MicrobiologyBytes

Ebolavirus (EBOV) and Marburgvirus (MARV) that compose the filovirus family of negative strand RNA viruses infect a broad range of mammalian cells. Recent studies indicate that cellular entry of this family of viruses requires a series of cellular protein interactions and molecular mechanisms, some of which are unique to filoviruses and others are commonly used by all viral glycoproteins. Details of their cell entry pathway are highlighted in a new paper.

Filovirus Entry: A Novelty in the Viral Fusion World. (2012) Viruses 4(2): 258-275; doi:10.3390/v4020258
Fliovirus entry into cells is initiated by the interaction of the viral glycoprotein1 subunit (GP1) with both adherence factors and one or more receptors on the surface of host cells. On epithelial cells, we recently demonstrated that TIM-1 serves as a receptor for this family of viruses, but the cell surface receptors in other cell types remain unidentified. Upon receptor binding, the virus is internalized into endosomes primarily via macropinocytosis, but perhaps by other mechanisms as well. Within the acidified endosome, the heavily glycosylated GP1 is cleaved to a smaller form by the low pH-dependent cellular proteases Cathepsin L and B, exposing residues in the receptor binding site (RBS). Details of the molecular events following cathepsin-dependent trimming of GP1 are currently incomplete; however, the processed GP1 specifically interacts with endosomal/lysosomal membranes that contain the Niemann Pick C1 (NPC1) protein and expression of NPC1 is required for productive infection, suggesting that GP/NPC1 interactions may be an important late step in the entry process. Additional events such as further GP1 processing and/or reducing events may also be required to generate a fusion-ready form of the glycoprotein. Once this has been achieved, sequences in the filovirus GP2 subunit mediate viral/cellular membrane fusion via mechanisms similar to those previously described for other enveloped viruses. This multi-step entry pathway highlights the complex and highly orchestrated path of internalization and fusion that appears unique for filoviruses.

MicrobiologyBytes: Yes folks, it’s that naughty Niemann Pick C1 (NPC1) protein again!
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New research shows that Niemann-Pick C1–like 1 (NPC1L1) cholesterol uptake receptor is an HCV cell entry factor that functions after binding, at or before fusion. Together with the facts that NPC1L1 is a cellular cholesterol receptor, the HCV particle is enriched in cholesterol, and relative dependence on NPC1L1 is correlated with HCV particle cholesterol levels, supports and expands on previous reports suggesting that virion cholesterol is involved in HCV cell entry. Whether NPC1L1 directly interacts with HCV or indirectly participates in HCV entry by removing virion-associated cholesterol to perhaps reveal protected viral glycoprotein binding sites or confer a required conformational change remains to be determined. As NPC1L1 is expressed only on human and primate hepatocytes, this discovery additionally highlights NPC1L1 as a potential HCV tropism determinant, which may facilitate the future development of animal models of HCV infection.

Identification of the Niemann-Pick C1–like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor. Nature Medicine 08 January 2012 doi:10.1038/nm.2581
Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. With ~170 million individuals infected and current interferon-based treatment having toxic side effects and marginal efficacy, more effective antivirals are crucially needed. Although HCV protease inhibitors were just approved by the US Food and Drug Administration (FDA), optimal HCV therapy, analogous to HIV therapy, will probably require a combination of antivirals targeting multiple aspects of the viral lifecycle. Viral entry represents a potential multifaceted target for antiviral intervention; however, to date, FDA-approved inhibitors of HCV cell entry are unavailable. Here we show that the cellular Niemann-Pick C1–like 1 (NPC1L1) cholesterol uptake receptor is an HCV entry factor amendable to therapeutic intervention. Specifically, NPC1L1 expression is necessary for HCV infection, as silencing or antibody-mediated blocking of NPC1L1 impairs cell culture–derived HCV (HCVcc) infection initiation. In addition, the clinically available FDA-approved NPC1L1 antagonist ezetimibe potently blocks HCV uptake in vitro via a virion cholesterol–dependent step before virion-cell membrane fusion. Moreover, ezetimibe inhibits infection by all major HCV genotypes in vitro and in vivo delays the establishment of HCV genotype 1b infection in mice with human liver grafts. Thus, we have not only identified NPC1L1 as an HCV cell entry factor but also discovered a new antiviral target and potential therapeutic agent.

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Arenaviruses comprise a diverse family of enveloped negative-strand RNA viruses that are endemic to specific rodent hosts worldwide. Several arenaviruses cause severe hemorrhagic fevers in humans, including Junín and Machupo viruses in South America and Lassa fever virus in western Africa.

