MicrobiologyBytes

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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When it comes to nasty pathogens, Marburg virus is among the nastiest. Cousin to Ebola virus, Marburg causes fever, rash, delirium, and severe hemorrhaging, often ending in organ failure and death. It is rare in the wild, but was a central focus of weaponization by the Soviet Union, and remains a concern for terrorism experts who fear its lethal potential and resistance to treatment.

One reason that treatments have proved so elusive is because the virus is so hard to work with – hazmat suits, self-contained breathing gear, and electronically secured airlocks are all required for even the simplest of studies with live virus. But another reason is that the virion (the virus particle) is heterogeneous in shape, and that heterogeneity has confounded standard imaging techniques (X-ray crystallography, cryo-electron microscopy), which require purified, identical particles to obtain their highest resolution. Researchers have now got around that problem by using a sophisticated combination of imaging techniques that provide the first clear three-dimensional picture of the intact Marburg virion structure.

Marburg Virus Structure Revealed in Detail. (2011) PLoS Biol 9(11): e1001198. doi:10.1371/journal.pbio.1001198
and:
Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells. (2011) PLoS Biol 9(11): e1001196. doi:10.1371/journal.pbio.1001196
Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible “Velcro-like” manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.

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Epstein-Barr virus (EBV) and Kaposi’s Sarcoma Associated Herpesvirus (KSHV) are DNA tumor viruses that provide risk factors for Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma, Kaposi’s Sarcoma and post-transplant lymphoproliferative disease. EBV infection has also been associated with multiple sclerosis. Healthy carriers consistently shed virus in saliva that infects naïve individuals despite being exposed to virus-specific antibody. This lack of neutralization contrasts completely with non-persistent mucosal infections such as that of poliovirus, and implies that gammaherpesviruses have evolved specific antibody evasion mechanisms.

Our understanding of EBV and KSHV is limited by their narrow species tropisms. Related animal viruses are therefore an important source of information. Two of the best established experimental models are provided by Murid herpesvirus 4 (MuHV-4) and Bovine herpesvirus 4 (BoHV-4). The homologs of gp350 are gp150 in MuHV-4 and gp180 in BoHV-4 are diverse in sequence but seem to be related in function, being involved in both binding to a cellular receptor and in blocking the infection of cells that do not express this receptor. So a non-essential glycoprotein hides some epitopes on cell-free virions from neutralization.

Antibody Evasion by a Gammaherpesvirus O-Glycan Shield. (2011) PLoS Pathog 7(11): e1002387. doi:10.1371/journal.ppat.1002387
All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus gp350. These glycoproteins are often involved in cell binding, and some provide neutralization targets. However, the capacity of gammaherpesviruses for long-term transmission from immune hosts implies that in vivo neutralization is incomplete. In this study, we used Bovine Herpesvirus 4 (BoHV-4) to determine how its gp350 homolog – gp180 – contributes to virus replication and neutralization. A lack of gp180 had no impact on the establishment and maintenance of BoHV-4 latency, but markedly sensitized virions to neutralization by immune sera. Antibody had greater access to gB, gH and gL on gp180-deficient virions, including neutralization epitopes. Gp180 appears to be highly O-glycosylated, and removing O-linked glycans from virions also sensitized them to neutralization. It therefore appeared that gp180 provides part of a glycan shield for otherwise vulnerable viral epitopes. Interestingly, this O-glycan shield could be exploited for neutralization by lectins and carbohydrate-specific antibody. The conservation of O-glycosylation sites in all gp350 homologs suggests that this is a general evasion mechanism that may also provide a therapeutic target.

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Cowpox virus (CPXV) is one of the earliest described members of the genus Orthopoxvirus (OPV). Historically, researchers referred to the ailment known as cowpox and even suggested that it could provide immunity against smallpox. It was Edward Jenner’s publications in 1798 and 1799 which provided the first scientific description of vaccination by detailing the efficacy of CPXV “scarification” in inducing protective immunity against challenge with variola (smallpox) virus (VARV). The common name “cowpox virus” refers to the association with pustular lesions on the teats of cows and historic zoonotic transmission of this disease to humans (milkers) through contact with infected cows. Human infections are generally mild and self-limiting with localized skin lesions healing after 3–4 weeks, however, systemic involvement and fatal outcome have been reported in immunocompromised individuals.

