Influence of extracellular and cytoplasmic domains of M2 ion channel of influenza a virus on its activity.

Co-expression of M2 protein of Influenza A virus (IAV) with pH-sensitive hemagglutinin (HA) reduces the conversion of HA to its low pH conformation during transport to the cell surface. Mutational analysis of extracellular domain of M2 protein showed that single aa substitution W 15A did not influence the ion channel activity of M2 protein. Replacement of first 24 aa of M2 protein with first 18 aa of NB protein of Influenza B virus in chimera BAA resulted in the loss of ion channel activity. The chimera B(SSD)AA had only first 21 aa replaced with first 18 aa of NB protein The remaining aa at positions 22, 23, and 24 were preserved from the wtM2 protein. The ion channel activity of B(SSD)AA chimera was fully restored and showed that the aa S22, S23, and D24 from wtM2 were important for the ion channel activity. However, these aa did not contribute to the activity of M2 protein directly, since they could be substituted by A, R, or H without change in ion channel activity. Mutational analysis of cytoplasmic domain of M2 protein showed that substitutions C50S, C50P, and H90S did not change the ion channel activity. The extracellular and cytoplasmic domains of M2 protein were not essential for ion channel activity of M2 protein.

Two biologically distinct isolates of Zucchini yellow mosaic virus lack seed transmissibility in cucumber.

The seed transmission of the Zucchini yellow mosaic virus (ZYMV) was studied in cucumber using two isolates unrelated in their biological characteristics. Although the virus could be readily detected in mature seeds harvested from infected cucumbers, the seedlings obtained from infected germinated seeds tested negative for ZYMV using both ELISA and RT-PCR assays. No evidence was obtained for transmission of two ZYMV isolates through seeds.

Electrophoretic mobility of the capsid protein of the Plum pox virus strain PPV-Rec indicates its partial phosphorylation.

A double-band SDS-PAGE profile was found reproducible for capsid protein (CP) of Plum pox virus (PPV) isolates belonging to the strain PPV-Rec. The double-band was also present in the virus population multiplied in various plants. A single-lesion passage in a hypersensitive host Chenopodium foetidum showed that its presence was not a result of a mixed infection. We found that the two electrophoretic forms of CP shared identical N-terminus. Therefore, they did not originate from an alternative proteolytic processing, but were different in their posttranslational modification. The slower band of CP could be converted to the faster one by the phosphatase treatment. We assumed that CP protein was present in both phosphorylated and dephosphorylated forms in the infected plants.

pH modulating activity of ion channels of influenza A, B, and C viruses.

The BM2 and NB proteins of Influenza B virus (the B virus) and the CM2 protein of Influenza C virus (the C virus) are structural homologs of the M2 protein of Influenza A virus (the A virus). It was shown recently that CM2 in vitro forms a voltage-activated ion channel permeable to chloride ion (Hongo et al., Arch. Virol. 149, 35-50, 2004). To demonstrate a possible pH modulating activity of BM2, NB and CM2, the latters were co-expressed with a pH-sensitive hemagglutinin (HA) of the A virus. BM2 was able to replace functionally M2 and prevented the A virus HA from adopting its low-pH conformation during transport to the cell surface. In contrast, NB had a negative effect on the quality of the co-expressed HA and was unable to modulate the pH in the trans-Golgi network (TGN) and to protect HA. A pH modulating activity was also demonstrated for CM2, but it was much lower than that of M2.

Expression of a part of the Potato virus A non-structural protein P3 in Escherichia coli for the purpose of antibody preparation and P3 immunodetection in plant material.

The N-terminal part of the Potato virus A (PVA) P3 protein was cloned into two E. coli fusion expression systems. An overexpression of the P3 fragment fused with thioredoxin was observed between 2 and 21 h after induction. The protein formed insoluble inclusions. Decreasing the cultivation temperature did not enhance its solubility. To obtain antigen for antibody preparation, inclusions were concentrated and purified by sucrose gradient centrifugation, and subjected to SDS-polyacrylamide gel electrophoresis. The band specific for the protein was excised from the gel and used for rabbit immunization. Obtained antibody tested positive with high specificity in immunoblots of expressed PVA P3 fused with either thioredoxin or GST. The antibody was also applied for the detection of P3 protein in plant material by immunoblot. Previous plant sap concentration was essential for most samples. Three concentration methods were tested: simple centrifugal size-exclusion filtration, the same preceded with high-speed centrifugation at 250,000 x g, and differential ammonium sulfate precipitation. The last approach was the most convenient. Plants tested included PVA P3-transgenic tobacco lines as well as PVA-infected wild-type tobacco. In all cases, mature P3 with a molecular mass of 40 kDa was detected.

