Residues T48 and A49 in HIV-1 NL4-3 Nef are responsible for the counteraction of autophagy initiation, which prevents the ubiquitin-dependent degradation of Gag through autophagosomes

Background Autophagy plays an important role as a cellular defense mechanism against intracellular pathogens, like viruses. Specifically, autophagy orchestrates the recruitment of specialized cargo, including viral components needed for replication, for lysosomal degradation. In addition to this primary role, the cleavage of viral structures facilitates their association with pattern recognition receptors and MHC-I/II complexes, which assists in the modulation of innate and adaptive immune responses against these pathogens. Importantly, whereas autophagy restricts the replicative capacity of human immunodeficiency virus type 1 (HIV-1), this virus has evolved the gene nef to circumvent this process through the inhibition of early and late stages of the autophagy cascade. Despite recent advances, many details of the mutual antagonism between HIV-1 and autophagy still remain unknown. Here, we uncover the genetic determinants that drive the autophagy-mediated restriction of HIV-1 as well as the counteraction imposed by Nef. Additionally, we also examine the implications of autophagy antagonism in HIV-1 infectivity. Results We found that sustained activation of autophagy potently inhibits HIV-1 replication through the degradation of HIV-1 Gag, and that this effect is more prominent for nef-deficient viruses. Gag re-localizes to autophagosomes where it interacts with the autophagosome markers LC3 and SQSTM1. Importantly, autophagy-mediated recognition and recruitment of Gag requires the myristoylation and ubiquitination of this virus protein, two post-translational modifications that are essential for Gag’s central role in virion assembly and budding. We also identified residues T48 and A49 in HIV-1 NL4-3 Nef as responsible for impairing the early stages of autophagy. Finally, a survey of pandemic HIV-1 transmitted/founder viruses revealed that these isolates are highly resistant to autophagy restriction. Conclusions This study provides evidence that autophagy antagonism is important for virus replication and suggests that the ability of Nef to counteract autophagy may have played an important role in mucosal transmission. Hence, disabling Nef in combination with the pharmacological manipulation of autophagy represents a promising strategy to prevent HIV spread.

Complement-Opsonized HIV Modulates Pathways Involved in Infection of Cervical Mucosal Tissues: A Transcriptomic and Proteomic Study

Genital mucosal transmission is the most common route of HIV spread. The initial responses triggered at the site of viral entry are reportedly affected by host factors, especially complement components present at the site, and this will have profound consequences on the outcome and pathogenesis of HIV infection. We studied the initial events associated with host-pathogen interactions by exposing cervical biopsies to free or complement-opsonized HIV. Opsonization resulted in higher rates of HIV acquisition/infection in mucosal tissues and emigrating dendritic cells. Transcriptomic and proteomic data showed a significantly more pathways and higher expression of genes and proteins associated with viral replication and pathways involved in different aspects of viral infection including interferon signaling, cytokine profile and dendritic cell maturation for the opsonized HIV. Moreover, the proteomics data indicate a general suppression by the HIV exposure. This clearly suggests that HIV opsonization alters the initial signaling pathways in the cervical mucosa in a manner that promotes viral establishment and infection. Our findings provide a foundation for further studies of the role these early HIV induced events play in HIV pathogenesis.

Capsaicin activates TRPV1 in human Langerhans cells and inhibits mucosal HIV-1 transmission via secreted CGRP