New analysis shows that the smallpox vaccine is known to have originated in the United Kingdom, however the vaccine strains were most closely allied to CPXV isolates from Russia and from Finland. The most likely scenario is that most of the commercially produced smallpox vaccines were not made from the original Jenner strain, but instead from isolates found in other regions of Europe.

Chasing Jenner’s Vaccine: Revisiting Cowpox Virus Classification. (2011) PLoS ONE 6(8): e23086. doi:10.1371/journal.pone.0023086
Cowpox virus (CPXV) is described as the source of the first vaccine used to prevent the onset and spread of an infectious disease. It is one of the earliest described members of the genus Orthopoxvirus, which includes the viruses that cause smallpox and monkeypox in humans. Both the historic and current literature describe “cowpox” as a disease with a single etiologic agent. Genotypic data presented herein indicate that CPXV is not a single species, but a composite of several (up to 5) species that can infect cows, humans, and other animals. The practice of naming agents after the host in which the resultant disease manifests obfuscates the true taxonomic relationships of “cowpox” isolates. These data support the elevation of as many as four new species within the traditional “cowpox” group and suggest that both wild and modern vaccine strains of Vaccinia virus are most closely related to CPXV of continental Europe rather than the United Kingdom, the homeland of the vaccine.

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Parvoviruses are small non-enveloped, icosahedral DNA viruses with a diameter of 18–26 nm that encapsidate a single-stranded genome of approximately (~)5–6 kb. To date, there are a number of parvoviruses known to infect humans, including adeno-associated viruses (AAVs), parvovirus B19 (B19V), and two newly identified human parvoviruses, which are the human bocavirus (HBoV) and human parvovirus 4 (PARV4).

HBoV was firstly identified in respiratory samples from children with lower respiratory tract infections and subsequently proven epidemiologically to be associated with the diseases. PARV4 was initially found in a blood sample from an intravenous drug user with acute viral infection syndrome. Subsequently, the PARV4 genome was detected in human plasma pools at a low titer. PARV4 had also been found in the livers of hepatitis C virus-positive individuals and the bone marrow of HIV-positive individuals. Recently, PARV4 DNA was detected in cerebrospinal fluid of two children with encephalitis of unknown etiology – however, the disease association of PARV4 remains unclear.

HBoV has been classified as a member in the genus Bocavirus based on the similarity of its genome sequence with those of the two animal bocaviruses. However, the known PARV4 incomplete genome, which lacks information of the terminal repeats, does not show a close relationship to any of the known parvoviruses in the genera of the family Parvoviridae that have been classified to date. This has led to the proposed classification of the PARV4 and PARV4-like viruses as members in a new genus called Partetravirus in the family Parvoviridae by the International Committee on Taxonomy of Viruses (ICTV).

Little is known about the gene expression of PARV4 and the function of PARV4 proteins. Since the PARV4 has not been cultured in vitro, and the full-length genome with terminal repeats has not been sequenced, researchers profiled the gene expression of PARV4 by transfecting a replication-competent PARV4 genome. This study has revelealed for the first time the detailed transcription map of PARV4, which can be beneficial for subsequent study of PARV4 infection.

Molecular characterization of the newly identified human parvovirus 4 in the family Parvoviridae. Virology. Oct 30 2011
Human parvovirus 4 (PARV4) is an emerging human virus, and little is known about the molecular aspects of PARV4 apart from its incomplete genome sequence, which lacks information of the termini. We analyzed the gene expression profile of PARV4 using a nearly full-length HPV4 genome in a replication competent system in 293 cells. We found that PARV4 utilizes two promoters to transcribe non-structural protein- and structural protein-encoding mRNAs, respectively, which were polyadenylated at the right end of the genome. Three major proteins, including the large non-structural protein NS1a, whose mRNA is spliced, and capsid proteins VP1 and VP2, were detected. Additional functional analysis of the NS1a revealed its capability to induce cell cycle arrest at G2/M phase in ex vivo-generated human hematopoietic stem cells. Taken together, our characterization of the molecular features of PARV4 suggests that PARV4 represents a new genus in the family Parvoviridae.

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The native conformations of viruses and toxins are assembled to withstand harsh extracellular environments, yet they efficiently disassemble upon engaging a host cell. These reactions invariably allow the virus and toxin to gain host entry. How a stably assembled virus or toxin unravels as it encounters a host cell is remarkable. What driving force harbored in a host cell untangles the numerous covalent and noncovalent forces holding these toxic agents together? What precise function does disassembly serve?

