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The latest outbreak of Zika virus (ZIKV) in the Americas was associated with significant neurologic complications, including microcephaly of newborns. We evaluated mechanisms that regulate ZIKV entry into human fetal astrocytes (HFAs). Astrocytes are key players in maintaining brain homeostasis. We show that the central mediator of canonical Wnt signaling, β-catenin, regulates Axl, a receptor for ZIKV infection of HFAs, at the transcriptional level. In turn, ZIKV inhibited β-catenin, potentially as a mechanism to overcome its restriction of ZIKV internalization through regulation of Axl. This was evident with three ZIKV strains tested but not with a laboratory-adapted strain which has a large deletion in its envelope gene. Finally, we show that β-catenin-mediated Axl-dependent internalization of ZIKV may be of increased importance for brain cells, as it regulated ZIKV infection of astrocytes and human brain microvascular cells but not kidney epithelial (Vero) cells. Collectively, our studies reveal a role for β-catenin in ZIKV infection and highlight a dynamic interplay between ZIKV and β-catenin to modulate ZIKV entry into susceptible target cells. IMPORTANCE ZIKV is an emerging pathogen with sporadic outbreaks throughout the world. The most recent outbreak in North America was associated with small brains (microcephaly) in newborns. We studied the mechanism(s) that may regulate ZIKV entry into astrocytes. Astrocytes are a critical resident brain cell population with diverse functions that maintain brain homeostasis, including neurogenesis and neuronal survival. We show that three ZIKV strains (and not a heavily laboratory-adapted strain with a large deletion in its envelope gene) require Axl for internalization. Most importantly, we show that β-catenin, the central mediator of canonical Wnt signaling, negatively regulates Axl at the transcriptional level to prevent ZIKV internalization into human fetal astrocytes. To overcome this restriction, ZIKV downregulates β-catenin to facilitate Axl expression. This highlights a dynamic host-virus interaction whereby ZIKV inhibits β-catenin to promote its internalization into human fetal astrocytes through the induction of Axl.

CD4dimCD8bright T cells, a genuine population of CD8+ T cells, are highly activated and cytolytic. Recently, the low affinity IgG Fc fragment receptor CD32a was described as marker of HIV latency while others reported that CD32a is associated with T cell activation. Given that we have previously established that CD4dimCD8bright T cells are highly activated, mediate anti-HIV responses, and are infected by HIV, we assessed here CD32 expression on CD4dimCD8bright T cells in context of HIV. CD32 frequency on peripheral CD4dimCD8bright and CD4+ T cells was determined by flow cytometry among HIV negative and HIV positive patients. We report that among HIV- individuals, mean CD32 percent expression was 60% on CD4dimCD8bright T cells and 17% on CD4+ T cells (p

Latency is the main obstacle towards an HIV cure, with cure strategies aiming to either elicit or prevent viral reactivation. While these strategies have shown promise, they have only succeeded in modulating latency in a fraction of the latent HIV reservoir, suggesting that the mechanisms controlling HIV latency are not completely understood, and that comprehensive latency modulation will require targeting of multiple latency maintenance pathways. We show here that the transcriptional co-activator and the central mediator of canonical Wnt signaling, β-catenin, inhibits HIV transcription in CD4+ T cells via TCF-4 LTR binding sites. Further, we show that inhibiting the β-catenin pathway reactivates HIV in a primary TCM cell model of HIV latency, primary cells from cART-controlled HIV donors, and in CD4+ latent cell lines. β-catenin inhibition or activation also enhanced or inhibited the activity of several classes of HIV latency reversing agents, respectively, in these models, with significant synergy of β-catenin and each LRA class tested. In sum, we identify β-catenin as a novel regulator of HIV latency in vitro and ex vivo, adding new therapeutic targets that may be combined for comprehensive HIV latency modulation in HIV cure efforts.

