Specific binding sites on Rhesus rotavirus capsid protein dictate the method of endocytosis inducing the murine model of biliary atresia.

Biliary atresia (BA) is the leading indication for pediatric liver transplantation. Rhesus rotavirus (RRV)-induced murine BA develops an obstructive cholangiopathy that mirrors the human disease. We have previously demonstrated the "SRL" motif on RRV's VP4 protein binds to heat shock cognate 70 protein (Hsc70) facilitating entry into cholangiocytes. In this study, we analyzed how binding to Hsc70 affects viral endocytosis, intracellular trafficking, and uniquely activates the signaling pathway that induces murine BA. Inhibition of clathrin- and dynamin-mediated endocytosis in cholangiocytes following infection demonstrated that blocking dynamin decreased the infectivity of RRV, whereas clathrin inhibition had no effect. Blocking early endosome trafficking resulted in decreased viral titers of RRV, whereas late endosome inhibition had no effect. After infection, TLR3 expression and p-NF-κB levels increased in cholangiocytes, leading to increased release of CXCL9 and CXCL10. Infected mice knocked out for TLR3 had decreased levels of CXCL9 and CXCL10, resulting in reduced NK cell numbers. Human patients with BA experienced an increase in CXCL10 levels, suggesting this as a possible pathway leading to biliary obstruction. Viruses that use Hsc70 for cell entry exploit a clathrin-independent pathway and traffic to the early recycling endosome uniquely activating NF-κB through TLR3, leading to the release of CXCL9 and CXCL10 and inducing NK cell recruitment. These results define how the "SRL" peptide found on RRV's VP4 protein modulates viral trafficking, inducing the host response leading to bile duct obstruction.NEW & NOTEWORTHY In this study, we have determined that the presence of the "SRL" peptide on RRV alters its method of endocytosis and intracellular trafficking through viral binding to heat shock cognate 70 protein. This initiates an inflammatory pathway that stimulates the release of cytokines associated with biliary damage and obstruction.

Tryptophan-based motifs in the LLP3 region of the HIV-1 envelope glycoprotein cytoplasmic tail direct trafficking to the endosomal recycling compartment and mediate particle incorporation.

IMPORTANCE: The HIV-1 envelope glycoprotein (Env) is an essential component of the virus and has an exceedingly long cytoplasmic tail (CT). Previous studies have suggested that trafficking signals in the CT interact with host factors to regulate the incorporation of Env into particles. One particular area of interest is termed lentiviral lytic peptide 3 (LLP3), as small deletions in this region have been shown to disrupt Env incorporation. In this study, we identify a small region within LLP3 that regulates how Env associates with cellular recycling compartments. Mutants that reduced or eliminated Env from the recycling compartment also reduced Env incorporation into particles. These findings emphasize the importance of two tryptophan motifs in LLP3 for the incorporation of Env into particles and provide additional support for the idea that the CT interacts with host recycling pathways to determine particle incorporation.

KIF16B Mediates Anterograde Transport and Modulates Lysosomal Degradation of the HIV-1 Envelope Glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is incorporated into virions at the site of particle assembly on the plasma membrane (PM). The route taken by Env to reach the site of assembly and particle incorporation remains incompletely understood. Following initial delivery to the PM through the secretory pathway, Env is rapidly endocytosed, suggesting that recycling is required for particle incorporation. Endosomes marked by the small GTPase Rab14 have been previously shown to play a role in Env trafficking. Here, we examined the role of KIF16B, the molecular motor protein that directs outward movement of Rab14-dependent cargo, in Env trafficking. Env colocalized extensively with KIF16B+ endosomes at the cellular periphery, while expression of a motor-deficient mutant of KIF16B redistributed Env to a perinuclear location. The half-life of Env labeled at the cell surface was markedly reduced in the absence of KIF16B, while a normal half-life was restored through inhibition of lysosomal degradation. In the absence of KIF16B, Env expression on the surface of cells was reduced, leading to a reduction in Env incorporation into particles and a corresponding reduction in particle infectivity. HIV-1 replication in KIF16B knockout cells was substantially reduced compared to that in wild-type cells. These results indicated that KIF16B regulates an outward sorting step involved in Env trafficking, thereby limiting lysosomal degradation and enhancing particle incorporation. IMPORTANCE The HIV-1 envelope glycoprotein is an essential component of HIV-1 particles. The cellular pathways that contribute to incorporation of envelope into particles are not fully understood. Here, we have identified KIF16B, a motor protein that directs movement from internal compartments toward the plasma membrane, as a host factor that prevents envelope degradation and enhances particle incorporation. This is the first host motor protein identified that contributes to HIV-1 envelope incorporation and replication.

