Potential Antiviral Targets Research Articles

Promotion of Endoplasmic Reticulum-Associated Degradation of Procathepsin D by Human Herpesvirus 8-Encoded Viral Interleukin-6.

The interleukin-6 homologue (viral interleukin-6 [vIL-6]) of human herpesvirus 8 is implicated in viral pathogenesis due to its proproliferative, inflammatory, and angiogenic properties, effected through gp130 receptor signaling. In primary effusion lymphoma (PEL) cells, vIL-6 is expressed latently and is essential for normal cell growth and viability. This is mediated partly via suppression of proapoptotic cathepsin D (CatD) via cocomplexing of the endoplasmic reticulum (ER)-localized CatD precursor, pro-CatD (pCatD), and vIL-6 with the previously uncharacterized ER membrane protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2). vIL-6 suppression of CatD occurs also during reactivated productive replication in PEL cells and is likely to contribute to proreplication functions of vIL-6. Here, we report that vIL-6 suppresses CatD through vIL-6, VKORC1v2, and pCatD association with components of the ER-associated degradation (ERAD) machinery. In transfected cells, expression of vIL-6 along with CatD led to proteasome-dependent (inhibitor-sensitive) decreases in CatD levels and the promotion of pCatD polyubiquitination. Depletion of particular ERAD-associated isomerases, lectins, and translocon components, including ERAD E3 ubiquitin ligase HRD1, diminished suppression of CatD by vIL-6. Coprecipitation assays identified direct or indirect interactions of VKORC1v2, vIL-6, and pCatD with translocon proteins (SEL1L and/or HRD1) and ERAD-associated lectins OS9 and XTP3-B. Endogenous CatD expression in PEL cells was increased by depletion of ERAD components, and suppression of CatD by vIL-6 overexpression in PEL cells was dependent on HRD1. Our data reveal a new mechanism of ER-localized vIL-6 activity and further characterize VKORC1v2 function. Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6), unlike cellular IL-6 proteins, is secreted inefficiently and sequestered mainly in the endoplasmic reticulum (ER), from where it can signal through the gp130 receptor. We have recently reported that vIL-6 also associates with a novel membrane protein termed vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2) and mediates suppression of VKORC1v2-cointeracting cathepsin D, a stress-released proapoptotic protein negatively impacting HHV-8 latently infected primary effusion lymphoma (PEL) cell viability and reactivated virus productive replication. Here, we have examined the mechanistic basis of the VKORC1v2-vIL-6 interaction-dependent suppression of cathepsin D and have found that this novel activity of vIL-6 is mediated through coassociation of VKORC1v2, procathepsin D, and vIL-6 with components of the ER-associated degradation (ERAD) machinery. Our findings provide information of significance for potential antiviral and therapeutic targeting of VKORC1v2-mediated vIL-6 activities and also indicate the nature of VKORC1v2 function in normal cell biology.

A broadly protective anti-influenza neuraminidase monoclonal antibody (VAC11P.1100)

Abstract Hemagglutinin (HA) and neuraminidase (NA) are the major surface glycoproteins of influenza viruses and the main targets of vaccine-induced antibodies (Abs). While several broadly neutralizing anti-HA Abs can cross-protect against diverse influenza subtypes, NA-specific Abs could only protect partially against strains from the same subtype. Through comprehensive bioinformatics analyses of all publicly available influenza A and B NA sequences, we found a universally conserved 9-mer peptide (ILRTQESEC) amongst all influenza NA proteins (amino acids 222-230). Growth kinetics of recombinant viruses with single alanine substitutions within this epitope proved its crucial roles in viral fitness and replication. Importantly, a monoclonal Ab (HCA-2 mAb) raised against this sequence showed broad in vitro inhibition against multiple strains from all influenza A NA subtypes (N1-N9) and influenza B lineages. It also provided in vivo heterosubtypic protection against lethal doses of H1N1 and H3N2 strains. Amino acid residues I222 and E227, located in close proximity to the active site, were found to be indispensable for inhibition by HCA-2 mAb. These findings reveal the essential role of this highly-conserved sequence in NA function and viral replication and show that it is sufficiently exposed to allow access of inhibitory Abs during the course of infection. Thus, it could represent a potential target for novel antivirals or vaccines against diverse strains of influenza A and B viruses.

