MicrobiologyBytes

Studies of virus particles and the steps in their life cycle have spearheaded our understanding of biological systems at the molecular level. These studies, however, relied on virus specimens isolated from nature. This dependency changed forever in 2002 when the chemical synthesis of poliovirus, in the absence of any natural template, was published. The work caused a shock wave because it led to excitement as well as revulsion, reflecting the new reality that, for better or worse, all of the more than 2,000 viruses whose genome sequences are deposited by the National Center for Biotechnology Information can be recreated in the laboratory in the absence of natural isolates. So what have we learned?

Synthetic poliovirus and other designer viruses: what have we learned from them? (2011) Annu Rev Microbiol. 65:583-609
Owing to known genome sequences, modern strategies of DNA synthesis have made it possible to recreate in principle all known viruses independent of natural templates. We describe the first synthesis of a virus (poliovirus) in 2002 that was accomplished outside living cells. We comment on the reaction of laypeople and scientists to the work, which shaped the response to de novo syntheses of other viruses. We discuss those viruses that have been synthesized since 2002, among them viruses whose precise genome sequence had to be established by painstakingly stitching together pieces of sequence information, and viruses involved in zoonosis. Synthesizing viral genomes provides a powerful tool for studying gene function and the pathogenic potential of these organisms. It also allows modification of viral genomes to an extent hitherto unthinkable. Recoding of poliovirus and influenza virus to develop new vaccine candidates and refactoring the phage T7 DNA genome are discussed as examples.

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The transmissible agent of prion disease consists of a prion protein in its abnormal conformation (PrP^Sc), which replicates itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrP^Sc. This new study reports that infectious prions and transmissible prion disease can be triggered in wild type animals by amyloid fibrils produced from recombinant prion prtotein, which are structurally different from PrP^Sc and lacks any detectable PrP^Sc particles.

This work introduces a new hypothesis that transmissible prion diseases can be induced by prion protein structures different from that of authentic PrP^Sc and suggests that a new mechanism for triggering PrP^Sc formation different from the classical templating exists. The current work provides important new insight into the mechanisms underlying genesis and evolution of the transmissible states of the prion protein and has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.

Genesis of Mammalian Prions: From Non-infectious Amyloid Fibrils to a Transmissible Prion Disease. (2011) PLoS Pathog 7(12): e1002419. doi:10.1371/journal.ppat.1002419
The transmissible agent of prion disease consists of a prion protein in its abnormal, β-sheet rich state (PrP^Sc), which is capable of replicating itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrP^Sc template. Here we report that authentic PrP^Sc and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP) amyloid fibrils, which are structurally different from PrP^Sc and lack any detectable PrP^Sc particles. When induced by rPrP fibrils, a long silent stage that involved two serial passages preceded development of the clinical disease. Once emerged, the prion disease was characterized by unique clinical, neuropathological, and biochemical features. The long silent stage to the disease was accompanied by significant transformation in neuropathological properties and biochemical features of the proteinase K-resistant PrP material (Pr^Pres) before authentic PrP^Sc evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrP^Sc and suggests that a new mechanism different from the classical templating exists. This new mechanism designated as “deformed templating” postulates that a change in the PrP folding pattern from the one present in rPrP fibrils to an alternative specific for PrP^Sc can occur. The current work provides important new insight into the mechanisms underlying genesis of the transmissible protein states and has numerous implications for understanding the etiology of neurodegenerative diseases.

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To deliver their genomes into host cells during entry, enveloped viruses contain glycoproteins that bind to cellular receptors and cause fusion of viral and cellular membranes. The influenza virus Hemagglutinin (HA) protein is the archetypal viral fusion glycoprotein, promoting entry by undergoing irreversible structural changes that drive membrane merger. HA trimers on the surfaces of infectious influenza virions are trapped in a metastable, high-energy conformation and are triggered to refold and cause membrane fusion after the virus is internalized and exposed to low pH.

This paper provides biochemical and x-ray crystallographic evidence that naturally occurring amino-acid variations at the interface of the esterase and fusogenic domains alter HA acid stability for highly pathogenic H5N1 influenza, resulting in a shift in the threshold pH required to activate HA protein structural changes that cause membrane fusion. Furthermore, the data reveals that an increased HA activation pH correlates with increased H5N1 virulence in chickens. Overall, the acid stability of the HA protein is identified as a novel virulence factor for emerging H5N1 influenza viruses. A major implication of this work is that the fitness of enveloped viruses may be fine-tuned by mutations that alter the activation energy thresholds of their fusion glycoproteins.

Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity. (2011) PLoS Pathog 7(12): e1002398. doi:10.1371/journal.ppat.1002398
Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 Å resolution and two structures of HP HA at 2.95 and 3.10 Å resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

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Viruses infecting higher plants are typically small RNA viruses that encode only a few genes. Although small viruses have recently been discovered that infect algae, many viruses infecting eukaryotic algae are huge dsDNA viruses with genomes ranging from 160 to 560 kb with up to 600 protein-encoding genes and are the subject of this review. These large viruses (family Phycodnaviridae), are found in aqueous environments throughout the world and play dynamic, albeit largely undocumented, roles in regulating algal communities such as the termination of massive algal blooms commonly referred to as red and brown tides.

This review focuses on one genus in the Phycodnaviridae, the chloroviruses, which are large, icosahedral, plaque-forming, dsDNA-containing viruses that replicate in certain unicellular, chlorella-like green algae. Their structure, their initial stages of infection, and many of their genes resemble bacteriophages more than viruses that infect eukaryotes – i.e. they are not your everyday plant virus.

Chloroviruses: not your everyday plant virus. Trends Plant Sci. Nov 17 2011
Viruses infecting higher plants are among the smallest viruses known and typically have four to ten protein-encoding genes. By contrast, many viruses that infect algae (classified in the virus family Phycodnaviridae) are among the largest viruses found to date and have up to 600 protein-encoding genes. This brief review focuses on one group of plaque-forming phycodnaviruses that infect unicellular chlorella-like green algae. The prototype chlorovirus PBCV-1 has more than 400 protein-encoding genes and 11 tRNA genes. About 40% of the PBCV-1 encoded proteins resemble proteins of known function including many that are completely unexpected for a virus. In many respects, chlorovirus infection resembles bacterial infection by tailed bacteriophages.

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