Tetherin is as tetherin does.

Tetherin is a cellular restriction factor that inhibits the release of HIV and other enveloped viruses from host cells. A paper from Perez-Caballero et al. (2009) in this issue of Cell clarifies how this factor works. The authors show that the aptly named tetherin directly tethers viral particles to the plasma membrane.

Warp Speed for Coronavirus Disease 2019 (COVID-19) Vaccines: Why Are Children Stuck in Neutral?

While adult clinical trials of coronavirus disease 2019 (COVID-19) vaccines have moved quickly into phase 3 clinical trials, clinical trials have not started in children in the United States. The direct COVID-19 impact upon children is greater than that observed for a number of other pathogens for which we now have effective pediatric vaccines. Additionally, the role of children in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission has clearly been underappreciated. Carefully conducted phase 2 clinical trials can adequately address potential COVID-19 vaccine safety concerns. Delaying phase 2 vaccine clinical trials in children will delay our recovery from COVID-19 and unnecessarily prolong its impact upon children's education, health, and emotional well-being, and equitable access to opportunities for development and social success. Given the potential direct and indirect benefits of pediatric vaccination, implementation of phase 2 clinical trials for COVID-19 vaccines should begin now.

A Bivalent, Spherical Virus-Like Particle Vaccine Enhances Breadth of Immune Responses against Pathogenic Ebola Viruses in Rhesus Macaques.

The 2013-2016 Ebola outbreak in West Africa led to accelerated efforts to develop vaccines against these highly virulent viruses. A live, recombinant vesicular stomatitis virus-based vaccine has been deployed in outbreak settings and appears highly effective. Vaccines based on replication-deficient adenovirus vectors either alone or in combination with a multivalent modified vaccinia Ankara (MVA) Ebola vaccine also appear promising and are progressing in clinical evaluation. However, the ability of current live vector-based approaches to protect against multiple pathogenic species of Ebola is not yet established, and eliciting durable responses may require additional booster vaccinations. Here, we report the development of a bivalent, spherical Ebola virus-like particle (VLP) vaccine that incorporates glycoproteins (GPs) from Zaire Ebola virus (EBOV) and Sudan Ebola virus (SUDV) and is designed to extend the breadth of immunity beyond EBOV. Immunization of rabbits with bivalent Ebola VLPs produced antibodies that neutralized all four pathogenic species of Ebola viruses and elicited antibody-dependent cell-mediated cytotoxicity (ADCC) responses against EBOV and SUDV. Vaccination of rhesus macaques with bivalent VLPs generated strong humoral immune responses, including high titers of binding, as well as neutralizing antibodies and ADCC responses. VLP vaccination led to a significant increase in the frequency of Ebola GP-specific CD4 and CD8 T cell responses. These results demonstrate that a novel bivalent Ebola VLP vaccine elicits strong humoral and cellular immune responses against pathogenic Ebola viruses and support further evaluation of this approach as a potential addition to Ebola vaccine development efforts.IMPORTANCE Ebola outbreaks result in significant morbidity and mortality in affected countries. Although several leading candidate Ebola vaccines have been developed and advanced in clinical testing, additional vaccine candidates may be needed to provide protection against different Ebola species and to extend the durability of protection. A novel approach demonstrated here is to express two genetically diverse glycoproteins on a spherical core, generating a vaccine that can broaden immune responses against known pathogenic Ebola viruses. This approach provides a new method to broaden and potentially extend protective immune responses against Ebola viruses.

Diagnostic testing for SARS-CoV-2/COVID19.