Herpes simplex virus type-1: replication, latency, reactivation and its antiviral targets.

Infection by herpes simplex virus type-1 (HSV-1) causes several diseases, ranging from cutaneous, oral and genital infections to fatal encephalitis. Despite the availability of antiviral therapies on the market, their efficacies are incomplete, and new cases of resistant strains arise, mainly in the immunocompromised, but also recently documented in immunocompetent patients. Over the last decades a lot has been discovered about the molecular basis of infection which has been of great benefit to the investigation of new anti-HSV-1 molecules. In this review we summarize replication, latency and reactivation highlighting potential antiviral targets and new molecules described in the past several years in the literature.

Antiviral therapies and prospects for a cure of chronic hepatitis B.

Current therapies of chronic hepatitis B (CHB) remain limited to either pegylated interferon-α (Peg-IFN-α), or one of the five approved nucleoside analog (NA) treatments. Although viral suppression can be achieved in the majority of patients with high-barrier-to-resistance new-generation NAs (i.e., entecavir and tenofovir), HBsAg loss is achieved in only 10% of patients with both classes of drugs after a follow-up of 5 years. Attempts to improve the response by administering two different NAs or a combination of NA and Peg-IFN-α have been unsuccessful. Therefore, there is a renewed interest to investigate a number of steps in the hepatitis B virus (HBV) replication cycle and specific virus-host cell interactions as potential targets for new antivirals. Novel targets and compounds could readily be evaluated using both relevant in vitro and newly developed in vivo models of HBV infection. The addition of one or several new drugs to current regimens should offer the prospect of markedly improving the response to therapy, thus reducing the burden of drug resistance, as well as the incidence of cirrhosis and hepatocellular carcinoma (HCC).

The interface between hepatitis B virus capsid proteins affects self-assembly, pregenomic RNA packaging, and reverse transcription.

Hepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication. The hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target. The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry of Cp caused defects in pgRNA packaging and synthesis of the second strand of DNA. These results suggest that the HBV Cp assembly domain potentially regulates reverse transcription, extending the activities of the capsid protein beyond its presumed role as an inert compartment.

Analysis of different HCV NS4B domains for the development of global consensus sequence.

The non-structural 4B (NS4B) protein of hepatitis C virus (HCV) is ahydrophobic protein implicated recently in the formation of membranous web, aplatform for the formation of replication complex and thus is potential target for antivirals. The CLC main workbench was used to generate genotype-specific consensus sequence, global consensus sequence and arepresentative phylogenetic tree from non-structural 4 B (NS4B) protein sequences of seven different HCV genotypes reported from all over the world. The C-terminal domain (CTD) of NS4B protein especially the residues involved in interaction with ER membrane were found to be highly conserved. Other residues found to be highly conserved across all HCV genotypes included; 5 aromatic residues of N-terminal domain (NTD) (F49, W50, W55, F57, and Y63), 3 hydrophobic leucine residues (L237, L240, L245), and 2 positively charged residues of CTD (R248 and H250), dimerization motif of transmembrane domain 3 (TMD3) (G143YGAG147) and its surrounding residues (F118 and F155) and TMD1 Ser/Thr cluster residues (T87, S88 and T95) involved in the hydrogen (H) bond interactions. In short, amino acids of NTD, TMD and CTD domains involved in the membrane association/anchoring of NS4B and formation of membranous web are highly conserved and can serve as potential targets for antivirals and peptide vaccines.These conserved residues formed the basis for the development of five short peptides proposed to serve as potential therapeutic target. The phylogenetic analysis was particularly interesting for NS4B sequences of 3a Pakistani isolates. The high degree of variability prevented the clustering of Pakistani isolates with other sequences in phylogenetic tree, revealing geographical disparity.

