Macrophage-Derived Factors with the Potential to Contribute to Pathogenicity of HIV-1 and HIV-2: Role of CCL-2/MCP-1.

Human immunodeficiency virus type 2 (HIV-2) is known to be less pathogenic than HIV-1. However, the mechanism(s) underlying the decreased HIV-2 pathogenicity is not fully understood. Herein, we report that ß-chemokine CCL2 expression was increased in HIV-1-infected human monocyte-derived macrophages (MDM) but decreased in HIV-2-infected MDM when compared to uninfected MDM. Inhibition of CCL2 expression following HIV-2 infection occurred at both protein and mRNA levels. By microarray analysis, quantitative PCR, and Western blotting, we identified that Signal Transducer and Activator of Transcription 1 (STAT1), a critical transcription factor for inducing CCL2 gene expression, was also reduced in HIV-2-infected MDM. Blockade of STAT1 in HIV-infected MDM using a STAT1 inhibitor significantly reduced the production of CCL2. In contrast, transduction of STAT1-expressing pseudo-retrovirus restored CCL2 production in HIV-2-infected MDM. These findings support the concept that CCL2 inhibition in HIV-2-infected MDM is meditated by reduction of STAT1. Furthermore, we showed that STAT1 reduction in HIV-2-infected MDM was regulated by the CUL2/RBX1 ubiquitin E3 ligase complex-dependent proteasome pathway. Knockdown of CUL2 or RBX1 restored the expression of STAT1 and CCL2 in HIV-2-infected MDM. Taken together, our findings suggest that differential regulation of the STAT1-CCL2 axis may be one of the mechanisms underlying the different pathogenicity observed for HIV-1 and HIV-2.

Uses and Challenges of Antiviral Polyclonal and Monoclonal Antibody Therapies.

Viral diseases represent a major public health concerns and ever-present risks for developing into future pandemics. Antiviral antibody therapeutics, either alone or in combination with other therapies, emerged as valuable preventative and treatment options, including during global emergencies. Here we will discuss polyclonal and monoclonal antiviral antibody therapies, focusing on the unique biochemical and physiological properties that make them well-suited as therapeutic agents. We will describe the methods of antibody characterization and potency assessment throughout development, highlighting similarities and differences between polyclonal and monoclonal products as appropriate. In addition, we will consider the benefits and challenges of antiviral antibodies when used in combination with other antibodies or other types of antiviral therapeutics. Lastly, we will discuss novel approaches to the characterization and development of antiviral antibodies and identify areas that would benefit from additional research.

Variable Induction of Pro-Inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo.

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.

Cytokine Effects on the Entry of Filovirus Envelope Pseudotyped Virus-Like Particles into Primary Human Macrophages.

Macrophages are one of the first and also a major site of filovirus replication and, in addition, are a source of multiple cytokines, presumed to play a critical role in the pathogenesis of the viral infection. Some of these cytokines are known to induce macrophage phenotypic changes in vitro, but how macrophage polarization may affect the cell susceptibility to filovirus entry remains largely unstudied. We generated different macrophage subsets using cytokine pre-treatment and subsequently tested their ability to fuse with beta-lactamase containing virus-like particles (VLP), pseudotyped with the surface glycoprotein of Ebola virus (EBOV) or the glycoproteins of other clinically relevant filovirus species. We found that pre-incubation of primary human monocyte-derived macrophages (MDM) with interleukin-10 (IL-10) significantly enhanced filovirus entry into cells obtained from multiple healthy donors, and the IL-10 effect was preserved in the presence of pro-inflammatory cytokines found to be elevated during EBOV disease. In contrast, fusion of IL-10-treated macrophages with influenza hemagglutinin/neuraminidase pseudotyped VLPs was unchanged or slightly reduced. Importantly, our in vitro data showing enhanced virus entry are consistent with the correlation established between elevated serum IL-10 and increased mortality in filovirus infected patients and also reveal a novel mechanism that may account for the IL-10-mediated increase in filovirus pathogenicity.

Electron-Beam-Lithographed Nanostructures as Reference Materials for Label-Free Scattered-Light Biosensing of Single Filoviruses.

Optical biosensors based on scattered-light measurements are being developed for rapid and label-free detection of single virions captured from body fluids. Highly controlled, stable, and non-biohazardous reference materials producing virus-like signals are valuable tools to calibrate, evaluate, and refine the performance of these new optical biosensing methods. To date, spherical polymer nanoparticles have been the only non-biological reference materials employed with scattered-light biosensing techniques. However, pathogens like filoviruses, including the Ebola virus, are far from spherical and their shape strongly affects scattered-light signals. Using electron beam lithography, we fabricated nanostructures resembling individual filamentous virions attached to a biosensing substrate (silicon wafer overlaid with silicon oxide film) and characterized their dimensions with scanning electron and atomic force microscopes. To assess the relevance of these nanostructures, we compared their signals across the visible spectrum to signals recorded from Ebola virus-like particles which exhibit characteristic filamentous morphology. We demonstrate the highly stable nature of our nanostructures and use them to obtain new insights into the relationship between virion dimensions and scattered-light signal.

Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection.

BACKGROUND: The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process. RESULTS: We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets--primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. CONCLUSIONS: Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences among previously published studies. More importantly, from an in vivo perspective, the preferential utilization of PDI may be relevant to the HIV-1 entry and establishment of virus reservoirs in resting CD4+ cells, while the elevated levels of Trx reported in the chronic stages of HIV-1 infection may facilitate the virus entry in macrophages and help to sustain high viremia during the decline of T lymphocytes.

Human TOP1 residues implicated in species specificity of HIV-1 infection are required for interaction with BTBD2, and RNAi of BTBD2 in old world monkey and human cells increases permissiveness to HIV-1 infection.

BACKGROUND: Host determinants of HIV-1 viral tropism include factors from producer cells that affect the efficiency of productive infection and factors in target cells that block infection after viral entry. TRIM5α restricts HIV-1 infection at an early post-entry step through a mechanism associated with rapid disassembly of the retroviral capsid. Topoisomerase I (TOP1) appears to play a role in HIV-1 viral tropism by incorporating into or otherwise modulating virions affecting the efficiency of a post-entry step, as the expression of human TOP1 in African Green Monkey (AGM) virion-producing cells increased the infectivity of progeny virions by five-fold. This infectivity enhancement required human TOP1 residues 236 and 237 as their replacement with the AGM counterpart residues abolished the infectivity enhancement. Our previous studies showed that TOP1 interacts with BTBD1 and BTBD2, two proteins which co-localize with the TRIM5α splice variant TRIM5δ in cytoplasmic bodies. Because BTBD1 and BTBD2 interact with one HIV-1 viral tropism factor, TOP1, and co-localize with a splice variant of another, we investigated the potential involvement of BTBD1 and BTBD2 in HIV-1 restriction. RESULTS: We show that the interaction of BTBD1 and BTBD2 with TOP1 requires hu-TOP1 residues 236 and 237, the same residues required to enhance the infectivity of progeny virions when hu-TOP1 is expressed in AGM producer cells. Additionally, interference with the expression of BTBD2 in AGM and human 293T target cells increased their permissiveness to HIV-1 infection two- to three-fold. CONCLUSIONS: These results do not exclude the possibility that BTBD2 may modestly restrict HIV-1 infection via colocation with TRIM5 variants in cytoplasmic bodies.

Cell adhesion-dependent membrane trafficking of a binding partner for the ebolavirus glycoprotein is a determinant of viral entry.

Ebolavirus is a hemorrhagic fever virus associated with high mortality. Although much has been learned about the viral lifecycle and pathogenesis, many questions remain about virus entry. We recently showed that binding of the receptor binding region (RBR) of the ebolavirus glycoprotein (GP) and infection by GP pseudovirions increase on cell adhesion independently of mRNA or protein synthesis. One model to explain these observations is that, on cell adhesion, an RBR binding partner translocates from an intracellular vesicle to the cell surface. Here, we provide evidence for this model by showing that suspension 293F cells contain an RBR binding site within a membrane-bound compartment associated with the trans-Golgi network and microtubule-organizing center. Consistently, trafficking of the RBR binding partner to the cell surface depends on microtubules, and the RBR binding partner is internalized when adherent cells are placed in suspension. Based on these observations, we reexamined the claim that lymphocytes, which are critical for ebolavirus pathogenesis, are refractory to infection because they lack an RBR binding partner. We found that both cultured and primary human lymphocytes (in suspension) contain an intracellular pool of an RBR binding partner. Moreover, we identified two adherent primate lymphocytic cell lines that bind RBR at their surface and strikingly, support GP-mediated entry and infection. In summary, our results reveal a mode of determining viral entry by a membrane-trafficking event that translocates an RBR binding partner to the cell surface, and they suggest that this process may be operative in cells important for ebolavirus pathogenesis (e.g., lymphocytes and macrophages).

Expression of Human CD4 and chemokine receptors in cotton rat cells confers permissiveness for productive HIV infection.

BACKGROUND: Current small animal models for studying HIV-1 infection are very limited, and this continues to be a major obstacle for studying HIV-1 infection and pathogenesis, as well as for the urgent development and evaluation of effective anti-HIV-1 therapies and vaccines. Previously, it was shown that HIV-1 can infect cotton rats as indicated by development of antibodies against all major proteins of the virus, the detection of viral cDNA in spleen and brain of challenged animals, the transmission of infectious virus, albeit with low efficiency, from animal to animal by blood, and an additional increase in the mortality in the infected groups. RESULTS: Using in vitro experiments, we now show that cotton rat cell lines engineered to express human receptor complexes for HIV-1 (hCD4 along with hCXCR4 or hCCR5) support virus entry, viral cDNA integration, and the production of infectious virus. CONCLUSION: These results further suggest that the development of transgenic cotton rats expressing human HIV-1 receptors may prove to be useful small animal model for HIV infection.