PURPOSE OF REVIEW: SARS-CoV-2 is the novel human coronavirus responsible for the COVID19 pandemic. Accurate detection of infection with SARS-CoV-2 is an essential component of efforts to treat individual patients and to contain spread of the virus in the community. The purpose of this review is to describe current diagnostic modalities for SARS-CoV-2 and outline their use. Special considerations for pediatric age groups are included. RECENT FINDINGS: RNA PCR from the upper respiratory tract remains the gold standard for detection of infection with SARS-CoV-2. Antigen testing is being widely deployed as a faster and more convenient alternative to PCR, but is less sensitive and should only be used for diagnosis early after symptom onset. Serologic assays can document prior infection and are helpful in diagnosing multisystem inflammatory syndrome in children. Serologic testing should not be used to diagnose acute or active infection. Immune assays are likely to provide a useful measure of protection against COVID19 in the future as knowledge of protective responses improves. SUMMARY: A variety of SARS-CoV-2 diagnostics have recently been developed and deployed. Clinicians should understand the appropriate use and interpretation of RNA PCR, antigen testing and immune assays for SARS-CoV-2 in order to diagnose and treat patients in this evolving pandemic.

Comparative analysis of human microglial models for studies of HIV replication and pathogenesis.

BACKGROUND: HIV associated neurocognitive disorders cause significant morbidity and mortality despite the advent of highly active antiretroviral therapy. A deeper understanding of fundamental mechanisms underlying HIV infection and pathogenesis in the central nervous system is warranted. Microglia are resident myeloid cells of the brain that are readily infected by HIV and may constitute a CNS reservoir. We evaluated two microglial model cell lines (C20, HMC3) and two sources of primary cell-derived microglia (monocyte-derived microglia [MMG] and induced pluripotent stem cell-derived microglia [iPSC-MG]) as potential model systems for studying HIV-microglia interactions. RESULTS: All four microglial model cells expressed typical myeloid markers with the exception of low or absent CD45 and CD11b expression by C20 and HMC3, and all four expressed the microglia-specific markers P2RY12 and TMEM119. Marked differences were observed upon gene expression profiling, however, indicating that MMG and iPSC-MG cluster closely together with primary human microglial cells, while C20 and HMC3 were similar to each other but very different from primary microglia. Expression of HIV-relevant genes also revealed important differences, with iPSC-MG and MMG expressing relevant genes at levels more closely resembling primary microglia. iPSC-MG and MMG were readily infected with R5-tropic HIV, while C20 and HMC3 lack CD4 and require pseudotyping for infection. Despite many similarities, HIV replication dynamics and HIV-1 particle capture by Siglec-1 differed markedly between the MMG and iPSC-MG. CONCLUSIONS: MMG and iPSC-MG appear to be viable microglial models that are susceptible to HIV infection and bear more similarities to authentic microglia than two transformed microglia cell lines. The observed differences in HIV replication and particle capture between MMG and iPSC-MG warrant further study.

Rapid Boosting of HIV-1 Neutralizing Antibody Responses in Humans Following a Prolonged Immunologic Rest Period.

BACKGROUND: The durability and breadth of human immunodeficiency virus type 1 (HIV-1)-specific immune responses elicited through vaccination are important considerations in the development of an effective HIV-1 vaccine. Responses to HIV-1 envelope subunit protein (Env) immunization in humans are often described as short-lived. METHODS: We enrolled 16 healthy volunteers who had received priming with an HIV-1 subtype B Env vaccine given with MF59 adjuvant 5-17 years previously and 20 healthy unprimed volunteers. Three booster immunizations with a heterologous subtype C trimeric gp140 protein vaccine were administered to the primed group, and the same subtype C gp140 protein vaccination regimen was administered to the unprimed subjects. RESULTS: Binding antibodies and neutralizing antibodies to tier 1 viral isolates were detected in the majority of previously primed subjects. Remarkably, a single dose of protein boosted binding and neutralizing antibody titers in 100% of primed subjects following this prolonged immunologic rest period, and CD4+ T-cell responses were boosted in 75% of primed individuals. CONCLUSIONS: These results demonstrate that HIV-1 protein immunogens can elicit durable memory T- and B-cell responses and that strong tier 1 virus neutralizing responses can be elicited by a single booster dose of protein following a long immunologic rest period. However, we found no evidence that cross-clade boosting led to a significantly broadened neutralizing antibody response.