Arenavirus entry into the host cell is mediated by the envelope glycoprotein complex, GPC. The virion is endocytosed on binding to a cell-surface receptor, and membrane fusion is initiated in response to physiological acidification of the endosome. As with other class I virus fusion proteins, GPC-mediated membrane fusion is promoted through a regulated sequence of conformational changes leading to formation of the classical postfusion trimer-of-hairpins structure. GPC is, however, unique among the class I fusion proteins in that the mature complex retains a stable signal peptide (SSP) as a third subunit, in addition to the canonical receptor-binding and fusion proteins.

This review describes the properties of the tripartite GPC complex and the evidence that SSP interacts with the fusion subunit to modulate pH-induced activation of membrane fusion. This unusual solution to maintaining the metastable prefusion state of GPC on the virion and activating the class I fusion cascade at acidic pH provides novel targets for antiviral intervention.

The Curious Case of Arenavirus Entry, and Its Inhibition. (2012) Viruses 4(1), 83-101 doi:10.3390/v4010083

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An unfailing observation over the past 70 years is that resistance to all antibiotics emerges eventually after use in the clinic. Where does this resistance come from? Recent work has shown that antibiotic resistance genes are common in metagenomes of ancient sediments. This prevalence of resistance, well before the use of antibiotics, denotes the importance of taking microbial chemical ecology and deep metagenomic profiling into account in the development and use of antibiotics.

Antibiotic resistance is ancient: implications for drug discovery. Trends Microbiol. 25 Jan 2012

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How bacteria grow and divide while retaining a defined shape is a fundamental question in microbiology, but technological advances are now driving a new understanding of how the shape-maintaining bacterial peptidoglycan sacculus grows. This review highlights the relationship between peptidoglycan synthesis complexes and cytoskeletal elements, as well as recent evidence that peptidoglycan growth is regulated from outside the sacculus in Gram-negative bacteria. It also discusses how growth of the sacculus is sensitive to mechanical force and nutritional status, and describe the roles of peptidoglycan hydrolases in generating cell shape and of D-amino acids in sacculus remodelling.

From the regulation of peptidoglycan synthesis to bacterial growth and morphology. (2012) Nature Reviews Microbiology 10: 123-136 doi:10.1038/nrmicro2677

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At the 64th gathering of the World Health Assembly in May 2011, a majority of delegates reaffirmed the view that the remaining stocks of variola virus, the causative agent of smallpox, should be destroyed. But consideration of precisely when that action should be taken was postponed for several years pending completion of crucial research addressing the development and assessment of tools for the treatment and control of the disease should it ever reoccur. This agenda of research includes the development and testing of vaccines and therapeutics, at the heart of which involves understanding the mechanisms that govern variola’s virulence and its capability to evade host defenses. But how can these questions be addressed when naturally occurring disease has been eliminated and no satisfactory animal model exists?

In the absence of smallpox, human infections with monkeypox virus constitute the most significant communicable Orthopoxvirus-associated illnesses extant in the world today. Monkeypox is not a direct research-proxy or substitute for smallpox (monkeypox is a zoonosis that can affect a broad range of animal taxa, whereas smallpox was an exclusive disease of humans) but vaccines and therapies developed for smallpox may be useful for the prevention and treatment of monkeypox, and many of the cornerstones of pathogenesis and immunomodulation for both variola and monkeypox probably emanate from conserved homologous processes. Both variola and monkeypox viruses are considered threat agents that could be used unlawfully for acts of bioterrorism. With monkeypox, there is the added question of its persistence in nature, and its potential for expansion in a world that is now more than 30 years without smallpox. A significant expansion of monkeypox in the world today could pose many of the same challenges as a resurgence of smallpox.

Outbreaks of human monkeypox after cessation of smallpox vaccination. Trends Microbiol. 10 Jan 2012
The recent observation of a surge in human monkeypox in the Democratic Republic of the Congo (DRC) prompts the question of whether cessation of smallpox vaccination is driving the phenomenon, and if so, why is re-emergence not universal throughout the historic geographic range of the virus? Research addressing the virus’s mechanisms for immune evasion and induction, as well as that directed at elucidating the genes involved in pathogenesis in different viral lineages (West African vs Congo Basin), provide insights to help explain why emergence appears to be geographically limited. Novel vaccines offer one solution to curtail the spread of this disease.

