Defective HIV-1 genomes and their potential impact on HIV pathogenesis.

Defective HIV-1 proviruses represent a population of viral genomes that are selected for by immune pressures, and clonally expanded to dominate the persistent HIV-1 proviral genome landscape. There are examples of RNA and protein expression from these compromised genomes which are generated by a variety of mechanisms. Despite the evidence that these proviruses are transcribed and translated, their role in HIV pathogenesis has not been fully explored. The potential for these genomes to participate in immune stimulation is particularly relevant considering the accumulation of cells harboring these defective proviruses over the course of antiretroviral therapy in people living with HIV. The expression of defective proviruses in different cells and tissues could drive innate sensing mechanisms and inflammation. They may also alter antiviral T cell responses and myeloid cell functions that directly contribute to HIV-1 associated chronic comorbidities. Understanding the impact of these defective proviruses needs to be considered as we advance cure strategies that focus on targeting the diverse population of HIV-1 proviral genomes.

Antibody-dependent cellular cytotoxicity responses and susceptibility influence HIV-1 mother-to-child transmission.

HIV-1 vaccine efforts are primarily directed toward eliciting neutralizing antibodies (nAbs). However, vaccine trials and mother-to-child natural history cohort investigations indicate that antibody-dependent cellular cytotoxicity (ADCC), not nAbs, correlate with prevention. The ADCC characteristics associated with lack of HIV-1 acquisition remain unclear. Here, we examine ADCC and nAb properties in pretransmission plasma from HIV-1-exposed infants and from the corresponding transmitting and nontransmitting mothers' breast milk and plasma. Breadth and potency (BP) were assessed against a panel of heterologous, nonmaternal variants. ADCC and neutralization sensitivity were estimated for the strains in the infected mothers. Infants who eventually acquired HIV-1 and those who remained uninfected had similar pretransmission ADCCBP. Viruses circulating in the transmitting and nontransmitting mothers had similar ADCC susceptibility. Infants with higher pretransmission ADCCBP and exposure to more ADCC-susceptible strains were less likely to acquire HIV-1. In contrast, higher preexisting infant neutralization BP and greater maternal virus neutralization sensitivity did not associate with transmission. Infants had higher ADCCBP closer to birth and in the presence of high plasma IgG relative to IgA levels. Mothers with potent humoral responses against their autologous viruses harbored more ADCC-sensitive strains. ADCC sensitivity of the exposure variants and preexisting ADCCBP influenced mother-to-child HIV-1 transmission during breastfeeding. Vaccination strategies that enhance ADCC are likely insufficient to prevent HIV-1 transmission because some strains may have low ADCC susceptibility.

Intragenic proviral elements support transcription of defective HIV-1 proviruses.

HIV-1 establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV proviruses has revealed that greater than 90% of integrated HIV genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, we observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription droplet digital PCR identified env and nef transcripts which lacked 5' untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5'UTR-deficient env transcripts were also identified in a cohort of people living with HIV (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5' rapid amplification of cDNA ends (RACE), we mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH.

HIV-1 Transcription but Not Intact Provirus Levels are Associated With Systemic Inflammation.

Individuals infected with human immunodeficiency virus (HIV) 1 have increased inflammation, which has been associated with age-associated diseases. Plasma markers, cell-associated virus levels, and ability to stimulate RNA transcription in latently infected cell lines was examined in younger and older HIV-1-infected individuals with suppressed virus. Cell-associated RNA, but not intact provirus level, had positive correlation with plasma D-dimer levels. Compared with the younger group, the older group had higher D-dimer levels and a trend toward more cell-associated RNA but similar levels of intact proviruses. Even though all measured inflammatory markers were relatively higher in the older group, this greater inflammation did not induce more HIV-1 transcription in latently infected cell lines. Inflammation and HIV-1 RNA expression increase with age despite similar levels of intact infectious HIV DNA. While plasma inflammation is correlated with HIV-1 RNA expression in peripheral blood mononuclear cells, it does not induce HIV-1 transcription in latently infected cell lines.

A new cell line for assessing HIV-1 antibody dependent cellular cytotoxicity against a broad range of variants.

Human immunodeficiency virus type 1 (HIV-1) studies suggest that antibody-dependent cellular cytotoxicity (ADCC) influences both virus acquisition and subsequent disease outcome. Technical issues with currently available assays, however, have limited the ability to comprehensively assess the impact of ADCC on transmission and disease progression. Commonly used ADCC assays use a target cell line, CEM.NKr-CCR5-Luc, that often does not support replication of relevant HIV-1 variants. Thus, the extent of ADCC responses against a large panel of HIV-1 strains often cannot be assessed using the currently available methods. We developed two new reporter cell-lines (MT4-CCR5-Luc and PM1-CCR5-Luc) to overcome these issues. MT4-CCR5-Luc cells are resistant, whereas PM1-CCR5-Luc cells are susceptible, to killing by a natural killer cell line, CD16+KHYG-1, in the absence of antibody. Polyclonal HIVIG gave similar ADCC estimates against HIV-1 isolate, NL4-3, regardless of which of the three cell lines were used as the targets. In contrast to CEM.NKr-CCR5-Luc and PM1-CCR5-Luc, however, MT4-CCR5-Luc target cells produce significantly higher luciferase after exposure to various HIV-1 strains, including transmitted founder variants and viruses incorporating specific envelopes of interest. This higher luciferase expression does not yield spurious results because ADCC estimates are similar when killing is assessed by both reporter protein expression and flow cytometry. Furthermore, ADCC estimates derived from MT4-CCR5-Luc cells are not skewed by non-antibody contents present in human plasma. In aggregate, the MT4-CCR5-Luc cell line can be used to estimate monoclonal antibody or plasma-induced ADCC responses against a diverse range of HIV-1 envelopes relevant for transmission and disease progression studies.

The Period protein homolog LIN-42 regulates germline development in C. elegans.

Germline stem cells are maintained in the distal region of the C. elegans gonad. These cells undergo mitotic divisions, and GLP-1/Notch signaling dictates whether they remain in this state. The somatic distal tip cell (DTC) caps the end of the distal gonad and is essential for maintenance of the germline mitotic zone. As germ cells move away from the DTC they exit mitosis and enter early meiotic prophase. Here we identify the Period protein homolog LIN-42 as a new regulator of germline development in C. elegans. LIN-42 is expressed in almost all somatic cells including the DTC, and LIN-42 functions as a transcription factor in the heterochronic pathway and to regulate molting. We found that the mitotic proliferative zone size in the distal gonad was significantly reduced by ~25% in lin-42 mutants compared to WT N2 worms. A lin-42 mutation also reduced the mitotic proliferative zone size caused by glp-1 partial loss-of-function and gain-of-function alleles. LIN-42 mediates this effect, at least in part, by regulating expression of the GLP-1/Notch ligand LAG-2. We further show that lin-42 expression itself is regulated by ATX-2, which promotes germline proliferation and is the homolog of the RNA binding protein ataxin-2 that is implicated in human neurodegenerative diseases. Altogether our results establish a new role for the conserved, important Period protein homolog LIN-42 in regulating early germline development. These results also suggest that in addition to regulating behavioral rhythms, the circadian clock plays an important role in communicating environmental signals to essential reproductive pathways.