Infiltration of inflammatory macrophages and neutrophils and widespread pyroptosis in lung drive influenza lethality in nonhuman primates.

Severe influenza kills tens of thousands of individuals each year, yet the mechanisms driving lethality in humans are poorly understood. Here we used a unique translational model of lethal H5N1 influenza in cynomolgus macaques that utilizes inhalation of small-particle virus aerosols to define mechanisms driving lethal disease. RNA sequencing of lung tissue revealed an intense interferon response within two days of infection that resulted in widespread expression of interferon-stimulated genes, including inflammatory cytokines and chemokines. Macaques with lethal disease had rapid and profound loss of alveolar macrophages (AMs) and infiltration of activated CCR2+ CX3CR1+ interstitial macrophages (IMs) and neutrophils into lungs. Parallel changes of AMs and neutrophils in bronchoalveolar lavage (BAL) correlated with virus load when compared to macaques with mild influenza. Both AMs and IMs in lethal influenza were M1-type inflammatory macrophages which expressed neutrophil chemotactic factors, while neutrophils expressed genes associated with activation and generation of neutrophil extracellular traps (NETs). NETs were prominent in lung and were found in alveolar spaces as well as lung parenchyma. Genes associated with pyroptosis but not apoptosis were increased in lung, and activated inflammatory caspases, IL-1ß and cleaved gasdermin D (GSDMD) were present in bronchoalveolar lavage fluid and lung homogenates. Cleaved GSDMD was expressed by lung macrophages and alveolar epithelial cells which were present in large numbers in alveolar spaces, consistent with loss of epithelial integrity. Cleaved GSDMD colocalized with viral NP-expressing cells in alveoli, reflecting pyroptosis of infected cells. These novel findings reveal that a potent interferon and inflammatory cascade in lung associated with infiltration of inflammatory macrophages and neutrophils, elaboration of NETs and cell death by pyroptosis mediates lethal H5N1 influenza in nonhuman primates, and by extension humans. These innate pathways represent promising therapeutic targets to prevent severe influenza and potentially other primary viral pneumonias in humans.

Widespread Virus Replication in Alveoli Drives Acute Respiratory Distress Syndrome in Aerosolized H5N1 Influenza Infection of Macaques.

Human infections with highly pathogenic avian influenza A (H5N1) virus are frequently fatal but the mechanisms of disease remain ill-defined. H5N1 infection is associated with intense production of proinflammatory cytokines, but whether this cytokine storm is the main cause of fatality or is a consequence of extensive virus replication that itself drives disease remains controversial. Conventional intratracheal inoculation of a liquid suspension of H5N1 influenza virus in nonhuman primates likely results in efficient clearance of virus within the upper respiratory tract and rarely produces severe disease. We reasoned that small particle aerosols of virus would penetrate the lower respiratory tract and blanket alveoli where target cells reside. We show that inhalation of aerosolized H5N1 influenza virus in cynomolgus macaques results in fulminant pneumonia that rapidly progresses to acute respiratory distress syndrome with a fatal outcome reminiscent of human disease. Molecular imaging revealed intense lung inflammation coincident with massive increases in proinflammatory proteins and IFN-α in distal airways. Aerosolized H5N1 exposure decimated alveolar macrophages, which were widely infected and caused marked influx of interstitial macrophages and neutrophils. Extensive infection of alveolar epithelial cells caused apoptosis and leakage of albumin into airways, reflecting loss of epithelial barrier function. These data establish inhalation of aerosolized virus as a critical source of exposure for fatal human infection and reveal that direct viral effects in alveoli mediate H5N1 disease. This new nonhuman primate model will advance vaccine and therapeutic approaches to prevent and treat human disease caused by highly pathogenic avian influenza viruses.

The anti-promyelocytic leukemia mode of action of two endophytic secondary metabolites unveiled by a proteomic approach.

As a result of a program to find antitumor compounds of endophytes from medicinal Asteraceae, the steroid (22E,24R)-8,14-epoxyergosta-4,22-diene-3,6-dione (a) and the diterpene aphidicolin (b) were isolated from the filamentous fungi Papulaspora immersa and Nigrospora sphaerica, respectively, and exhibited strong cytotoxicity against HL-60 cells. A proteomic approach was used in an attempt to identify the drugs' molecular targets and their respective antiproliferative mode of action. Results suggested that the (a) growth inhibition effect occurs by G2/M cell cycle arrest via reduction of tubulin alpha and beta isomers and 14-3-3 protein gamma expression, followed by a decrease of apoptotic and inflammatory proteins, culminating in mitochondrial oxidative damage that triggered autophagy-associated cell death. Moreover, the decrease observed in the expression levels of several types of histones indicated that (a) might be disarming oncogenic pathways via direct modulation of the epigenetic machinery. Effects on cell cycle progression and induction of apoptosis caused by (b) were confirmed. In addition, protein expression profiles also revealed that aphidicolin is able to influence microtubule dynamics, modulate proteasome activator complex expression, and control the inflammatory cascade through overexpression of thymosin beta 4, RhoGDI2, and 14-3-3 proteins. Transmission electron micrographs of (b)-treated cells unveiled dose-dependent morphological characteristics of autophagy- or oncosis-like cell death.