Cell adhesion promotes Ebola virus envelope glycoprotein-mediated binding and infection.

Ebola virus infects a wide variety of adherent cell types, while nonadherent cells are found to be refractory. To explore this correlation, we compared the ability of pairs of related adherent and nonadherent cells to bind a recombinant Ebola virus receptor binding domain (EboV RBD) and to be infected with Ebola virus glycoprotein (GP)-pseudotyped particles. Both human 293F and THP-1 cells can be propagated as adherent or nonadherent cultures, and in both cases adherent cells were found to be significantly more susceptible to both EboV RBD binding and GP-pseudotyped virus infection than their nonadherent counterparts. Furthermore, with 293F cells the acquisition of EboV RBD binding paralleled cell spreading and did not require new mRNA or protein synthesis.

Identification of Hendra virus G glycoprotein residues that are critical for receptor binding.

Hendra virus (HeV) is an emerging paramyxovirus capable of infecting and causing disease in a variety of mammalian species, including humans. The virus infects its host cells through the coordinated functions of its fusion (F) and attachment (G) glycoproteins, the latter of which is responsible for binding the virus receptors ephrinB2 and ephrinB3. In order to identify the receptor binding site, a panel of G glycoprotein constructs containing mutations was generated using an alanine-scanning mutagenesis strategy. Based on a predicted G structure, charged amino acids residing in regions that could be homologous to those in the measles virus H attachment glycoprotein known to be involved in its protein receptor interaction were targeted. Using a coprecipitation-based assay, seven single-amino-acid substitutions in HeV G were identified as having significantly impaired binding to both the ephrinB2 and ephrinB3 viral receptors: D257A, D260A, G439A, K443A, G449A, K465A, and D468A. The impairment of receptor interaction conferred a concomitant diminution in their abilities to promote membrane fusion when coexpressed with F. The G glycoprotein mutants were also recognized by three or more conformation-dependent monoclonal antibodies of a panel of five, were expressed on the cell surface, and retained their abilities to bind and coprecipitate F. Interestingly, some of these mutant G glycoproteins coprecipitated with F more efficiently than wild-type G. Taken together, these data provide strong biochemical and functional evidence that some of these residues could be part of a conformation-dependent, discontinuous, and overlapping ephrinB2 and -B3 binding domain within the HeV G glycoprotein.

The tyrosine kinase inhibitor genistein blocks HIV-1 infection in primary human macrophages.

Binding of HIV-1 envelope glycoprotein (Env) to its cellular receptors elicits a variety of signaling events, including the activation of select tyrosine kinases. To evaluate the potential role of such signaling, we examined the effects of the tyrosine kinase inhibitor, genistein, on HIV-1 entry and infection of human macrophages using a variety of assays. Without altering cell viability, cell surface expression of CD4 and CCR5 or their abilities to interact with Env, genistein inhibited infection of macrophages by reporter gene-encoding, beta-lactamase containing, or wild type virions, as well as Env-mediated cell-fusion. The observation that genistein blocked virus infection if applied before, during or immediately after the infection period, but not 24h later; coupled with a more pronounced inhibition of infection in the reporter gene assays as compared to both beta-lactamase and p24 particle entry assays, imply that genistein exerts its inhibitory effects on both entry and early post-entry steps. These findings suggest that other exploitable targets, or steps, of the HIV-1 infection process may exist and could serve as additional opportunities for the development of new therapeutics.

Thiol/disulfide exchange is a prerequisite for CXCR4-tropic HIV-1 envelope-mediated T-cell fusion during viral entry.

Attachment of gp120 to CD4 during HIV-1 entry triggers structural rearrangement in gp120 that enables binding to an appropriate coreceptor. Following coreceptor engagement, additional conformational changes occur in the envelope (Env), resulting in fusion of virion and cell membranes. Catalysts with redox-isomerase activity, such as protein disulfide isomerase (PDI), facilitate Env conversion from its inactive to its fusion-competent conformation. We report here that anti-PDI agents effectively block CXCR4 Env-mediated fusion and spread of virus infection. Exogenously added PDI, in turn, can rescue fusion from this blockade. We further find that PDI facilitates thiol/disulfide rearrangement in gp120 during conformational change, whereas inhibition of this redox shuffling prevents gp41 from assuming the fusogenic 6-helix bundle conformation. At the virus-cell contact site, gp120 induces assembly of PDI, CD4, and CXCR4 into a tetramolecular protein complex serving as a portal for viral entry. Our findings support the hypothesis that Env conformational change depends on a well-coordinated action of a tripartite system in which PDI works in concert with the receptor and the coreceptor to effectively lower the activation energy barrier required for Env conformational rearrangement.