Tracking the Evolution in Phylogeny, Structure and Function of H5N1 Influenza Virus PA Gene

Highly pathogenic avian influenza (HPAI) H5N1 viruses have severely affected the poultry industry of Vietnam and Indonesia. The outbreaks of HPAI H5N1 viruses continue to pose a serious threat to public health, which have profound impacts on public health. In this study, we presented phylogenetic evidences for five reassortants among HPAI H5N1 viruses sampled from Vietnam and Indonesia during 2003-2013 and found that reassortment events occurred more frequently in the three gene segments (PB1, PA and HA) than in the remaining five gene segments (PB2, NA, NP, NS and MP). The sequence-based analyses have revealed that the PA protein displays high levels of DNA sequence polymorphism and variability than other internal proteins. Seven positive selection sites were detected in PA proteins, which ranked second only to the surface glycoproteins. Structure-based comparative analysis of PA proteins showed a remarkable sequence conservation between the high-pathogenic, low-pathogenic and reassortant viruses, indicating that PA appears to be a potential antiviral target. Furthermore, by analysing the published data, we compared the differential expression of genes involved in RIG-I- and MAVS-mediated intracellular type I interferon (IFN)-inducing pathway between the VN3028IIcl2-infected, IDN3006-infected and IDN3006/PA-infected groups. Our analyses indicated that the inhibitory effect of the PA protein on MAVS was not strong. In addition, transcriptional levels of 33 mitochondrial proteins involved in the induction of apoptosis have significantly increased, suggesting that PA may play an important role in apoptosis signalling pathway.

Emerging antiviral resistant strains of influenza A and the potential therapeutic targets within the viral ribonucleoprotein (vRNP) complex.

Emerging antiviral resistant strains of influenza A virus are greatly limiting the therapies available to stop aggressive infections. Genome changes that confer resistance to the two classes of approved antivirals have been identified in circulating influenza A viruses. It is only a matter of time before the currently approved influenza A antivirals are rendered ineffective, emphasizing the need for additional influenza antiviral therapies. This review highlights the current state of antiviral resistance in circulating and highly pathogenic influenza A viruses and explores potential antiviral targets within the proteins of the influenza A virus ribonucleoprotein (vRNP) complex, drawing attention to the viral protein activities and interactions that play an indispensable role in the influenza life cycle. Investigation of small molecule inhibition, accelerated by the use of crystal structures of vRNP proteins, has provided important information about viral protein domains and interactions, and has revealed many promising antiviral drug candidates discussed in this review.

Advances in targeting nucleocapsid–nucleic acid interactions in HIV-1 therapy

The continuing challenge of HIV-1 treatment resistance in patients creates a need for the development of new antiretroviral inhibitors. The HIV nucleocapsid (NC) protein is a potential therapeutic target. NC is necessary for viral RNA packaging and in the early stages of viral infection. The high level of NC amino acid conservation among all HIV-1 clades suggests a low tolerance for mutations. Thus, NC mutations that could arise during inhibitor treatment to provide resistance may render the virus less fit. Disruption of NC function provides a unique opportunity to strongly dampen replication at multiple points during the viral life cycle with a single inhibitor. Although NC exhibits desirable features for a potential antiviral target, the structural flexibility, size, and the presence of two zinc fingers makes small molecule targeting of NC a challenging task. In this review, we discuss the recent advances in strategies to develop inhibitors of NC function and present a perspective on potential novel approaches that may help to overcome some of the current challenges in the field.

A novel life cycle modeling system for Ebola virus shows a genome length-dependent role of VP24 in virus infectivity.

