Yukiko Muramoto

In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses

Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.

Avian flu: isolation of drug-resistant H5N1 virus.

The persistence of H5N1 avian influenza viruses in many Asian countries and their ability to cause fatal infections in humans have raised serious concerns about a global flu pandemic. Here we report the isolation of an H5N1 virus from a Vietnamese girl that is resistant to the drug oseltamivir, which is an inhibitor of the viral enzyme neuraminidase and is currently used for protection against and treatment of influenza. Further investigation is necessary to determine the prevalence of oseltamivir-resistant H5N1 viruses among patients treated with this drug.

Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors.

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-α2,6-galactose (SAα2,6Gal), whereas the latter prefer those ending in SAα2,3Gal (refs 3–6). A conversion from SAα2,3Gal to SAα2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.

Contributions of Two Nuclear Localization Signals of Influenza A Virus Nucleoprotein to Viral Replication

ABSTRACT The RNA genome of influenza A virus, which forms viral ribonucleoprotein complexes (vRNPs) with viral polymerase subunit proteins (PA, PB1, and PB2) and nucleoprotein (NP), is transcribed and replicated in the nucleus. NP, the major component of vRNPs, has at least two amino acid sequences that serve as nuclear localization signals (NLSs): an unconventional NLS (residues 3 to 13; NLS1) and a bipartite NLS (residues 198 to 216; NLS2). Although both NLSs are known to play a role in nuclear transport, their relative contributions to viral replication are poorly understood. We therefore investigated their contributions to NP subcellular/subnuclear localization, viral RNA (vRNA) transcription, and viral replication. Abolishing the unconventional NLS caused NP to localize predominantly to the cytoplasm and affected its activity in vRNA transcription. However, we were able to create a virus whose NP contained amino acid substitutions in NLS1 known to abolish its nuclear localization function, although this virus was highly attenuated. These results indicate that while the unconventional NLS is not essential for viral replication, it is necessary for efficient viral mRNA synthesis. On the other hand, the bipartite NLS, whose contribution to the nuclear transport of NP is limited, was essential for vRNA transcription and NPs nucleolar accumulation. A virus with nonfunctional NLS2 could not be generated. Thus, the bipartite NLS, but not the unconventional NLS, of NP is essential for influenza A virus replication.

Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions

ABSTRACT The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3′ coding region are critical for efficient NS vRNA incorporation and that deletion of the 3′ segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3′ NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3′ end) form a structure that is required for efficient influenza A virus vRNA packaging.

Identification of Novel Influenza A Virus Proteins Translated from PA mRNA

ABSTRACT Many replication events are involved in the influenza A virus life cycle, and they are accomplished by different virus proteins with specific functions. However, because the size of the influenza virus genome is limited, the virus uses different mechanisms to express multiple viral proteins from a single gene segment. The M2 and NS2 proteins are produced by splicing, and several novel influenza A virus proteins, such as PB1-F2, PB1-N40, and PA-X, have recently been identified. Here, we identified novel PA-related proteins in influenza A virus-infected cells. These newly identified proteins are translated from the 11th and 13th in-frame AUG codons in the PA mRNA and are, therefore, N-terminally truncated forms of PA, which we named PA-N155 and PA-N182, respectively. The 11th and 13th AUG codons are highly conserved among influenza A viruses, and the PA-N155 and PA-N182 proteins were detected in cells infected with various influenza A viruses isolated from different host species, suggesting the expression of these N-truncated PAs is universal in nature among influenza A viruses. These N-truncated PAs did not show polymerase activity when expressed together with PB1 and PB2; however, mutant viruses lacking the N-truncated PAs replicated more slowly in cell culture and had lower pathogenicity in mice than did wild-type virus. These results suggest that these novel PA-related proteins likely possess important functions in the replication cycle of influenza A virus.

Hierarchy among Viral RNA (vRNA) Segments in Their Role in vRNA Incorporation into Influenza A Virions

ABSTRACT The genome of influenza A viruses comprises eight negative-strand RNA segments. Although all eight segments must be present in cells for efficient viral replication, the mechanism(s) by which these viral RNA (vRNA) segments are incorporated into virions is not fully understood. We recently found that sequences at both ends of the coding regions of the HA, NA, and NS vRNA segments of A/WSN/33 play important roles in the incorporation of these vRNAs into virions. In order to similarly identify the regions of the PB2, PB1, and PA vRNAs of this strain that are critical for their incorporation, we generated a series of mutant vRNAs that possessed the green fluorescent protein gene flanked by portions of the coding and noncoding regions of the respective segments. For all three polymerase segments, deletions at the ends of their coding regions decreased their virion incorporation efficiencies. More importantly, these regions not only affected the incorporation of the segment in which they reside, but were also important for the incorporation of other segments. This effect was most prominent with the PB2 vRNA. These findings suggest a hierarchy among vRNA segments for virion incorporation and may imply intersegment association of vRNAs during virus assembly.

