Schematic representation of the functional domains with mutated residues in the 2009 H1N1pdm proteins.

<p><b>Left panels</b>: bird's eye view of protein structures of 2009 H1N1pdm collected at the pre-epidemic period in 2009; <b>Middle panels</b>: close-up view of the mutated amino acid residues in proteins of 2009 H1N1pdm collected at the pre-epidemic period in 2009; <b>Right panels</b>: close-up view of the mutated amino acid residues in proteins of 2009 H1N1pdm collected at the late period in 2009. The amino acid numberings were based on influenza virus A/Puerto Rico/8/1934 (H1N1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009549#pone.0009549-Chen1" target="_blank">[6]</a>. The residues in viruses collected in the pre-epidemic period are colored in red, and those in viruses collected in the late period are colored in yellow. <b>A–C:</b> NP trimer and monomer. <b>D–F:</b> NA tetramer and monomer. Drug target domain (DTD) is highlighted in dark blue. H260 [274 in A/Vietnam/1203/04(H5N1)] is a critical residue for the NA inhibitor, oseltamivir. NA H274Y mutation results in resistance of 2009 H1N1pdm and other influenza viruses to oseltamivir. <b>G–I</b>: HA trimer and monomer. Receptor binding domain (RBD) was highlighted in wheat color, while other part is in green color. <b>J–L</b>: Dimer and monomer of effector domain (ED) in NS1.</p

Mutation trend analysis of signature and non-signature amino acid residues in the functional domains of the proteins of 2009 H1N1pdm during the influenza pandemic in 2009.

<p>Note: the number in brackets indicates the percent (%) of sequences with the mutated amino acid in total number of the sequences collected in the period; ‘-’ means unavailable in non-H1N1 virus.</p

Comparison of the amino acid signatures in the proteins of 2009 H1N1pdm with those in human, swine, and avian IAVs, as well as those causing past influenza pandemics.

<p>Note: Only the dominant residues were listed. The human-like amino acid signatures in the 2009 H1N1pdm were highlighted in bold and italic.</p

The identity of amino acid signatures in the proteins of pandemic IAVs and human, swine, and avian IAVs.

<p>The identity of amino acid signatures in the proteins of pandemic IAVs and human, swine, and avian IAVs.</p

CD spectrographic analysis of the complexes formed between N36 and C34 or their mutants.

<p>(<b>A</b>) The secondary structures of N36 and its mutants; (<b>B</b>) The secondary structures of the complexes formed by C34 and N36 or N36’s mutants; (<b>C</b>) The secondary structures of the complexes formed by N36 and C34 or C34’s mutants; and (<b>D</b>) The stability of complexes formed by C34 with N36 and its mutants, as measured by thermal denaturation analysis.</p

Analysis of putative interactions of R46 in the gp41 NHR with the residues in the 6-HB formed between C34 and N36 or their mutants.

<p>(<b>A</b>) The 6-HB formed between C34 and N36; (<b>B</b>) The 6-HB formed between C34 and N36 R46A; (<b>C</b>) The 6-HB formed between C34 and R46E; (<b>D</b>) The 6-HB formed between C34 E137A and N36; and (<b>E</b>) The 6-HB formed between C34 E137R and N36. The putative interactions via hydrogen bond or salt bridge were predicted by using the MOE program. NHRs are colored in blue; CHR is colored in green.</p

Effect of R46 mutations in gp41 NHR region on viral infectivity and Env expression.

<p>(<b>A</b>) Infectivity of HIV-1 pseudoviruses carrying wild-type N36 and its mutants. Luciferase assay was used to measure the single-cycle infection of pseudovirus on TZM-bl cells. (<b>B</b>) Expression of gp120 and gp160 on pseudoviruses bearing wild-type N36 sequence and its mutants as determined by Western blot.</p

Using the MOE program to predict hydrogen bonds and salt bridges in 6-HB formed by C34 and N36, or N36’s mutants, or by N36 and C34, or C34’s mutants.

*<p>“0” indicates the absence of hydrogen bond or salt bridge between the two corresponding residues.</p

Interaction of C34 with N36 and its mutants to form 6-HB, as determined by FN-PAGE.

<p>C34 was labeled with FAM (carboxyfluorescein, Molecular Probes, Eugene, OR), which could be viewed under UV light.</p

Inhibition of the peptides N36, C34 and their mutants on HIV-1_IIIB-mediated cell-cell fusion and HIV-1_IIIB infection.

*<p>Compared with the corresponding wild-type peptide N36 or C34 (Student <i>t</i>-test was performed using Excel).</p