Unique functional properties of conserved arginine residues in the lentivirus lytic peptide domains of the C-terminal tail of HIV-1 gp41.

A previous study from our laboratory reported a preferential conservation of arginine relative to lysine in the C-terminal tail (CTT) of HIV-1 envelope (Env). Despite substantial overall sequence variation in the CTT, specific arginines are highly conserved in the lentivirus lytic peptide (LLP) motifs and are scarcely substituted by lysines, in contrast to gp120 and the ectodomain of gp41. However, to date, no explanation has been provided to explain the selective incorporation and conservation of arginines over lysines in these motifs. Herein, we address the functions in virus replication of the most conserved arginines by performing conservative mutations of arginine to lysine in the LLP1 and LLP2 motifs. The presence of lysine in place of arginine in the LLP1 motif resulted in significant impairment of Env expression and consequently virus replication kinetics, Env fusogenicity, and incorporation. By contrast, lysine exchanges in LLP2 only affected the level of Env incorporation and fusogenicity. Our findings demonstrate that the conservative lysine substitutions significantly affect Env functional properties indicating a unique functional role for the highly conserved arginines in the LLP motifs. These results provide for the first time a functional explanation to the preferred incorporation of arginine, relative to lysine, in the CTT of HIV-1 Env. We propose that these arginines may provide unique functions for Env interaction with viral or cellular cofactors that then influence overall Env functional properties.

Detailed topology mapping reveals substantial exposure of the "cytoplasmic" C-terminal tail (CTT) sequences in HIV-1 Env proteins at the cell surface.

Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT). While few studies have been designed to directly address the topology of the CTT, results from envelope (Env) protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20-50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected) cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model.

Topology of the C-terminal tail of HIV-1 gp41: differential exposure of the Kennedy epitope on cell and viral membranes.

The C-terminal tail (CTT) of the HIV-1 gp41 envelope (Env) protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the "intracytoplasmic domain" based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical "Kennedy epitope" (KE) of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs). Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.

Reactivation of latent tuberculosis in cynomolgus macaques infected with SIV is associated with early peripheral T cell depletion and not virus load.

HIV-infected individuals with latent Mycobacterium tuberculosis (Mtb) infection are at significantly greater risk of reactivation tuberculosis (TB) than HIV-negative individuals with latent TB, even while CD4 T cell numbers are well preserved. Factors underlying high rates of reactivation are poorly understood and investigative tools are limited. We used cynomolgus macaques with latent TB co-infected with SIVmac251 to develop the first animal model of reactivated TB in HIV-infected humans to better explore these factors. All latent animals developed reactivated TB following SIV infection, with a variable time to reactivation (up to 11 months post-SIV). Reactivation was independent of virus load but correlated with depletion of peripheral T cells during acute SIV infection. Animals experiencing reactivation early after SIV infection (<17 weeks) had fewer CD4 T cells in the periphery and airways than animals reactivating in later phases of SIV infection. Co-infected animals had fewer T cells in involved lungs than SIV-negative animals with active TB despite similar T cell numbers in draining lymph nodes. Granulomas from these animals demonstrated histopathologic characteristics consistent with a chronically active disease process. These results suggest initial T cell depletion may strongly influence outcomes of HIV-Mtb co-infection.

HIV-1 matrix dependent membrane targeting is regulated by Gag mRNA trafficking.

Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC) assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV) post-transcriptional regulatory element (PRE) mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA) with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

Comparison of the effects of pathogenic simian human immunodeficiency virus strains SHIV-89.6P and SHIV-KU2 in cynomolgus macaques.

