The benefits of integration.

Retroviruses, including the human immunodeficiency virus (HIV), are notorious for two essential steps of their viral replication: reverse transcription and integration. This latter property is considered to be essential for productive replication and ensures the stable long-term insertion of the viral genome sequence in the host chromatin, thereby leading to the life-long association of the virus with the infected cell. Using HIV as a prototypic example, the present review aims to provide an overview of how and where integration occurs, as well as presenting general consequences for both the virus and the infected host

The use of single-cell RNA-Seq to understand virus-host interactions.

Single-cell analyses allow uncovering cellular heterogeneity, not only per se, but also in response to viral infection. Similarly, single cell transcriptome analyses (scRNA-Seq) can highlight specific signatures, identifying cell subsets with particular phenotypes, which are relevant in the understanding of virus-host interactions

Identification of HIV integration sites in infected host genomic DNA.

The integration of the Human Immunodeficiency Virus (HIV) genetic information into the host genome is fundamental for its replication and long-term persistence in the host. Isolating and characterizing the integration sites can be useful for obtaining data such as identifying the specific genomic location of integration or understanding the forces dictating HIV integration site selection. The methods outlined in this article describe a highly efficient and precise technique for identifying HIV integration sites in the host genome on a small scale using molecular cloning techniques and standard sequencing or on a massive scale using 454 pyrosequencing

Dynamic models of viral replication and latency.

PURPOSE OF REVIEW: HIV targets primary CD4(+) T cells. The virus depends on the physiological state of its target cells for efficient replication, and, in turn, viral infection perturbs the cellular state significantly. Identifying the virus-host interactions that drive these dynamic changes is important for a better understanding of viral pathogenesis and persistence. The present review focuses on experimental and computational approaches to study the dynamics of viral replication and latency. RECENT FINDINGS: It was recently shown that only a fraction of the inducible latently infected reservoirs are successfully induced upon stimulation in ex-vivo models while additional rounds of stimulation make allowance for reactivation of more latently infected cells. This highlights the potential role of treatment duration and timing as important factors for successful reactivation of latently infected cells. The dynamics of HIV productive infection and latency have been investigated using transcriptome and proteome data. The cellular activation state has shown to be a major determinant of viral reactivation success. Mathematical models of latency have been used to explore the dynamics of the latent viral reservoir decay. SUMMARY: Timing is an important component of biological interactions. Temporal analyses covering aspects of viral life cycle are essential for gathering a comprehensive picture of HIV interaction with the host cell and untangling the complexity of latency. Understanding the dynamic changes tipping the balance between success and failure of HIV particle production might be key to eradicate the viral reservoir

Exploring viral infection using single-cell sequencing.

Single-cell sequencing (SCS) has emerged as a valuable tool to study cellular heterogeneity in diverse fields, including virology. By studying the viral and cellular genome and/or transcriptome, the dynamics of viral infection can be investigated at single cell level. Most studies have explored the impact of cell-to-cell variation on the viral life cycle from the point of view of the virus, by analyzing viral sequences, and from the point of view of the cell, mainly by analyzing the cellular host transcriptome. In this review, we will focus on recent studies that use single-cell sequencing to explore viral diversity and cell variability in response to viral replication

HIV-1 latent reservoir: size matters.

More than 35 million people remain infected with HIV-1. Upon antiretroviral therapy cessation, HIV-1-positive individuals systematically fail to achieve sustained virological remission, revealing the presence of a reservoir. This reservoir takes into account anatomical sanctuaries where HIV-1 continues to replicate, and latently infected cells also known as the latent reservoir (LR). A better understanding of the nature and features of the LR and its quantification are crucial to evaluate the efficiency of therapeutic strategies aiming at purging HIV-1. Culture- and PCR-based assays have already been implemented to measure the LR, and new assays are continuously being developed. In this review, we will discuss these methods highlighting the difficulties to accurately measure the LR, one main obstacle in curing HIV-1

LEDGF/p75 TATA-less promoter is driven by the transcription factor Sp1.

PSIP1 (PC4 and SFRS1 interacting protein 1) encodes two splice variants: lens epithelium-derived growth factor or p75 (LEDGF/p75) and p52. PSIP1 gene products were shown to be involved in transcriptional regulation, affecting a plethora of cellular processes, including cell proliferation, cell survival, and stress response. Furthermore, LEDGF/p75 has implications for various diseases and infections, including autoimmunity, leukemia, embryo development, psoriasis, and human immunodeficiency virus integration. Here, we reported the first characterization of the PSIP1 promoter. Using 5' RNA ligase-mediated rapid amplification of cDNA ends, we identified novel transcription start sites in different cell types. Using a luciferase reporter system, we identified regulatory elements controlling the expression of LEDGF/p75 and p52. These include (i) minimal promoters (-112/+59 and +609/+781) that drive the basal expression of LEDGF/p75 and of the shorter splice variant p52, respectively; (ii) a sequence (+319/+397) that may control the ratio of LEDGF/p75 expression to p52 expression; and (iii) a strong enhancer (-320/-207) implicated in the modulation of LEDGF/p75 transcriptional activity. Computational, biochemical, and genetic approaches enabled us to identify the transcription factor Sp1 as a key modulator of the PSIP1 promoter, controlling LEDGF/p75 transcription through two binding sites at -72/-64 and -46/-36. Overall, our results provide initial data concerning LEDGF/p75 promoter regulation, giving new insights to further understand its biological function and opening the door for new therapeutic strategies in which LEDGF/p75 is involved

Innate immune defects in HIV permissive cell lines.

Primary CD4+ T cells and cell lines differ in their permissiveness to HIV infection. Impaired innate immunity may contribute to this different phenotype. We used transcriptome profiling of 1503 innate immunity genes in primary CD4+ T cells and permissive cell lines. Two clusters of differentially expressed genes were identified: a set of 249 genes that were highly expressed in primary cells and minimally expressed in cell lines and a set of 110 genes with the opposite pattern. Specific to HIV, HEK293T, Jurkat, SupT1 and CEM cell lines displayed unique patterns of downregulation of genes involved in viral sensing and restriction. Activation of primary CD4+ T cells resulted in reversal of the pattern of expression of those sets of innate immunity genes. Functional analysis of prototypical innate immunity pathways of permissive cell lines confirmed impaired responses identified in transcriptome analyses. Integrity of innate immunity genes and pathways needs to be considered in designing gain/loss functional genomic screens of viral infection

"From womb to tomb; we're bound to others": Microbiome in forensic science

Microbiome is a new field of interest in clinical medicine with high potential in forensic medicine. It could be used in several applications, such as post-mortem interval (PMI) estimation, personal identification, differential diagnosis of cause of death and toxicology. Regarding PMI, during the decomposition of a corpse, the passage of time involves changing in microbial population both outside and inside the corpse but also in surrounding soil (cadaver decomposition island). These variations could be hypothetically used as PMI indicators (microbial clock), even thanks to the development of machine learning approach. Another potential use of skin and saliva microbiome is personal identification thanks to its inter-individual variability and tendency to remain unvarying over time. It may also be helpful to link a person to a specific object that has been touched (microbial fingerprint). Furthermore, we could infer some information about health state of human subjects, comparing post-mortem and ante-mortem microbiome, but this field of research is quite new and needs further studies. Moreover, we have to consider the influence of microbiome metabolism in post-mortem toxicological evaluation; microbes could alter substances concentrations - for example of ethanol, gamma-hydroxybutyric acid (GHB) and nitrobenzodiazepines - due to enzymatic degradation and individual microbial metabolism. Finally, integration of microbiome and human being's transcriptomic analysis may be helpful to depict their complex interactions even after death