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C560-649) and examined the consequences on Env trafficking and incorporation into particles. FIP1C560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane.IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes trapped inside the cell within the endosomal recycling compartment. Intracellular trapping resulted in a loss of envelope protein on released particles and a corresponding loss of infectivity. Mutations of specific trafficking motifs in the envelope protein tail prevented its trapping in the recycling compartment. These results establish that trafficking to the endosomal recycling compartment is an essential step in HIV envelope protein particle incorporation.

Siglec-1 initiates formation of the virus-containing compartment and enhances macrophage-to-T cell transmission of HIV-1.

HIV-1 particles assemble and bud from the plasma membrane of infected T lymphocytes. Infected macrophages, in contrast, accumulate particles within an apparent intracellular compartment known as the virus-containing compartment or VCC. Many aspects of the formation and function of the VCC remain unclear. Here we demonstrate that VCC formation does not actually require infection of the macrophage, but can be reproduced through the exogenous addition of non-infectious virus-like particles or infectious virions to macrophage cultures. Particles were captured by Siglec-1, a prominent cell surface lectin that attaches to gangliosides on the lipid envelope of the virus. VCCs formed within infected macrophages were readily targeted by the addition of ganglioside-containing virus-like particles to the extracellular media. Depletion of Siglec-1 from the macrophage or depletion of gangliosides from viral particles prevented particle uptake into the VCC and resulted in substantial reductions of VCC volume. Furthermore, Siglec-1-mediated virion capture and subsequent VCC formation was required for efficient trans-infection of autologous T cells. Our results help to define the nature of this intracellular compartment, arguing that it is a compartment formed by particle uptake from the periphery, and that this compartment can readily transmit virus to target T lymphocytes. Inhibiting or eliminating the VCC may be an important component of strategies to reduce HIV transmission and to eradicate HIV reservoirs.

The future of careers in pediatric infectious diseases: a call to action.

PURPOSE OF REVIEW: The purpose of this review is to describe some current challenges facing the field of pediatric infectious diseases and discuss strategies for enhancing recognition of the value of infectious disease services and for recruiting new talent to the field. RECENT FINDINGS: Pediatric infectious disease programs are currently filling approximately half of their fellowship positions, and salaries are among the lowest in medical subspecialties. Research-intensive careers in pediatric infectious diseases are threatened by low the National Institutes of Health paylines for career development awards. Despite this, there are new opportunities in pediatric infectious diseases in growing areas, such as transplant infectious diseases and antimicrobial stewardship. SUMMARY: Pediatric infectious disease practitioners are concerned that infectious disease services are often undervalued by the healthcare system. Some of the contributions made by this cognitive specialty to overall patient outcomes are difficult to quantify and are not fully reimbursed. Strategies to enhance value and program support are needed, including sharing individual success stories and collecting data from programs nationally to generate some standards for support of pediatric infectious disease programs in areas, such as antimicrobial stewardship, transplant infectious diseases, and infection prevention. Recruitment of top talent to the field can be enhanced by a number of initiatives that can be implemented at the local level with encouragement and leadership from the pediatric infectious diseases society.

Challenges and solutions for instituting an efficient maintenance program for laboratory equipment in Central Asian, and developing world, countries.

We review the current state of quality assurance in laboratories of the five Central Asia Republics (CARs), focusing on laboratory equipment, and compare quality assurance approaches with CLSI standards. The laboratories of the CARs faced exceptional challenges including highly-structured laboratory systems that retain centralized and outmoded Soviet-era approaches to quality assurance, considerably jeopardizing the validity of laboratory tests. The relative isolation of the CARs, based on geography and almost exclusive use of the Russian language, further hamper change. CARs must make high-level government decisions to widely implement quality assurance programs within their laboratory systems, within which approaches to the management of laboratory equipment will be a prominent part.