Work with infectious Ebola viruses is restricted to biosafety level 4 (BSL4) laboratories, presenting a significant barrier for studying these viruses. Life cycle modeling systems, including minigenome systems and transcription- and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus life cycle under BSL2 conditions; however, all current systems model only certain aspects of the virus life cycle, rely on plasmid-based viral protein expression, and have been used to model only single infectious cycles. We have developed a novel life cycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP1,2. This system is ideally suited for studying morphogenesis, budding, and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ∼ 500-fold higher than that in previous systems. Using this system for functional studies of VP24, we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings, we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription. Ebola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently being available, and must be studied in maximum-containment laboratories. Only a few of these laboratories exist worldwide, limiting our ability to study Ebola viruses and develop countermeasures. Here we report the development of a novel reverse genetics-based system that allows the study of Ebola viruses without maximum-containment laboratories. We used this system to investigate the Ebola virus protein VP24, showing that, contrary to previous reports, it only modestly inhibits virus genome replication and transcription but is important for packaging of genomes into virus particles, which constitutes a previously unknown function of VP24 and a potential antiviral target. We further propose a comprehensive model for the function of VP24 in nucleocapsid assembly. Importantly, on the basis of this approach, it should easily be possible to develop similar experimental systems for other viruses that are currently restricted to maximum-containment laboratories.

Transactivation of programmed ribosomal frameshifting by a viral protein.

Programmed -1 ribosomal frameshifting (-1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes -1 frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual -2 frameshifting (-2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of -1 PRF, yielding a third, truncated nsp2 variant named "nsp2N." Remarkably, we now show that both -2 and -1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1β). Embedded in nsp1β's papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1β with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of ribosomal frameshifting. The newly identified frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection.

The ubiquitin-proteasome system is required for the early stages of porcine circovirus type 2 replication

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases (PCVAD). It has been shown that the ubiquitin-proteasome system (UPS) is correlated with viral infection, but its role in PCV2 replication remains unknown. In the present study, we explored the interplay between PCV2 replication and the UPS in PK15 cells and found that treatment with a proteasome inhibitor (MG132 and lactacystin) significantly decreased the PCV2 titer at the early infection stage. We further revealed that inhibition of the UPS did not affect virus entry but decreased viral protein expression and RNA transcription potentially in a cell cycle-dependent manner. PCV2 infection has little effect on the chymotrypsin-like activity, and the gene-silencing of ubiquitin reduced the PCV2 titer, which indicates that the effective replication of PCV2 may be related to protein ubiquitination. Taken together, our data suggested that PCV2 replication requires the UPS machinery, which may represent a potential antiviral target against PCV2.

In silico study on indole derivatives as anti HIV-1 agents: a combined docking, molecular dynamics and 3D-QSAR study

The HIV-1 envelope glycoprotein gp120 plays a vital role in the entry of virus into the host cells and is a potential antiviral drug target. Recently, indole derivatives have been reported to inhibit HIV-1 through binding to gp120, and this prevents gp120 and CD4 interaction to inhibit the infectivity of HIV-1. In this work, molecular docking, molecular dynamics (MD) and three-dimensional quantitative structure-activity relationship studies were carried out. Molecular docking studies of the most active and the least active compounds were performed to identify important residues in the binding pocket. We refined the docked poses by MD simulations which resulted in conformational changes. After equilibration, the structure of the ligand and receptor complex was stable. Therefore, we just took the last snapshot as the representative binding pose for this study. This pose for the most active inhibitor was used as a template for receptor-based alignment which was subsequently used for comparative molecular field analysis. Resultant 3D contour maps suggested smaller substituents are desirable at the 7-position of indole ring to avoid steric interactions with Ser375, Phe382 and Tyr384 residues in the active site. These results can be exploited to develop potential leads and for structure-based drug design of novel HIV-1 inhibitors.

Cellular vimentin regulates construction of dengue virus replication complexes through interaction with NS4A protein.