Upon its mucosal transmission, human immunodeficiency virus type 1 (HIV-1) rapidly targets resident antigen-presenting Langerhans cells (LCs) in genital epithelia, which subsequently trans-infect CD4+ T-cells. We previously described an inhibitory neuro-immune sensory mucosal crosstalk, whereby peripheral pain-sensing nociceptor neurons, innervating all mucosal epithelia and associating with LCs, secret the neuropeptide calcitonin gene-related peptide (CGRP) that strongly inhibits HIV-1 trans-infection. Moreover, we reported that LCs secret low levels of CGRP that are further increased by CGRP itself via an autocrine/paracrine mechanism. As nociceptors secret CGRP following activation of their Ca2+ ion channel transient receptor potential vanilloid 1 (TRPV1), we investigated whether LCs also express functional TRPV1. We found that human LCs expressed TRPV1 mRNA and protein. TRPV1 in LCs was functional, as the TRPV1 agonists capsaicin (CP) and resiniferatoxin (RTX) induced Ca2+ influx in a dose-dependent manner. Treatment of LCs with CP and the TRPV1 agonist rutaecarpine (Rut) increased CGRP secretion, reaching concentrations close to its IC50 for inhibition of HIV-1 trans-infection. Accordingly, CP significantly inhibited HIV-1 trans-infection, which was abrogated by antagonists of both TRPV1 and the CGRP receptor. Finally, pre-treatment of inner foreskin tissue explants with CP markedly increased CGRP secretion, and upon subsequent polarized exposure to HIV-1, inhibited increase in LC-T-cell conjugate formation and T-cell infection. Together, our results reveal that alike nociceptors, LCs express functional TRPV1, whose activation induces CGRP secretion that inhibits mucosal HIV-1 transmission. Our studies could permit re-positioning of formulations containing TRPV1 agonists, already approved for pain relief, as novel topical microbicides against HIV-1.Significance StatementUpon its sexual transmission, HIV-1 targets different types of mucosal immune cells, such as antigen-presenting Langerhans cells (LCs). In turn, LCs transfer HIV-1 to its principal cellular targets, namely CD4+ T-cells, in a process termed trans-infection. We previously discovered that the mucosal neuropeptide CGRP strongly inhibits trans-infection. CGRP is principally secreted from pain-sensing peripheral neurons termed nociceptors, once activated via their TRPV1 ion channel. Herein, we reveal that LCs also express functional TRPV1, whose activation induces secretion of CGRP that inhibits mucosal HIV-1 transmission. Accordingly, molecules activating TRPV1 and inducing CGRP secretion could be used to prevent mucosal HIV-1 transmission. This approach represents an original neuro-immune strategy to fight HIV-1.

Poly(dA:dT) Suppresses HSV-2 Infection of Human Cervical Epithelial Cells Through RIG-I Activation

Epithelial cells of the female reproductive tract (FRT) participate in the initial innate immunity against viral infections. Poly(dA:dT) is a synthetic analog of B form double-stranded (ds) DNA which can activate the interferon (IFN) signaling pathway-mediated antiviral immunity through DNA-dependent RNA Polymerase III. Here we investigated whether poly(dA:dT) could inhibit herpes simplex virus type 2 (HSV-2) infection of human cervical epithelial cells (End1/E6E7). We demonstrated that poly(dA:dT) treatment of End1/E6E7 cells could significantly inhibit HSV-2 infection. Mechanistically, poly(dA:dT) treatment of the cells induced the expression of the intracellular IFNs and the multiple antiviral IFN-stimulated genes (ISGs), including IFN-stimulated gene 15 (ISG15), IFN-stimulated gene 56 (ISG56), 2’-5’-oligoadenylate synthetase 1 (OAS1), 2’-5’-oligoadenylate synthetase 2 (OAS2), myxovirus resistance protein A (MxA), myxovirus resistance protein B (MxB), virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible (Viperin), and guanylate binding protein 5 (GBP5). Further investigation showed that the activation of RIG-I was largely responsible for poly(dA:dT)-mediated HSV-2 inhibition and IFN/ISGs induction in the cervical epithelial cells, as RIG-I knockout abolished the poly(dA:dT) actions. These observations demonstrate the importance for design and development of AT-rich dsDNA-based intervention strategies to control HSV-2 mucosal transmission in FRT.

