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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The C-C chemokine receptor type 5 (CCR5) is a key player in HIV infection due to its involvement in the infection process. Investigations into the role of the CCR5 coreceptor first focused on its binding to the virus and the molecular mechanisms leading to the entry and spread of HIV. The identification of naturally occurring CCR5 mutations has allowed scientists to address the CCR5 molecule as a promising target to prevent or limit HIV infection in vivo. Naturally occurring CCR5-specific antibodies have been found in exposed but uninfected people, and in a subset of HIV seropositive people who show long-term control of the infection. This suggests that natural autoimmunity to the CCR5 coreceptor exists and may play a role in HIV control. Such natural immunity has prompted strategies aimed at achieving anti-HIV humoral responses through CCR5 targeting.

From Natural Resistance to a New Anti-HIV Strategy. Viruses 2010, 2(2), 574-600 doi:10.3390/v2020574

Related:

• Chemokines, receptors and virus infection
• Escape of HIV-1 from a small molecule CCR5 inhibitor is not associated with a fitness loss

Highly pathogenic avian H5N1 influenza viruses have spread throughout Asia, Europe, and Africa, raising serious worldwide concern about their pandemic potential. Although more than 250 people have been infected with these viruses, with a high rate of mortality, the molecular mechanisms responsible for the efficient transmission of H5N1 viruses among humans remain elusive. We used a mouse model to examine the role of the amino acid at position 627 of the PB2 viral protein in efficient replication of H5N1 viruses in the mammalian respiratory tract. Viruses possessing Lys at position 627 of PB2 replicated efficiently in lungs and nasal turbinates, as well as in cells, even at the lower temperature of 33°C. Those viruses possessing Glu at this position replicated less well in nasal turbinates than in lungs, and less well in cells at the lower temperature. These results suggest that Lys at PB2–627 confers to avian H5N1 viruses the advantage of efficient growth in the upper and lower respiratory tracts of mammals. Therefore, efficient viral growth in the upper respiratory tract may provide a platform for the adaptation of avian H5N1 influenza viruses to humans and for efficient person-to-person virus transmission, in the context of changes in other viral properties including specificity for human receptors.

Growth of H5N1 Influenza A Viruses in the Upper Respiratory Tracts of Mice
PLoS Pathogens 2007 3 (10) e133

What does this all mean?
The H5N1 influenza virus (formerly known as “bird flu”) has mutated to infect people more easily, although it still has not transformed into a pandemic strain. In order to acquire the capacity for efficient human-to-human transmission, H5N1 avian influenza viruses must undergo a series of genetic changes resulting in the ability to replicate at lower temperatures, in a wider range of cell types, to recognize human receptors, and other unknown phenotypic changes controlled by virus proteins. Birds usually have a body temperature of 41°C, and humans 37°C. The human nose and throat, where flu viruses usually enter, is usually around 33°C, so the virus doesn’t grow well in the nose or throat of humans. This research identifes a mutation which allows H5N1 to replicate in the cooler temperatures of the human upper respiratory tract. The H5N1 viruses circulating in Europe and Africa all have this mutation. What we don’t know at present is how many additional mutations are needed for these humanized viruses to become a pandemic strain, or how long that process will take.