Dengue virus (DENV) interacts with host cellular factors to construct a more favorable environment for replication, and the interplay between DENV and the host cellular cytoskeleton may represent one of the potential antiviral targeting sites. However, the involvement of cellular vimentin intermediate filaments in DENV replication has been explored less. Here, we revealed the direct interaction between host cellular vimentin and DENV nonstructural protein 4A (NS4A), a known component of the viral replication complex (RC), during DENV infection using tandem affinity purification, coimmunoprecipitation, and scanning electron microscopy. Furthermore, the dynamics of vimentin-NS4A interaction were demonstrated by using confocal three-dimensional (3D) reconstruction and proximity ligation assay. Most importantly, we report for the first time the discovery of the specific region of NS4A that interacts with vimentin lies within the first 50 amino acid residues at the cytosolic N-terminal domain of NS4A (N50 region). Besides identifying vimentin-NS4A interaction, vimentin reorganization and phosphorylation by calcium calmodulin-dependent protein kinase II occurs during DENV infection, signifying that vimentin reorganization is important in maintaining and supporting the DENV RCs. Interestingly, we found that gene silencing of vimentin by small interfering RNA induced a significant alteration in the distribution of RCs in DENV-infected cells. This finding further supports the crucial role of intact vimentin scaffold in localizing and concentrating DENV RCs at the perinuclear site, thus facilitating efficient viral RNA replication. Collectively, our findings implicate the biological and functional significance of vimentin during DENV replication, as we propose that the association of DENV RCs with vimentin is mediated by DENV NS4A.

The Intracellular Cargo Receptor ERGIC-53 Is Required for the Production of Infectious Arenavirus, Coronavirus, and Filovirus Particles

SummaryArenaviruses and hantaviruses cause severe human disease. Little is known regarding host proteins required for their propagation. We identified human proteins that interact with the glycoproteins (GPs) of a prototypic arenavirus and hantavirus and show that the lectin endoplasmic reticulum (ER)-Golgi intermediate compartment 53 kDa protein (ERGIC-53), a cargo receptor required for glycoprotein trafficking within the early exocytic pathway, associates with arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus GPs. ERGIC-53 binds to arenavirus GPs through a lectin-independent mechanism, traffics to arenavirus budding sites, and is incorporated into virions. ERGIC-53 is required for arenavirus, coronavirus, and filovirus propagation; in its absence, GP-containing virus particles form but are noninfectious, due in part to their inability to attach to host cells. Thus, we have identified a class of pathogen-derived ERGIC-53 ligands, a lectin-independent basis for their association with ERGIC-53, and a role for ERGIC-53 in the propagation of several highly pathogenic RNA virus families.

Unmasking the information encoded as structural motifs of viral RNA genomes: a potential antiviral target.

SUMMARYRNA viruses show enormous capacity to evolve and adapt to new cellular and molecular contexts, a consequence of mutations arising from errors made by viral RNA‐dependent RNA polymerase during replication. Sequence variation must occur, however, without compromising functions essential for the completion of the viral cycle. RNA viruses are safeguarded in this respect by their genome carrying conserved information that does not code only for proteins but also for the formation of structurally conserved RNA domains that directly perform these critical functions. Functional RNA domains can interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. They are therefore potential targets for novel therapeutic strategies. This review summarises our knowledge of the functional RNA domains of human RNA viruses and examines the achievements made in the design of antiviral compounds that interfere with their folding and therefore their function. Copyright © 2013 John Wiley & Sons, Ltd.

