HSV Usurps Eukaryotic Initiation Factor 3 Subunit M for Viral Protein Translation: Novel Prevention Target

Prevention of genital herpes is a global health priority. B5, a recently identified ubiquitous human protein, was proposed as a candidate HSV entry receptor. The current studies explored its role in HSV infection. Viral plaque formation was reduced by ∼90% in human cells transfected with small interfering RNA targeting B5 or nectin-1, an established entry receptor. However, the mechanisms were distinct. Silencing of nectin-1 prevented intracellular delivery of viral capsids, nuclear transport of a viral tegument protein, and release of calcium stores required for entry. In contrast, B5 silencing had no effect on these markers of entry, but inhibited viral protein translation. Specifically, viral immediate early genes, ICP0 and ICP4, were transcribed, polyadenylated and transported from the nucleus to the cytoplasm, but the viral transcripts did not associate with ribosomes or polysomes in B5-silenced cells. In contrast, immediate early gene viral transcripts were detected in polysome fractions isolated from control cells. These findings are consistent with sequencing studies demonstrating that B5 is eukaryotic initiation factor 3 subunit m (eIF3m). Although B5 silencing altered the polysome profile of cells, silencing had little effect on cellular RNA or protein expression and was not cytotoxic, suggesting that this subunit is not essential for host cellular protein synthesis. Together these results demonstrate that B5 plays a major role in the initiation of HSV protein translation and could provide a novel target for strategies to prevent primary and recurrent herpetic disease

The C Terminus of the B5 Receptor for Herpes Simplex Virus Contains a Functional Region Important for Infection

The expression of a previously uncharacterized human hfl-B5 cDNA confers susceptibility for herpes simplex virus (HSV) to porcine cells and fulfills criteria as an HSV entry receptor (A. Perez, Q.-X. Li, P. Perez-Romero, G. DeLassus, S. R. Lopez, S. Sutter, N. McLaren, and A. Oveta Fuller, J. Virol. 79:7419-7430, 2005). Heptad repeats found in the B5 C terminus are predicted to form an α-helix for coiled coil structure. We used mutagenesis and synthetic peptides with wild-type and mutant sequences to examine the function of the heptad repeat motif in HSV binding and entry into porcine cells that express B5 and for infection of naturally susceptible human HEp-2 cells. B5 with point mutations predicted to disrupt the putative C-terminal coiled coil failed to mediate HSV binding and entry into porcine cells. Synthetic peptides that contain the single amino acid changes lose the blocking activity of HSV entry. We concluded that the C terminus of B5 contains a functional region that is important for the B5 receptor to mediate events in HSV entry. Structural evidence that this functional region forms coiled coil structures is under investigation. Blocking of HSV interaction with the C-terminal region of the B5 receptor is a new potential target site to intervene in the virus infection of human cells

‘We can act different from what we used to’: Findings from experiences of religious leader participants in an HIV-prevention intervention in Zambia

Faith-based organisations (FBOs) have long been part of the fight against HIV and AIDS. International bodies continue to collaborate with FBOs to implement HIV-prevention programmes with mixed success. Zambia has been a target of such programmes in part due to its high HIV prevalence. The Trusted Messenger approach to provide religious leader networks with biomedical, science-focused education about HIV and AIDS was piloted in 2006, but participant experiences of the intervention have not been explored qualitatively. In 2016, in-depth interviews were conducted of 34 randomly chosen individuals who attended Trusted Messenger workshops between 2006 and 2016 in Livingstone, Lusaka, and the Copperbelt region. Findings indicate that the religious leader attendees gained scientific insights about HIV which motivated their action in personal, social, and religious contexts. Participants found the science comprehensible and empowering and identified workshop frequency and language as challenging. Utilising science-focused education within contextual settings of religious leader networks can combat the spread of HIV and the mistreatment of people living with HIV and AIDS

Herpes Simplex Virus Entry Mediator Associates in Infected Cells in a Complex with Viral Proteins gD and at Least gH

We examined herpes simplex virus (HSV)-infected human HEp-2 cells or porcine cells that express herpes virus entry mediator (HVEM) for virus and receptor protein interactions. Antibody to HVEM, or its viral ligand gD, coimmunoprecipitated several similar proteins. A prominent 110-kDa protein that coprecipitated was identified as gH. The HVEM/gD/gH complex was detected with mild or stringent cell lysis conditions. It did not form in cells infected with HSV-1(KOS)Rid1 virus or with null virus lacking gD, gH, or gL. Thus, in cells a complex forms through physical associations of HVEM, gD, and at least gH

A New Class of Receptor for Herpes Simplex Virus Has Heptad Repeat Motifs That Are Common to Membrane Fusion Proteins

We isolated a human cDNA by expression cloning and characterized its gene product as a new human protein that enables entry and infection of herpes simplex virus (HSV). The gene, designated hfl-B5, encodes a type II cell surface membrane protein, B5, that is broadly expressed in human primary tissue and cell lines. It contains a high-scoring heptad repeat at the extracellular C terminus that is predicted to form an α-helix for coiled coils like those in cellular SNAREs or in some viral fusion proteins. A synthetic 30-mer peptide that has the same sequence as the heptad repeat α-helix blocks HSV infection of B5-expressing porcine cells and human HEp-2 cells. Transient expression of human B5 in HEp-2 cells results in increased polykarocyte formation even in the absence of viral proteins. The B5 protein fulfills all criteria as a receptor or coreceptor for HSV entry. Use by HSV of a human cellular receptor, such as B5, that contains putative membrane fusion domains provides an example where a pathogenic virus with broad tropism has usurped a widely expressed cellular protein to function in infection at events that lead to membrane fusion

Silencing of eIF2α, but not B5, is cytotoxic.

<p>(A) Cells were transfected with HVEM, eIF2α or B5 specific siRNA or no transfection (media) and 48h later, evaluated for the expression of eIF2α and β-actin (control) by Western blot; blots are representative of 2 independent experiments. (B) Cells were treated with media or transfected with siB5, sieIF2α, or siHVEM and then 48h later, infected with HSV-2(G) and 4h pi, cell lysates were prepared and evaluated for expression of viral ICP4 and β-actin; a representative blot from 3 independent experiments is shown (left). The blots were scanned and results are presented as odu relative to cells treated with media (mean + sd from 3 experiments). (C). Cells were grown on 96-well plates, transfected with siB5 or sieIF2α or treated with 0.1% N-9, Effectene, or untreated and then quadruplicate wells were evaluated daily for 5 days for cell viability using an MTT assay. Results are presented as percent viability relative to untreated cells and are means ± sd obtained from 2 independent experiments.</p

B5 silencing has more modest effects on HIV or VSV infection.

<p>(A) Jurkat-TAT-CCR5 cells were transfected with siHVEM (control) or B5 specific siRNA and 48 h post-transfection, immunoblots of cell lysates were prepared and probed with anti-B5 polyclonal Ab (upper panel). Transfected cells were infected with HSV-1(KOS) and 4 h pi, cell lysates prepared and analyzed for ICP0 expression (middle panel) and β-actin as a loading control (lower panel). In parallel, the transfected cells were infected with HIV-1(BaL) and culture supernatants harvested 5 days pi and analyzed for p24 expression (right). (B) CaSki cells were transfected with HVEM or B5 specific siRNA and 48 h post-transfection immunoblots of cell lysates were prepared and probed with anti-B5 polyclonal Ab (upper) and β-actin as a loading control (lower panel). The transfected cells were infected with HSV-2(G) or VSV and plaques counted 48 h pi; results are means ± sd obtained from two independent experiments conducted in duplicate.</p