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Herpes simplex virus (HSV) is an important pathogenic agent that causes recurrent oral and genital lesions, blindness and encephalitis. It is a member of the family Herpesviridae, which contains three subfamilies (alpha- beta- and gammaherpesvirinae) whose members infect humans to cause a variety of ailments, from benign rashes to nasopharyngeal carcinoma. Although this review focuses on HSV, the assembly steps that occur in the nucleus and the proteins involved are highly conserved among all family members, which suggests that antiviral agents that block these steps might be effective against many different herpesviruses and their associated diseases. Despite this potential, a broadly effective compound has yet to be realized, in part because many of the processes are only poorly understood in sufficient molecular detail. This review outlines these intranuclear assembly steps and illustrate potential and existing antiviral strategies that exploit them.

Herpes simplex virus capsid assembly and DNA packaging: a present and future antiviral drug target. (2011) Trends Microbiol. 19(12) :606-613

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Recent evidence shows that the quadrivalent HPV vaccine prevents several HPV-related diseases in men. However, despite the licensure of the vaccine in the USA for men 9 through 26 years of age, rates of male vaccination are very low. Research on acceptability, in general, indicates strong interest in vaccination among men, parents, and healthcare providers, though female vaccination is typically seen as a higher priority. Cost-effectiveness studies indicate that in the context of modest female vaccination rates and with the specification of a broad range of disease outcomes (e.g. genital warts, anogenital cancers, and oropharyngeal cancers), male vaccination can be quite cost-effective.

This review describes the indications for vaccinating men with the quadrivalent human papillomavirus (HPV) vaccine, reports on the U.S. rates of male vaccination, and reviews the recent research on acceptability of vaccinating men and research on the cost-effectiveness of adding men to existing female HPV immunization programs.

Summary: Men are at high risk for HPV infection and can benefit from vaccination, but vaccination rates among men remain extremely low. More research needs to be done on the predictors of uptake of HPV vaccine among men and on the development of interventions to increase male vaccination.

Human papillomavirus vaccine and men: what are the obstacles and challenges? (2012) Curr Opin Infect Dis. 25(1): 86-91

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By convention, the human adenovirus replication cycle is divided into two phases, an early and a late phase, which are separated by the onset of viral DNA replication. Based on temporal changes of the gene expression pattern as revealed by DNA microarray analysis, adenovirus type 2 (Ad2) infection in human primary lung fibroblasts can be divided into four periods. The first period is from 0 to 12 h after infection before or shortly after adenoviral gene expression has commenced. During this time, changes in cellular gene expression are likely to be triggered by the virus entry process, such as attachment of virus to cell surface receptors, and its intracellular transport along microtubules.

The second period covers the time from 12 to 24 h after infection and follows activation of the immediate early E1A gene. During this period, there is an increase in the number of differentially expressed cellular genes. About 50% of these genes are involved in cell cycle regulation, cell proliferation and antiviral response. The third period extends from 24 to 42 h after infection. By this time, the virus has gained control of the cellular metabolic machinery, resulting in an efficient replication of the viral genome. Additional changes in cellular gene expression are modest during this phase. During the fourth and last period, when the cytopathic effect becomes apparent, the number of down-regulated genes increases dramatically including many genes involved in intra- and extracellular structure.

The most intensive battle between the adenovirus and its host takes place during the second period after adenovirus genes expression has started. The major functions of the early gene products are to force the host cell to enter the S phase in order to provide optimal conditions for viral DNA replication and to suppress the host antiviral response. Adenoviruses encode several regulatory proteins within the early regions E1A, E1B, E3, and E4. The immediate-early E1A gene encodes two regulators of viral and cellular gene expression, the E1A-243R and E1A-289R proteins. The E1A proteins act as promiscuous transcriptional activators or repressors of cellular genes. E1A proteins are essential for promoting the host cell to enter the S phase. This is achieved by the binding of the E1A proteins to members of the retinoblastoma tumor suppressor (pRB) family, thereby releasing the E2F transcription factors, which are activators of genes required in the S-phase.

The transcriptome of the adenovirus infected cell. Virology. 9 Jan 2012
Alternations of cellular gene expression following an adenovirus type 2 infection of human primary cells were studied by using superior sensitive cDNA sequencing. In total, 3791 cellular genes were identified as differentially expressed more than 2-fold. Genes involved in DNA replication, RNA transcription and cell cycle regulation were very abundant among the up-regulated genes. On the other hand, genes involved in various signaling pathways including TGF-β, Rho, G-protein, Map kinase, STAT and NF-κB stood out among the down-regulated genes. Binding sites for E2F, ATF/CREB and AP2 were prevalent in the up-regulated genes, whereas binding sites for SRF and NF-κB were dominant among the down-regulated genes. It is evident that the adenovirus has gained a control of the host cell cycle, growth, immune response and apoptosis at 24h after infection. However, efforts from host cell to block the cell cycle progression and activate an antiviral response were also observed.

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