Influenza viruses and their ion channels.

Influenza is an ancient disease that has infected humans in irregular intervals throughout recorded history. The most infamous pandemic was "Spanish Flu" which affected large parts of the world population and killed in 1918-1919, at a rough estimate, at least 50 million people. More recently, two influenza A pandemics occurred in 1957 ("Asian influenza") and 1968 ("Hong Kong influenza") and caused significant morbidity and mortality globally. Most recently, in 1997 and 2003, limited outbreaks caused by a new influenza A virus subtype H5N1 that was directly transmitted from birds to humans, occurred in the Hong Kong Special Administrative Region of China. Since 2003, the avian H5N1 strain has infected more then 130 persons in Vietnam, Thailand, and Cambodia and has killed more than half of them. To prevent the human and economical losses caused by human and avian influenza viruses, it is necessary to prepare safe universal influenza vaccines. In order to develop a broad-spectrum protection against different influenza virus strains or variants, some recent studies have were aimed at the M2 protein of Influenza A virus. This review compares the influenza A, B, and C viruses by focusing on their ion channels.

Plum Pox Virus Mixed Infection Detected on Apricot in Pakistan.

Sharka, caused by Plum pox virus (PPV), is the most detrimental viral disease of stone fruit trees. First reported from Bulgaria in 1917, the virus is now widespread in Europe, the Mediterranean Basin, and Asia Minor and is sporadically present in North and South America. On the basis of molecular and serological properties, six PPV subgroups are recognized, from which PPV-D, PPV-M, and PPV-Rec are the most common (1,2). Several apricot trees (Prunus armeniaca) showing mild, pale green rings and diffuse chlorotic spots on leaves were found in a small orchard in the Baltistan District in northern Pakistan at approximately 2,400 m above sea level. Dried leaf samples from one symptomatic tree randomly selected from the orchard were positive for PPV using double-antibody sandwich enzyme-linked immunosorbent assay with antisera prepared in the laboratory, immunoblot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) targeting the capsid protein (CP) gene using standard procedures (1). To check the subgroup affiliation and evaluate the molecular variability, the 562-bp variable region spanning the C-terminus of NIb and the N-terminus of the CP was amplified, the RT-PCR product was cloned into the pGEM-T Easy vector (Promega, Madison, WI), and positive clones were analyzed by restriction and sequence analyses. Interestingly, sequence analysis of four clones revealed mixed infection, i.e., the presence of two different PPV isolates in the apricot sample. One isolate belonged to PPV-D (GenBank Accession No. DQ422147) and the other belonged to the PPV-Rec subgroup (GenBank Accession No. DQ422148). Multiple alignment of the sequenced genome portion of the Pakistan PPV-D isolate indicated 96 to 99% nt identity with various PPV-D isolates without unique, clear-cut differences. Similarly, the PPV-Rec isolate had 98 to 99% identity with European PPV-Rec isolates and retained the cross-over at nucleotide position 8450 in the 3' terminus of NIb. This sequence had the amino acid signature at the N-terminus of the CP typical of the PPV-Rec subgroup (2). Moreover, no particular clustering of the Pakistan isolates within PPV-D and PPV-Rec could be observed after phylogenetic analysis. The DAG motif, essential for aphid transmission, was present in both sequences. To our knowledge, this is the first indication of PPV occurrence in Pakistan and first identification of the PPV-Rec isolate outside Europe. Together with previous reports on the PPV presence in China and Kazakhstan (3,4), this report indicates the need for more detailed epidemiological studies focusing the PPV spread and its molecular diversity in Asia. References: (1) T. Candresse et al. Phytopathology 88:198, 1998. (2) M. Glasa et al. J. Gen. Virol. 85:2671, 2004. (3) M. Navrátil et al. Plant Dis. 89:338, 2005. (4) S. Spiegel et al. Plant Dis. 88:973, 2004.