Factors explaining why human immunodeficiency virus (HIV) enhances the risk of reactivated tuberculosis (TB) are poorly understood. Unfortunately, experimental models of HIV-induced reactivated TB are lacking. We examined whether cynomolgus macaques, which accurately model latent TB in humans, could be used to model pathogenesis of HIV infection in the lungs and associated lymph nodes. These experiments precede studies modeling the effects of HIV infection on latent TB. We infected two groups of macaques with chimeric simian-human immunodeficiency viruses (SHIV-89.6P and SHIV-KU2) and followed viral titers and immunologic parameters including lymphocytes numbers and phenotype in the blood, bronchoalveolar lavage cells, and lymph nodes over the course of infection. Tissues from the lungs, liver, kidney, spleen, and lymph nodes were similarly examined at necropsy. Both strains produced dramatic CD4(+) T cell depletion. Plasma titers were not different between viruses, but we found more SHIV-89.6P in the lungs. Both viruses induced similar patterns of cell activation markers. SHIV-89.6P induced more IFN-gamma expression than SHIV-KU2. These results indicate SHIV-89.6P and SHIV-KU2 infect cynomolgus macaques and may be used to accurately model effects of HIV infection on latent TB.

Distinct intracellular trafficking of equine infectious anemia virus and human immunodeficiency virus type 1 Gag during viral assembly and budding revealed by bimolecular fluorescence complementation assays.

Retroviral Gag polyproteins are necessary and sufficient for virus budding. Numerous studies of human immunodeficiency virus type 1 (HIV-1) Gag assembly and budding mechanisms have been reported, but relatively little is known about these fundamental pathways among animal lentiviruses. While there may be a general assumption that lentiviruses share common assembly mechanisms, studies of equine infectious anemia virus (EIAV) have indicated alternative cellular pathways and cofactors employed among lentiviruses for assembly and budding. In the current study, we used bimolecular fluorescence complementation to characterize and compare assembly sites and budding efficiencies of EIAV and HIV-1 Gag in both human and rodent cells. The results of these studies demonstrated that replacing the natural RNA nuclear export element (Rev-response element [RRE]) used by HIV-1 and EIAV with the hepatitis B virus posttranscriptional regulatory element (PRE) altered HIV-1, but not EIAV, Gag assembly sites and budding efficiency in human cells. Consistent with this novel observation, different assembly sites were revealed in human cells for Rev-dependent EIAV and HIV-1 Gag polyproteins. In rodent cells, Rev-dependent HIV-1 Gag assembly and budding were blocked, but changing RRE to PRE rescued HIV-1 Gag assembly and budding. In contrast, EIAV Gag polyproteins synthesized from mRNA exported via either Rev-dependent or PRE-dependent mechanisms were able to assemble and bud efficiently in rodent cells. Taken together, our results suggest that lentivirus assembly and budding are regulated by the RNA nuclear export pathway and that alternative cellular pathways can be adapted for lentiviral Gag assembly and budding.

Differential functional phenotypes of two primary HIV-1 strains resulting from homologous point mutations in the LLP domains of the envelope gp41 intracytoplasmic domain.

We previously reported that selected mutations of highly conserved arginine residues within the LLP regions of HIV-1(ME46) gp41 had diverse effects on Env function. In the current study, we sought to test if the observed LLP mutant phenotypes would be similar in HIV-1(89.6). The results of the current studies revealed that the LLP-1 mutations conferred reduced Env incorporation, infectivity, and replication phenotypes in both viruses, while homologous LLP-2 mutations had differential phenotypical effects between the two strains. In particular, several of the 89.6 LLP-2 mutant viruses were replication defective in CEMX174 cells despite having increased levels of Env incorporation, and with both strains, there were differential effects on infectivity. This comparison of homologous point mutations in two different strains of HIV supports the role of LLPs as determinants of Env function, but reveals for the first time the influence of virus strain on LLP mutant phenotypes.

Association of gag multimers with filamentous actin during equine infectious anemia virus assembly.

A role for the actin cytoskeleton in retrovirus assembly has long been speculated. However, specific mechanisms by which actin facilitates the assembly process remain elusive. We previously demonstrated differential effects of experimentally modified actin dynamics on virion production of equine infectious anemia virus (EIAV), a lentivirus related to HIV-1, suggesting an involvement of actin dynamics in retrovirus production. In the current study, we used bimolecular fluorescence complementation (BiFC) to reveal intimate (<15 nm) and specific associations between EIAV Gag and actin, but not tubulin. Specific interaction between Gag and filamentous actin was also demonstrated by co-immunoprecipitation experiments combined with the actin severing protein gelsolin to solubilize F-actin. Deletion of capsid (CA) or nucleocapsid (NC) genes reduced Gag association with F-actin by 40% and 95%, respectively. Interestingly, GCN4, a leucine zipper motif, could substitute for the NC domain in mediating F-actin association. Furthermore, deficiency of the DeltaNC Gag in F-actin interaction was restored upon co-expression of Gag constructs containing both CA and NC or the GCN4, suggesting a requirement for Gag polyprotein multimerization prior to F-actin association. The observed Gag-F-actin association appeared to correlate with viral budding, as enhanced budding of the DeltaNC mutant was evident upon restoration of F-actin association. Intracellular association of Gag complexes with F-actin was also detected by immunoscanning electron microscopy of Triton-extracted EIAV-infected cells. Together, these data suggest that Gag multimers induced by CA and NC domains interact with F-actin and that this association is important for efficient virion production.