Adjuvanting a Simian Immunodeficiency Virus Vaccine with Toll-Like Receptor Ligands Encapsulated in Nanoparticles Induces Persistent Antibody Responses and Enhanced Protection in TRIM5α Restrictive Macaques.

Our previous work has shown that antigens adjuvanted with ligands specific for Toll-like receptor 4 (TLR4) and TLR7/8 encapsulated in poly(lactic-co-glycolic) acid (PLGA)-based nanoparticles (NPs) induce robust and durable immune responses in mice and macaques. We investigated the efficacy of these NP adjuvants in inducing protective immunity against simian immunodeficiency virus (SIV). Rhesus macaques (RMs) were immunized with NPs containing TLR4 and TLR7/8 agonists mixed with soluble recombinant SIVmac239-derived envelope (Env) gp140 and Gag p55 (protein) or with virus-like particles (VLPs) containing SIVmac239 Env and Gag. NP-adjuvanted vaccines induced robust innate responses, antigen-specific antibody responses of a greater magnitude and persistence, and enhanced plasmablast responses compared to those achieved with alum-adjuvanted vaccines. NP-adjuvanted vaccines induced antigen-specific, long-lived plasma cells (LLPCs), which persisted in the bone marrow for several months after vaccination. NP-adjuvanted vaccines induced immune responses that were associated with enhanced protection against repeated low-dose, intravaginal challenges with heterologous SIVsmE660 in animals that carried TRIM5α restrictive alleles. The protection induced by immunization with protein-NP correlated with the prechallenge titers of Env-specific IgG antibodies in serum and vaginal secretions. However, no such correlate was apparent for immunization with VLP-NP or alum as the adjuvant. Transcriptional profiling of peripheral blood mononuclear cells isolated within the first few hours to days after primary vaccination revealed that NP-adjuvanted vaccines induced a molecular signature similar to that induced by the live attenuated yellow fever viral vaccine. This systems approach identified early blood transcriptional signatures that correlate with Env-specific antibody responses in vaginal secretions and protection against infection. These results demonstrate the adjuvanticity of the NP adjuvant in inducing persistent and protective antibody responses against SIV in RMs with implications for the design of vaccines against human immunodeficiency virus (HIV). IMPORTANCE: The results of the RV144 HIV vaccine trial, which demonstrated a rapid waning of protective immunity with time, have underscored the need to develop strategies to enhance the durability of protective immune responses. Our recent work in mice has highlighted the capacity of nanoparticle-encapsulated TLR ligands (NP) to induce potent and durable antibody responses that last a lifetime in mice. In the present study, we evaluated the ability of these NP adjuvants to promote robust and durable protective immune responses against SIV in nonhuman primates. Our results demonstrate that immunization of rhesus macaques with NP adjuvants mixed with soluble SIV Env or a virus-like particle form of Env (VLP) induces potent and durable Env-specific antibody responses in the serum and in vaginal secretions. These responses were superior to those induced by alum adjuvant, and they resulted in enhanced protection against a low-dose intravaginal challenge with a heterologous strain of SIV in animals with TRIM5a restrictive alleles. These results highlight the potential for such NP TLR L adjuvants in promoting robust and durable antibody responses against HIV in the next generation of HIV immunogens currently being developed.

EGFR Interacts with the Fusion Protein of Respiratory Syncytial Virus Strain 2-20 and Mediates Infection and Mucin Expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from "mucogenic" strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease.

A tyrosine-based motif in the HIV-1 envelope glycoprotein tail mediates cell-type- and Rab11-FIP1C-dependent incorporation into virions.

