2 (red spots) was observed in the cells transfected with pPB1-F2 PR8 3KR in multiple focal planes orthogonal section reconstruction. The nuclei were stained with DAPI.</p

Subcellular localization of PR8- and HK97-derived PB1-F2 wt and the C-terminal K residue cluster mutants.

<p>MDCK cells were transfected transiently with the respective PB1-F2 expressing plasmid (1 μg pPB1-F2 PR8/pPB1-F2 PR8 3KR/pPB1-F2 HK97/ pPB1-F2 HK97 4KR). At 24 hr p.t., the cells were stained with Mitotracker RedCMXRos according to the manufacturer’s instructions (Life Technologies), fixed and stained for the presence of PB1-F2 variants (A). mAb AG55, specific to N-terminal region of the PB1-F2, and the FITC-conjugated secondary antibody were used to stain PB1-F2 (green, left column). Mitotracker RedCMXRos was used to stain mitochondria (red, second left column). Merge of PB1-F2 and mitochondrial signals (yellow, right column) showing clearly visible colocalization in PB1-F2 PR8 and PB1-F2 PR8 3KR-positive cells. The nuclei were stained with DAPI. (B) Orthogonal section reconstruction suggests the colocalization of the PB1-F2 with mitochondria signal throughout the entire cytoplasm for PR8-derived PB1-F2 and PB1-F2 PR8 3KR, but not for PB1-F2 HK97 and PB1-F2 HK97 4KR. The images were acquired at the same exposure, pinhole and optical slice conditions. (C) Image analysis software (Bitplane Imaris) was used to generate 2D histograms of the PB1-F2 (green) and mitochondria (red) signal intensities for volume pixels (voxels) and calculating Pearson’s coefficient in colocalized volume.</p

Effect of PB1-F2 stabilization on the activity of vRdRp.

<p>(A) A minigenome reporter assay was used for the quantification of the effect of PB1-F2C-terminal K cluster mutation on the activity of vRdRp. The 293T cells were transfected with a set of expression plasmids for the vRdRp subunits pPB1, pPB2, pPA, and pNP, pRLTK (transfection efficiency normalization) and the reporter plasmid pPolPR8MLuc, encoding the firefly luciferase gene under the control of the IAV matrix protein gene segment promoter. Additionally, pPB1-F2 PR8, pPB1-F2 PR8 3KR, pPB1-F2HK97, or pPB1-F2 HK97 4KR plasmids were co-transfected. At 48 hr p.t., the samples were harvested and the activities of luciferases were quantified using a Dual Luciferase Reporter Assay System kit (Promega) on a BIOTEK Synergy HT luminometer. The samples were measured in triplicate and normalized to renilla luciferase activity (transfection efficiency) and firefly luciferase activity (vRdRp activity) in samples co-transfected with pPB1-F2 PR8. The data represent 3 independent experiments. The bars indicate the average value of the independent experiments, and the error bars indicate the intra-experiment SD values. The asterisk indicates a significant difference between the groups compared (<i>P<0</i>.<i>05</i>). (B) PB1-F2 protein and Beta-actin were detected using western blotting to compare the relative activity of vRdRp with the relative amount of the PB1-F2 in the same samples.</p

Antibody responses to pPB1-F2 PR8 vs. pPB1-F2 PR8 3KR after DNA immunization.

<p>The mice (n═ 5) were immunized four times at two-week intervals with 50 μg of pPB1-F2 PR8 or pPB1-F2 PR8 3KR DNA. MDCK cells were transfected with pPB1-F2 PR8 and lysed directly in the wells at 24 hr p. t. using SDS sample buffer. The proteins were separated through SDS-PAGE in a single well and transferred to nitrocellulose membrane. The sera were pooled after each dose and assayed for the presence of anti-PB1-F2 antibodies using immunoblotting on a sliced nitrocellulose membrane.</p