HIV invades the brain during acute infection. Yet, it is unknown whether long-lived infected brain cells release productive virus that can egress from the brain to re-seed peripheral organs. This understanding has significant implication for the brain as a reservoir for HIV and most importantly HIV interplay between the brain and peripheral organs. Given the sheer number of astrocytes in the human brain and their controversial role in HIV infection, we evaluated their infection in vivo and whether HIV infected astrocytes can support HIV egress to peripheral organs. We developed two novel models of chimeric human astrocyte/human peripheral blood mononuclear cells: NOD/scid-IL-2Rgc null (NSG) mice (huAstro/HuPBMCs) whereby we transplanted HIV (non-pseudotyped or VSVg-pseudotyped) infected or uninfected primary human fetal astrocytes (NHAs) or an astrocytoma cell line (U138MG) into the brain of neonate or adult NSG mice and reconstituted the animals with human peripheral blood mononuclear cells (PBMCs). We also transplanted uninfected astrocytes into the brain of NSG mice and reconstituted with infected PBMCs to mimic a biological infection course. As expected, the xenotransplanted astrocytes did not escape/migrate out of the brain and the blood brain barrier (BBB) was intact in this model. We demonstrate that astrocytes support HIV infection in vivo and egress to peripheral organs, at least in part, through trafficking of infected CD4+ T cells out of the brain. Astrocyte-derived HIV egress persists, albeit at low levels, under combination antiretroviral therapy (cART). Egressed HIV evolved with a pattern and rate typical of acute peripheral infection. Lastly, analysis of human cortical or hippocampal brain regions of donors under cART revealed that astrocytes harbor between 0.4–5.2% integrated HIV gag DNA and 2–7% are HIV gag mRNA positive. These studies establish a paradigm shift in the dynamic interaction between the brain and peripheral organs which can inform eradication of HIV reservoirs., Author summary HIV latency and residual low-level HIV replication is a major obstacle towards an HIV cure. HIV infects the brain in acute disease yet it is unknown whether long lived-infected brain cells release productive virus that can egress from the brain to re-seed peripheral organs and whether astrocytes are productively infected in vivo. We demonstrate astrocyte-initiated HIV spread from the brain to the spleen and lymph nodes, likely through T cell trafficking out of CNS and into peripheral organs. Additionally, brain sections from patients on cART show HIV integration in astrocytes. Collectively, given that astrocytes constitute ~60% of brain cells and even with a conservative rate of infection at >3%, astrocytes can be a significant reservoir for HIV. As such, cure initiatives must consider the contribution of the CNS to ongoing HIV replication within and outside of the brain.

The sooty mangabey-derived simian immunodeficiency virus (SIV) strain E660 (SIVsmE660) is a genetically heterogeneous, pathogenic isolate that is commonly used as a vaccine challenge strain in the nonhuman primate (NHP) model of human immunodeficiency virus type 1 (HIV-1) infection. Though it is often employed to assess antibody-based vaccine strategies, its sensitivity to antibody-mediated neutralization has not been well characterized. Here, we utilize single-genome sequencing and infectivity assays to analyze the neutralization sensitivity of the uncloned SIVsmE660 isolate, individual viruses comprising the isolate, and transmitted/founder (T/F) viruses arising from low-dose mucosal inoculation of macaques with the isolate. We found that the SIVsmE660 isolate overall was highly sensitive to neutralization by SIV-infected macaque plasma samples (50% inhibitory concentration [IC 50 ] < 10 −5 ) and monoclonal antibodies targeting V3 (IC 50 < 0.01 μg/ml), CD4-induced (IC 50 < 0.1 μg/ml), CD4 binding site (IC 50 ∼ 1 μg/ml), and V4 (IC 50 , ∼5 μg/ml) epitopes. In comparison, SIVmac251 and SIVmac239 were highly resistant to neutralization by these same antibodies. Differences in neutralization sensitivity between SIVsmE660 and SIVmac251/239 were not dependent on the cell type in which virus was produced or tested. These findings indicate that in comparison to SIVmac251/239 and primary HIV-1 viruses, SIVsmE660 generally exhibits substantially less masking of antigenically conserved Env epitopes. Interestingly, we identified a minor population of viruses (∼10%) in both the SIVsmE660 isolate and T/F viruses arising from it that were substantially more resistant (>1,000-fold) to antibody neutralization and another fraction (∼20%) that was intermediate in neutralization resistance. These findings may explain the variable natural history and variable protection afforded by heterologous Env-based vaccines in rhesus macaques challenged by high-dose versus low-dose SIVsmE660 inoculation regimens.