Lentiviruses such as HIV-1 encode envelope glycoproteins (Env) with long cytoplasmic tails (CTs) that include motifs mediating interactions with host-cell-trafficking factors. We demonstrated recently that Rab11-family interacting protein 1C (FIP1C) is required for CT-dependent incorporation of Env into HIV-1 particles. Here, we used viruses bearing targeted substitutions within CT to map the FIP1C-dependent incorporation of Env. We identified YW795 as a critical motif mediating cell-type-dependent Env incorporation. Disruption of YW795 reproduced the cell-type-dependent particle incorporation of Env that had previously been observed with large truncations of CT. A revertant virus bearing a single amino acid change near the C terminus of CT restored wild-type levels of Env incorporation, Gag-Env colocalization on the plasma membrane, and viral replication. These findings highlight the importance of YW795 in the cell-type-dependent incorporation of Env and support a model of HIV assembly in which FIP1C/RCP mediates Env trafficking to the particle assembly site.

A Putative Cyclin-binding Motif in Human SAMHD1 Contributes to Protein Phosphorylation, Localization, and Stability.

SAMHD1 (sterile α motif and HD domain-containing protein 1) is a mammalian protein that regulates intracellular dNTP levels through its hydrolysis of dNTPs. SAMHD1 functions as an important retroviral restriction factor through a mechanism relying on its dNTPase activity. We and others have reported that human SAMHD1 interacts with the cell cycle regulatory proteins cyclin A, CDK1, and CDK2, which mediates phosphorylation of SAMHD1 at threonine 592, a post-translational modification that has been implicated in abrogating SAMHD1 restriction function and ability to form stable tetramers. Utilizing co-immunoprecipitation and co-localization approaches, we show that endogenous SAMHD1 is able to interact with the cyclin A-CDK1-CDK2 complexin monocytic THP-1 cells and primary monocyte-derived macrophages. Sequence analysis of SAMHD1 identifies a putative cyclin-binding motif found in many cyclin-CDK complex substrates. Using a mutagenesis-based approach, we demonstrate that the conserved residues in the putative cyclin-binding motif are important for protein expression, protein half-life, and optimal phosphorylation of SAMHD1 at Thr592 Furthermore, we observed that SAMHD1 mutants of the cyclin-binding motif mislocalized to a nuclear compartment and had reduced ability to interact with cyclin A-CDK complexes and to form the tetramer. These findings help define the mechanisms by which SAMHD1 is phosphorylated and suggest the contribution of cyclin binding to SAMHD1 expression and stability in dividing cells.

Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells.

UNLABELLED: Tetherin (BST2, CD317, or HM1.24) is a host cellular restriction factor that prevents the release of enveloped viruses by mechanically linking virions to the plasma membrane. The precise arrangement of tetherin molecules at the plasma membrane site of HIV-1 assembly, budding, and restriction is not well understood. To gain insight into the biophysical mechanism underlying tetherin-mediated restriction of HIV-1, we utilized cryo-electron tomography (cryo-ET) to directly visualize HIV-1 virus-like particles (VLPs) and virions tethered to human cells in three dimensions (3D). Rod-like densities that we refer to as tethers were seen connecting HIV-1 virions to each other and to the plasma membrane. Native immunogold labeling showed tetherin molecules located on HIV-1 VLPs and virions in positions similar to those of the densities observed by cryo-ET. The location of the tethers with respect to the ordered immature Gag lattice or mature conical core was random. However, tethers were not uniformly distributed on the viral membrane but rather formed clusters at sites of contact with the cell or other virions. Chains of tethered HIV-1 virions often were arranged in a linear fashion, primarily as single chains and, to a lesser degree, as branched chains. Distance measurements support the extended tetherin model, in which the coiled-coil ectodomains are oriented perpendicular with respect to the viral and plasma membranes. IMPORTANCE: Tetherin is a cellular factor that restricts HIV-1 release by directly cross-linking the virus to the host cell plasma membrane. We used cryo-electron tomography to visualize HIV-1 tethered to human cells in 3D. We determined that tetherin-restricted HIV-1 virions were physically connected to each other or to the plasma membrane by filamentous tethers that resembled rods ∼15 nm in length, which is consistent with the extended tetherin model. In addition, we found the position of the tethers to be arbitrary relative to the ordered immature Gag lattice or the mature conical cores. However, when present as multiple copies, the tethers clustered at the interface between virions. Tethered HIV-1 virions were arranged in a linear fashion, with the majority as single chains. This study advances our understanding of tetherin-mediated HIV-1 restriction by defining the spatial arrangement and orientation of tetherin molecules at sites of HIV-1 restriction.