Structure of the NS1 Protein N-Terminal Origin Recognition/Nickase Domain from the Emerging Human Bocavirus

Human bocavirus is a newly identified, globally prevalent, parvovirus that is associated with respiratory infection in infants and young children. Parvoviruses encode a large nonstructural protein 1 (NS1) that is essential for replication of the viral single-stranded DNA genome and DNA packaging and may play versatile roles in virus-host interactions. Here, we report the structure of the human bocavirus NS1 N-terminal domain, the first for any autonomous parvovirus. The structure shows an overall fold that is canonical to the histidine-hydrophobic-histidine superfamily of nucleases, which integrates two distinct DNA-binding sites: (i) a positively charged region mediated by a surface hairpin (residues 190 to 198) that is responsible for recognition of the viral origin of replication of the double-stranded DNA nature and (ii) the nickase active site that binds to the single-stranded DNA substrate for site-specific cleavage. The structure reveals an acidic-residue-rich subdomain that is present in bocavirus NS1 proteins but not in the NS1 orthologs in erythrovirus or dependovirus, which may mediate bocavirus-specific interaction with DNA or potential host factors. These results provide insights into recognition of the origin of replication and nicking of DNA during bocavirus genome replication. Mapping of variable amino acid residues of NS1s from four human bocavirus species onto the structure shows a scattered pattern, but the origin recognition site and the nuclease active site are invariable, suggesting potential targets for antivirals against this clade of highly diverse human viruses.

The L–VP35 and L–L interaction domains reside in the amino terminus of the Ebola virus L protein and are potential targets for antivirals

The Ebola virus (EBOV) RNA-dependent RNA polymerase (RdRp) complex consists of the catalytic subunit of the polymerase, L, and its cofactor VP35. Using immunofluorescence analysis and coimmunoprecipitation assays, we mapped the VP35 binding site on L. A core binding domain spanning amino acids 280–370 of L was sufficient to mediate weak interaction with VP35, while the entire N-terminus up to amino acid 380 was required for strong VP35–L binding. Interestingly, the VP35 binding site overlaps with an N-terminal L homo-oligomerization domain in a non-competitive manner. N-terminal L deletion mutants containing the VP35 binding site were able to efficiently block EBOV replication and transcription in a minigenome system suggesting the VP35 binding site on L as a potential target for the development of antivirals.

Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus

Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed.

Activation of CCR2+ human proinflammatory monocytes by human herpesvirus-6B chemokine N-terminal peptide

Human monocytes expressing CCR2 with CD14 and CD16 can mediate antigen presentation, and promote inflammation, brain infiltration and immunosenescence. Recently identified roles are in human immunodeficiency virus infection, tuberculosis and parasitic disease. Human herpesvirus 6B (HHV-6B) encodes a chemokine, U83B, which is monospecific for CCR2, and is distinct from the related HHV-6A U83A, which activates CCR1, CCR4, CCR5, CCR6 and CCR8 on immune effector cells and dendritic cells. These differences could alter leukocyte-subset recruitment for latent/lytic replication and associated neuroinflammatory pathology. Therefore, cellular interactions between U83A and U83B could help dictate potential tropism differences between these viruses. U83A specificity is maintained in the 38-residue N-terminal spliced-truncated form. Here, we sought to determine the basis for the chemokine receptor specificity differences and identify possible applications. To do this we first analysed variation in a natural host population in sub-Saharan Africa where both viruses are equally prevalent and compared these to global strains. Analyses of U83 N-terminal variation in 112 HHV-6A and HHV-6B infections identified 6/38 U83A or U83B-specific residues. We also identified a unique single U83A-specific substitution in one U83B sequence, 'U83BA'. Next, the variation effects were tested by deriving N-terminal (NT) 17-mer peptides and assaying activation of ex vivo human leukocytes, the natural host and cellular target. Chemotaxis of CCR2+ leukocytes was potently induced by U83B-NT, but not U83BA-NT or U83A-NT. Analyses of the U83B-NT activated population identified migrated CCR2+, but not CCR5+, leukocytes. The U83BA-NT asparagine-lysine14 substitution disrupted activity, thus defining CCR2 specificity and acting as a main determinant for HHV-6A/B differences in cellular interactions. A flow-cytometry-based shape-change assay was designed, and used to provide further evidence that U83B-NT could activate CCR2+CD14+CD16+ monocytes. This defines a potential antiviral target for HHV-6A/B disease and novel peptide immunomodulator for proinflammatory monocytes.