Sexual transmission of HIV-1 is an inefficient process, with only one or few variants of the donor quasispecies establishing the new infection. A critical, and as yet unresolved, question is whether the mucosal bottleneck selects for viruses with increased transmission fitness. Here, we characterized 300 limiting dilution-derived virus isolates from the plasma, and in some instances genital secretions, of eight HIV-1 donor and recipient pairs. Although there were no differences in the amount of virion-associated envelope glycoprotein, recipient isolates were on average 3-fold more infectious (P = 0.0001), replicated to 1.4-fold higher titers (P = 0.004), were released from infected cells 4.2-fold more efficiently (P < 0.00001), and were significantly more resistant to type I interferons (IFNs) than the corresponding donor isolates. Remarkably, transmitted viruses exhibited 7.8-fold higher IFNα2 (P < 0.00001) and 39-fold higher IFNβ (P < 0.00001) half-maximal inhibitory concentrations (IC50) than did donor isolates, and their odds of replicating in CD4+ T cells at the highest IFNα2 and IFNβ doses were 35-fold (P < 0.00001) and 250-fold (P < 0.00001) greater, respectively. Interestingly, pretreatment of CD4+ T cells with IFNβ, but not IFNα2, selected donor plasma isolates that exhibited a transmitted virus-like phenotype, and such viruses were also detected in the donor genital tract. These data indicate that transmitted viruses are phenotypically distinct, and that increased IFN resistance represents their most distinguishing property. Thus, the mucosal bottleneck selects for viruses that are able to replicate and spread efficiently in the face of a potent innate immune response.

Defining the virus–host interactions responsible for HIV-1 transmission, including the phenotypic requirements of viruses capable of establishing de novo infections, could be important for AIDS vaccine development. Previous analyses have failed to identify phenotypic properties other than chemokine receptor 5 (CCR5) and CD4+ T-cell tropism that are preferentially associated with viral transmission. However, most of these studies were limited to examining envelope (Env) function in the context of pseudoviruses. Here, we generated infectious molecular clones of transmitted founder (TF; n = 27) and chronic control (CC; n = 14) viruses of subtypes B ( n = 18) and C ( n = 23) and compared their phenotypic properties in assays specifically designed to probe the earliest stages of HIV-1 infection. We found that TF virions were 1.7-fold more infectious ( P = 0.049) and contained 1.9-fold more Env per particle ( P = 0.048) compared with CC viruses. TF viruses were also captured by monocyte-derived dendritic cells 1.7-fold more efficiently ( P = 0.035) and more readily transferred to CD4+ T cells ( P = 0.025). In primary CD4+ T cells, TF and CC viruses replicated with comparable kinetics; however, when propagated in the presence of IFN-α, TF viruses replicated to higher titers than CC viruses. This difference was significant for subtype B ( P = 0.000013) but not subtype C ( P = 0.53) viruses, possibly reflecting demographic differences of the respective patient cohorts. Together, these data indicate that TF viruses are enriched for higher Env content, enhanced cell-free infectivity, improved dendritic cell interaction, and relative IFN-α resistance. These viral properties, which likely act in concert, should be considered in the development and testing of AIDS vaccines.