The Cytoplasmic Tail of the Influenza A Virus M2 Protein Plays a Role in Viral Assembly

ABSTRACT The viral replication cycle concludes with the assembly of viral components to form progeny virions. For influenza A viruses, the matrix M1 protein and two membrane integral glycoproteins, hemagglutinin and neuraminidase, function cooperatively in this process. Here, we asked whether another membrane protein, the M2 protein, plays a role in virus assembly. The M2 protein, comprising 97 amino acids, possesses the longest cytoplasmic tail (54 residues) of the three transmembrane proteins of influenza A viruses. We therefore generated a series of deletion mutants of the M2 cytoplasmic tail by reverse genetics. We found that mutants in which more than 22 amino acids were deleted from the carboxyl terminus of the M2 tail were viable but grew less efficiently than did the wild-type virus. An analysis of the virions suggested that viruses with M2 tail deletions of more than 22 carboxy-terminal residues apparently contained less viral ribonucleoprotein complex than did the wild-type virus. These M2 tail mutants also differ from the wild-type virus in their morphology: while the wild-type virus is spherical, some of the mutants were filamentous. Alanine-scanning experiments further indicated that amino acids at positions 74 to 79 of the M2 tail play a role in virion morphogenesis and affect viral infectivity. We conclude that the M2 cytoplasmic domain of influenza A viruses plays an important role in viral assembly and morphogenesis.

Reassortment between avian H5N1 and human H3N2 influenza viruses creates hybrid viruses with substantial virulence

The spread of avian H5N1 influenza viruses around the globe has become a worldwide public health concern. To evaluate the pathogenic potential of reassortant viruses between currently cocirculating avian H5N1 and human H3N2 influenza viruses, we generated all the 254 combinations of reassortant viruses between A/chicken/South Kalimantan/UT6028/06 (SK06, H5N1) and A/Tokyo/Ut-Sk-1/07 (Tok07, H3N2) influenza viruses by reverse genetics. We found that the presence of Tok07 PB2 protein in the ribonucleoprotein (RNP) complex allowed efficient viral RNA transcription in a minigenome assay and that RNP activity played an essential role in the viability and replicative ability of the reassortant viruses. When the pathogenicity of 75 reassortant H5 viruses was tested in mice, 22 were more pathogenic than the parental SK06 virus, and three were extremely virulent. Strikingly, all 22 of these viruses obtained their PB2 segment from Tok07 virus. Further analysis showed that Tok07 PB1 alone lacked the ability to enhance the pathogenicity of the reassortant viruses but could do so by cooperating with Tok07 PB2. Our data demonstrate that reassortment between an avian H5N1 virus with low pathogenicity in mice and a human virus could result in highly pathogenic viruses and that the human virus PB2 segment functions in the background of an avian H5N1 virus, enhancing its virulence. Our findings highlight the importance of surveillance programs to monitor the emergence of human H5 reassortant viruses, especially those containing a PB2 segment of human origin.

Assembly and Budding of Ebolavirus

Ebolavirus is responsible for highly lethal hemorrhagic fever. Like all viruses, it must reproduce its various components and assemble them in cells in order to reproduce infectious virions and perpetuate itself. To generate infectious Ebolavirus, a viral genome-protein complex called the nucleocapsid (NC) must be produced and transported to the cell surface, incorporated into virions, and then released from cells. To further our understanding of the Ebolavirus life cycle, we expressed the various viral proteins in mammalian cells and examined them ultrastructurally and biochemically. Expression of nucleoprotein alone led to the formation of helical tubes, which likely serve as a core for the NC. The matrix protein VP40 was found to be critical for transport of NCs to the cell surface and for the incorporation of NCs into virions, where interaction between nucleoprotein and the matrix protein VP40 is likely essential for these processes. Examination of virus-infected cells revealed that virions containing NCs mainly emerge horizontally from the cell surface, whereas empty virions mainly bud vertically, suggesting that horizontal budding is the major mode of Ebolavirus budding. These data form a foundation for the identification and development of potential antiviral agents to combat the devastating disease caused by this virus.