Virus-Like Particles Displaying Trimeric Simian Immunodeficiency Virus (SIV) Envelope gp160 Enhance the Breadth of DNA/Modified Vaccinia Virus Ankara SIV Vaccine-Induced Antibody Responses in Rhesus Macaques.

UNLABELLED: The encouraging results of the RV144 vaccine trial have spurred interest in poxvirus prime-protein boost human immunodeficiency virus (HIV) vaccine modalities as a strategy to induce protective immunity. Because vaccine-induced protective immunity is critically determined by HIV envelope (Env) conformation, significant efforts are directed toward generating soluble trimeric Env immunogens that assume native structures. Using the simian immunodeficiency virus (SIV)-macaque model, we tested the immunogenicity and efficacy of sequential immunizations with DNA (D), modified vaccinia virus Ankara (MVA) (M), and protein immunogens, all expressing virus-like particles (VLPs) displaying membrane-anchored trimeric Env. A single VLP protein boost displaying trimeric gp160 adjuvanted with nanoparticle-encapsulated Toll-like receptor 4/7/8 (TLR4/7/8) agonists, administered 44 weeks after the second MVA immunization, induced up to a 3-fold increase in Env-specific IgG binding titers in serum and mucosa. Importantly, the VLP protein boost increased binding antibody against scaffolded V1V2, antibody-dependent phagocytic activity against VLP-coated beads, and antibody breadth and neutralizing antibody titers against homologous and heterologous tier 1 SIVs. Following 5 weekly intrarectal SIVmac251 challenges, two of seven DNA/MVA and VLP (DM+VLP)-vaccinated animals were completely protected compared to productive infection in all seven DM-vaccinated animals. Vaccinated animals demonstrated stronger acute viral pulldown than controls, but a trend for higher acute viremia was observed in the DM+VLP group, likely due to a slower recall of Gag-specific CD8 T cells. Our findings support immunization with VLPs containing trimeric Env as a strategy to augment protective antibody but underscore the need for optimal engagement of CD8 T cells to achieve robust early viral control. IMPORTANCE: The development of an effective HIV vaccine remains a global necessity for preventing HIV infection and reducing the burden of AIDS. While this goal represents a formidable challenge, the modest efficacy of the RV144 trial indicates that multicomponent vaccination regimens that elicit both cellular and humoral immune responses can prevent HIV infection in humans. However, whether protein immunizations synergize with DNA prime-viral vector boosts to enhance cellular and humoral immune responses remains poorly understood. We addressed this question in a nonhuman primate model, and our findings show benefit for sequential protein immunization combined with a potent adjuvant in boosting antibody titers induced by a preceding DNA/MVA immunization. This promising strategy can be further developed to enhance neutralizing antibody responses and boost CD8 T cells to provide robust protection and viral control.

Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition.

Although the biology of platelet adhesion on subendothelial matrix after vascular injury is well characterized, how the matrix biophysical properties affect platelet physiology is unknown. Here we demonstrate that geometric orientation of the matrix itself regulates platelet α-granule secretion, a key component of platelet activation. Using protein microcontact printing, we show that platelets spread beyond the geometric constraints of fibrinogen or collagen micropatterns with <5-µm features. Interestingly, α-granule exocytosis and deposition of the α-granule contents such as fibrinogen and fibronectin were primarily observed in those areas of platelet extension beyond the matrix protein micropatterns. This enables platelets to "self-deposit" additional matrix, provide more cellular membrane to extend spreading, and reinforce platelet-platelet connections. Mechanistically, this phenomenon is mediated by actin polymerization, Rac1 activation, and αIIbß3 integrin redistribution and activation, and is attenuated in gray platelet syndrome platelets, which lack α-granules, and Wiskott-Aldrich syndrome platelets, which have cytoskeletal defects. Overall, these studies demonstrate how platelets transduce geometric cues of the underlying matrix geometry into intracellular signals to extend spreading, which endows platelets spatial flexibility when spreading onto small sites of exposed subendothelium.