African green monkeys (AGMs) are naturally infected with a simian immunodeficiency virus (SIVagm) that is nonpathogenic in its host. Although SIVagm is common and widespread, little is known about the mechanisms that govern its transmission. Since the earliest virus-host interactions may provide key insights into the nonpathogenic phenotype of SIVagm, we developed a mucosal transmission model for this virus. Using plasma from an acutely infected AGM as the virus inoculum, we exposed adult and juvenile AGMs, as well as pigtailed macaques (PTMs) as a nonnatural host control, by mucosal routes to increasing titers of virus and compared the doses needed to establish a productive infection. Four juvenile and four adult AGMs as well as two PTMs were intrarectally (IR) exposed, while two additional adult female AGMs were intravaginally (IVAG) exposed. No animal became infected following exposure to 10 5 RNA copies. Both PTMs but none of the AGMs became infected following exposure to 10 6 RNA copies. Finally, all adult AGMs and two of the four juvenile AGMs became infected following exposure to 10 7 RNA copies, acquiring either one (2 IR infected juveniles, 1 IR infected adult, 2 IVAG infected adults) or two (3 IR infected adults) transmitted founder viruses. These results were consistent with immunophenotypic data, which revealed a significant correlation between the percentage of CD4 + T cells expressing CCR5 in the mucosa and the susceptibility to infection, in terms of both the viral dose and the numbers of transmitted founder viruses. Moreover, studies of uninfected AGMs showed that the fraction of CCR5-expressing CD4 + T cells increased significantly with age. These results indicate that (i) AGMs are readily infected with SIVagm by both intrarectal and intravaginal routes, (ii) susceptibility to infection is proportional to the number of available CCR5 + CD4 + target cells in the mucosa, and (iii) the paucity of CCR5 + CD4 + target cells in infant and juvenile AGMs may explain the near absence of vertical transmission.

Disease and other health hazards pose serious threats to the persistence of wild ape populations. The total chimpanzee population at Gombe National Park, Tanzania, has declined from an estimated 120 to 150 individuals in the 1960's to around 100 individuals by the end of 2013, with death associated with observable signs of disease as the leading cause of mortality. In 2004, we began a non-invasive health-monitoring program in the two habituated communities in the park (Kasekela and Mitumba) with the aim of understanding the prevalence of health issues in the population, and identifying the presence and impacts of various pathogens. Here we present prospectively collected data on clinical signs (observable changes in health) in the chimpanzees of the Kasekela (n = 81) and Mitumba (n = 32) communities over an 8-year period (2005-2012). First, we take a population approach and analyze prevalence of clinical signs in five different categories: gastrointestinal system (diarrhea), body condition (estimated weight loss), respiratory system (coughing, sneezing etc.), wounds/lameness, and dermatologic issues by year, month, and community membership. Mean monthly prevalence of each clinical sign per community varied, but typically affected10% of observed individuals. Secondly, we analyze the presence of clinical signs in these categories as they relate to individual demographic and social factors (age, sex, and dominance rank) and simian immunodeficiency virus (SIVcpz) infection status. Adults have higher odds of being observed with diarrhea, loss of body condition, and wounds or lameness when compared to immatures, while males have a higher probability of being observed with wounds or lameness than females. In contrast, signs of respiratory illness appear not to be related to chimpanzee-specific factors and skin abnormalities are very rare. For a subset of known-rank individuals, dominance rank predicts the probability of wounding/lameness in adult males, but does not predict any adverse clinical signs in adult females. Instead, adult females with SIVcpz infection are more likely to be observed with diarrhea, a finding that warrants further investigation. Comparable data are needed from other sites to determine whether the prevalence of clinical signs we observe are relatively high or low, as well as to more fully understand the factors influencing health of wild apes at both the population and individual level. Am. J. Primatol. 80:e22562, 2018. © 2016 Wiley Periodicals, Inc.