Immune suppression of challenged vaccinates as a rigorous assessment of sterile protection by lentiviral vaccines.

We previously reported that an experimental live-attenuated equine infectious anemia virus (EIAV) vaccine, containing a mutated S2 accessory gene, provided protection from disease and detectable infection after virulent virus (EIAV(PV)) challenge [Li F, Craigo JK, Howe L, Steckbeck JD, Cook S, Issel C, et al. A live-attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses. J Virol 2003;77(13):7244-53; Craigo JK, Li F, Steckbeck JD, Durkin S, Howe L, Cook SJ, et al. Discerning an effective balance between equine infectious anemia virus attenuation and vaccine efficacy. J Virol 2005;79(5):2666-77]. To determine if attenuated EIAV vaccines actually prevent persistent infection by challenge virus, we employed a 14-day dexamethasone treatment of vaccinated horses post-challenge to suppress host immunity and amplify replication levels of any infecting EIAV. At 2 months post-challenge the horses were all protected from virulent-virus challenge, evidenced by a lack of EIA signs and detectable challenge plasma viral RNA. Upon immune suppression, 6/12 horses displayed clinical EIA. Post-immune suppression characterizations demonstrated that the attenuated vaccine evidently prevented detectable challenge virus infection in 50% of horses. These data highlight the utility of post-challenge immune suppression for evaluating persistent viral vaccine protective efficacy.

Apparent elimination of EIAV ancestral species in a long-term inapparent carrier.

Equine infectious anemia virus (EIAV) envelope variation produces newly dominant quasispecies with each sequential disease cycle; new populations arise, and previous plasma quasispecies, including the original inoculum, become undetectable. The question remains whether these ancestral variants exist in tissue reservoirs or if the immune system eliminates quasispecies from persistent infections. To examine this, an EIAV long-term inapparent carrier was immune suppressed with dexamethasone. Immune suppression resulted in increased plasma viral loads by approximately 10(4) fold. Characterization of pre- and post-immune suppression populations demonstrated continual envelope evolution and revealed novel quasispecies distinct from defined populations from previous disease stages. Analysis of the tissue and plasma populations post-immune suppression indicated the original infectious inoculum and early populations were undetectable. Therefore, the host immune system apparently eliminated a diverse array of antigenic variants, but viral persistence was maintained by relentless evolution of new envelope populations from tissue reservoirs in response to ongoing immune pressures.

Epstein-Barr virus and human immunodeficiency virus serological responses and viral burdens in HIV-infected patients treated with HAART.

Epstein-Barr virus (EBV) associated non-Hodgkin lymphoma is recognized as a complication of human immunodeficiency virus (HIV) infection. Little is known regarding the influence of highly active antiretroviral therapy (HAART) on the biology of EBV in this population. To characterize the EBV- and HIV-specific serological responses together with EBV DNA levels in a cohort of HIV-infected adults treated with HAART, a study was conducted to compare EBV and HIV serologies and EBV DNA copy number (DNAemia) over a 12-month period after the commencement of HAART. All patients were seropositive for EBV at baseline. Approximately 50% of patients had detectable EBV DNA at baseline, and 27/30 had detectable EBV DNA at some point over the follow-up period of 1 year. Changes in EBV DNA copy number over time for any individual were unpredictable. Significant increases in the levels of Epstein-Barr nuclear antigen (EBNA) and Epstein-Barr early antigen (EA) antibodies were demonstrated in the 17 patients who had a good response to HAART. Of 29 patients with paired samples tested, four-fold or greater increases in titers were detected for EA in 12/29 (41%), for EBNA in 7/29 (24%), for VCA-IgG in 4/29 (14%); four-fold decreases in titers were detected in 2/29 (7%) for EA and 12/29 (41%) for EBNA. A significant decline in the titer of anti-HIV antibodies was also demonstrated. It was concluded that patients with advanced HIV infection who respond to HAART have an increase in their EBV specific antibodies and a decrease in their HIV-specific antibodies. For the cohort overall, there was a transient increase in EBV DNA levels that had declined by 12 months.