HIV-1 Entry and Prospects for Protecting against Infection

Human Immunodeficiency Virus type-1 (HIV-1) establishes a latent viral reservoir soon after infection, which poses a major challenge for drug treatment and curative strategies. Many efforts are therefore focused on blocking infection. To this end, both viral and host factors relevant to the onset of infection need to be considered. Given that HIV-1 is most often transmitted mucosally, strategies designed to protect against infection need to be effective at mucosal portals of entry. These strategies need to contend also with cell-free and cell-associated transmitted/founder (T/F) virus forms; both can initiate and establish infection. This review will discuss how insight from the current model of HIV-1 mucosal transmission and cell entry has highlighted challenges in developing effective strategies to prevent infection. First, we examine key viral and host factors that play a role in transmission and infection. We then discuss preventive strategies based on antibody-mediated protection, with emphasis on targeting T/F viruses and mucosal immunity. Lastly, we review treatment strategies targeting viral entry, with focus on the most clinically advanced entry inhibitors.

Use of Monoclonal Antibodies to Prevent the Sexual Transmission of Human Immunodeficiency Virus Type 1

<P&gt;Passive immunization has been used since the late 1800’s to prevent and treat human infectious diseases. Administration of animal immune sera and human immunoglobulin has given way to the use of monoclonal antibodies (mAbs) for passive immunization, and highly potent broadly neutralizing anti-HIV antibodies (bNAbs) are now being considered for HIV therapy and prophylaxis. Recent studies have shown that systemic and topical administration of bNAbs can effectively inhibit HIV/SHIV mucosal transmission in macaques and in humanized mice, and selected bNAbs are currently being tested in clinical trials for safety and efficacy in humans. In this review, we outline strategies for the selection, engineering and manufacture of human bNAbs to prevent the sexual transmission of HIV, describe the proof-of-concept animal studies that have demonstrated mAb-mediated protection against mucosal HIV transmission, and review clinical trials currently underway to test the safety and efficacy of mAb-based HIV prevention in humans.

Virologic Aspects of Mucosal Transmission

The transmission of HIV is generally inefficient. Despite the development of a diverse viral quasispecies in a chronically infected individual, a severe genetic bottleneck is observed during transmission, leading to only one or a few genetic variants establishing infection. This genetic bottleneck is the result of both stochastic events and selection pressures, such that viruses with specific traits are favored during transmission. This chapter discusses current models of HIV mucosal transmission, evidence for selection of specific viral traits during this process, and the biological characterization of transmitted founder viruses based on monkey models and human cohorts. The impact of transmitted viral phenotypes on disease progression is also described. Understanding in greater depth the key viral features required for transmission will be essential to the development of effective interventions for HIV prevention.

Signal peptide of HIV envelope protein impacts glycosylation and antigenicity of gp120

The HIV-1 envelope protein (Env) of early-replicating viruses encodes several distinct transmission signatures. One such signature involves a reduced number of potential N-linked glycosylation sites (PNGs). This transmission signature underscores the importance of posttranslational modifications in the fitness of early-replicating isolates. An additional signature in Env involves the overrepresentation of basic amino acid residues at a specific position in the Env signal peptide (SP). In this report, we investigated the potential impact of this SP signature on gp120 glycosylation and antigenicity. Two recombinant gp120s were constructed, one derived from an isolate that lacks this signature and a second from an early-replicating isolate that includes this signature. Chimeric gp120s were also constructed in which the two SPs were swapped between the isolates. All four gp120s were probed with glycan-, structure- and receptor- specific probes in a surface plasmon resonance binding assay. We found that the SP of Env influences qualitative aspects of Env glycosylation that in turn affect the antigenicity of Env in a major way. The SP impacts the affinity of Env for DC-SIGN, a lectin receptor expressed on dendritic cells that is believed to play a role in mucosal transmission. Additionally, affinity for the monoclonal antibodies 17b and A32, which recognize a CD4-induced, open conformation of Env is also altered. These results demonstrate that natural variation in the SP of HIV Env can significantly impact the antigenicity of mature gp120. Thus, the SP is likely subject to antibody-mediated immune pressure.