SUMMARY Densely arranged N-linked glycans shield the HIV-1 envelope (Env) trimer from antibody recognition. Strain-specific breaches in this shield (glycan holes) can be targets of vaccine-induced neutralizing antibodies that lack breadth. To understand the interplay between glycan holes and neutralization breadth in HIV-1 infection, we developed a sequence-and structure-based approach to identify glycan holes for individual Env sequences that are shielded in most M-group viruses. Applying this approach to 12 longitudinally followed individuals, we found that transmitted viruses with more intact glycan shields correlated with development of greater neutralization breadth. Within 2 years, glycan acquisition filled most glycan holes present at transmission, indicating escape from hole-targeting neutralizing antibodies. Glycan hole filling generally preceded the time to first detectable breadth, although time intervals varied across hosts. Thus, completely glycan-shielded viruses were associated with accelerated neutralization breadth development, suggesting that Env immunogens with intact glycan shields may be preferred components of AIDS vaccines., In Brief Wagh et al. show that transmitted viruses with more intact glycan shields are correlated with development of neutralization breadth in HIV-1-infected individuals. This is consistent with previous findings that glycan holes in Env immunogens are targeted by strain-specific neutralizing responses, and suggests that immunogens with intact glycan Shields may be advantageous., Graphical Abstract

Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Igmim2, CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4+ T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection., IMPORTANCE SIVcpz is widespread in wild-living chimpanzees and can cause AIDS-like immunopathology and clinical disease. HIV-1 infection of humans can be controlled by antiretroviral therapy; however, treatment of wild-living African apes with current drug regimens is not feasible. Nonetheless, it may be possible to curb the spread of SIVcpz in select ape communities using vectored immunoprophylaxis and/or therapy. Here, we show that antibodies and antibody-like inhibitors developed to combat HIV-1 infection in humans are capable of neutralizing genetically diverse SIVcpz and SIVgor strains with considerable breadth and potency, including in primary chimpanzee CD4+ T cells. These reagents provide an important first step toward translating intervention strategies currently developed to treat and prevent AIDS in humans to SIV-infected apes.

Age-associated skeletal muscle mass loss curtails quality of life and may contribute to defects in metabolic homeostasis in older persons. The onset of sarcopenia occurs in middle age in rhesus macaques although the trigger has yet to be identified. Here, we show that a shift in metabolism occurs in advance of the onset of sarcopenia in rhesus vastus lateralis. Multiphoton laser-scanning microscopy detects a shift in the kinetics of photon emission from autofluorescent metabolic cofactors NADH and FAD. Lifetime of both fluorophores is shortened at mid-age, and this is observed in both free and bound constituent pools. Levels of FAD and free NADH are increased and the NAD/NADH redox ratio is lower. Concomitant with this, expression of fiber-type myosin isoforms is altered resulting in a shift in fiber-type distribution, activity of cytochrome c oxidase involved in mitochondrial oxidative phosphorylation is significantly lower, and the subcellular organization of mitochondria in oxidative fibers is compromised. A regulatory switch involving the transcriptional coactivator PGC-1α directs metabolic fuel utilization and governs the expression of structural proteins. Age did not significantly impact total levels of PGC-1α; however, its subcellular localization was disrupted, suggesting that PGC-1α activities may be compromised. Consistent with this, intracellular lipid storage is altered and there is shift to larger lipid droplet size that likely reflects a decline in lipid turnover or a loss in efficiency of lipid metabolism. We suggest that changes in energy metabolism contribute directly to skeletal muscle aging in rhesus monkeys.

Disease and other health hazards pose serious threats to the persistence of wild ape populations. The total chimpanzee population at Gombe National Park, Tanzania, has declined from an estimated 120 to 150 individuals in the 1960's to around 100 individuals by the end of 2013, with death associated with observable signs of disease as the leading cause of mortality. In 2004, we began a non-invasive health-monitoring program in the two habituated communities in the park (Kasekela and Mitumba) with the aim of understanding the prevalence of health issues in the population, and identifying the presence and impacts of various pathogens. Here we present prospectively collected data on clinical signs (observable changes in health) in the chimpanzees of the Kasekela (n = 81) and Mitumba (n = 32) communities over an 8-year period (2005-2012). First, we take a population approach and analyze prevalence of clinical signs in five different categories: gastrointestinal system (diarrhea), body condition (estimated weight loss), respiratory system (coughing, sneezing etc.), wounds/lameness, and dermatologic issues by year, month, and community membership. Mean monthly prevalence of each clinical sign per community varied, but typically affected