Many viruses and toxins disassemble to enter host cells and cause disease. These conformational changes must be orchestrated temporally and spatially during entry to avoid premature disassembly leading to nonproductive pathways. Although viruses and toxins are evolutionarily distinct toxic agents, emerging findings in their respective fields have revealed that the cellular locations supporting disassembly, the host factors co-opted during disassembly, the nature of the conformational changes, and the physiological function served by disassembly are strikingly conserved. This review examines some of the shared disassembly principles observed in model viruses and toxins. Where appropriate, it underscores their differences, with the intention to draw together the fields of virus and toxin cell entry by using lessons gleaned from each field to inform and benefit one another.

How Viruses and Toxins Disassemble to Enter Host Cells. (2011) Annual Review of Microbiology 65: 287-305 doi: 10.1146/annurev-micro-090110-102855

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Herpes simplex virus (HSV) type-1 and type-2 have evolved numerous strategies to infect a wide range of hosts and cell types. The result is a very successful prevalence of the virus in the human population infecting 40-80% of people worldwide. HSV entry into host cell is a multistep process that involves the interaction of the viral glycoproteins with various cell surface receptors. Based on the cell type, HSV enter into host cell using different modes of entry. The combination of various receptors and entry modes has resulted in a virus that is capable of infecting virtually all cell types. Identifying the common rate limiting steps of the infection may help the development of antiviral agents that are capable of preventing the virus entry into host cell. This review describes the major features of HSV entry that have contributed to the wide susceptibility of cells to HSV infection.

Herpes simplex virus infects most cell types in vitro: clues to its succes. (2011) Virology Journal 8: 481 doi:10.1186/1743-422X-8-481

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Inflammatory responses generated during virus infections are critical for antiviral immune responses. The exact virus recognition elements that activate cells to induce pro-inflammatory signals are not completely characterized. Virus recognition elements such as dsRNA and 5′-triphosphate single-stranded RNA are recognized by several cellular pathways. The intracellular or extracellular interaction of cells with virus recognition elements results in activation of innate immune responses as indicated by expression of inflammatory cytokines and chemokines. In addition to the innate immune inflammation, virus recognition elements trigger establishment of an antiviral state, under which each cell resists virus infection. The resistance to virus infection is in part through inhibition of virus replication by perturbation of RNA and protein synthesis. Determining the exact mechanisms by which immune and antivirus responses are activated is essential for understanding virus pathogenesis.

RNA with high inosine content is not commonly found in normally growing eukaryotic cells but it is present during infections with DNA and RNA viruses such as polyomavirus, Rous-associated virus, vesicular stomatitis virus, measles virus, and respiratory syncytial virus. Extracellular Ino-RNA is generated during virus infections. Cell lysis occurs frequently during virus infections, which results in the release of cell content, including intracellular generated Ino-RNA, into the extracellular space. Extracellular dsRNA has been shown to be able to stimulate antiviral responses in neighboring, uninfected cells.

Using RSV infection as a model, this paper reports that the presence of inosines in ssRNA is a potent inducer of inflammatory cytokines and the antiviral state during virus infection and suggests that Ino-RNA, of virus or cellular origin, in the surrounding tissue after release from infected cells is a signal for the presence of virus infections.

Inosine-Containing RNA Is a Novel Innate Immune Recognition Element and Reduces RSV Infection. (2011) PLoS ONE 6(10): e26463. doi:10.1371/journal.pone.0026463
During viral infections, single- and double-stranded RNA (ssRNA and dsRNA) are recognized by the host and induce innate immune responses. The cellular enzyme ADAR-1 (adenosine deaminase acting on RNA-1) activation in virally infected cells leads to presence of inosine-containing RNA (Ino-RNA). Here we report that ss-Ino-RNA is a novel viral recognition element. We synthesized unmodified ssRNA and ssRNA that had 6% to16% inosine residues. The results showed that in primary human cells, or in mice, 10% ss-Ino-RNA rapidly and potently induced a significant increase in inflammatory cytokines, such as interferon (IFN)-β (35 fold), tumor necrosis factor (TNF)-α (9.7 fold), and interleukin (IL)-6 (11.3 fold) (p

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