Chromatin Immunoprecipitation of Retroviral Genomes with Antibodies Recognizing Modified Histones and Specific Viral Proteins.

Mammalian cells have developed and optimized defense mechanisms to prevent or hamper viral infection. The early transcriptional silencing of incoming viral DNAs is one such antiviral strategy and seems to be of fundamental importance, since most cell types silence unintegrated retroviral DNAs. In this chapter, a method for chromatin immunoprecipitation of unintegrated DNA is described.

Two lymphoid cell lines potently silence unintegrated HIV-1 DNAs.

Mammalian cells mount a variety of defense mechanisms against invading viruses to prevent or reduce infection. One such defense is the transcriptional silencing of incoming viral DNA, including the silencing of unintegrated retroviral DNA in most cells. Here, we report that the lymphoid cell lines K562 and Jurkat cells reveal a dramatically higher efficiency of silencing of viral expression from unintegrated HIV-1 DNAs as compared to HeLa cells.

CHAF1A/B mediate silencing of unintegrated HIV-1 DNAs early in infection.

Early events of the retroviral life cycle are the targets of many host restriction factors that have evolved to prevent establishment of infection. Incoming retroviral DNAs are transcriptionally silenced before integration in most cell types, and efficient viral gene expression occurs only after formation of the provirus. The molecular machinery for silencing unintegrated retroviral DNAs of HIV-1 remains poorly characterized.

Silencing and Transcriptional Regulation of Endogenous Retroviruses: An Overview.

Almost half of the human genome is made up of transposable elements (TEs), and about 8% consists of endogenous retroviruses (ERVs). ERVs are remnants of ancient exogenous retrovirus infections of the germ line. Most TEs are inactive and not detrimental to the host.

Unintegrated HIV-1 DNAs are loaded with core and linker histones and transcriptionally silenced.

Upon delivery into the nucleus of the host cell, linear double-stranded retroviral DNAs are either integrated into the host genome to form the provirus or act as a target of the DNA damage response and become circularized. Little is known about the chromatinization status of the unintegrated retroviral DNAs of the human immunodeficiency virus type 1 (HIV-1). In this study, we used chromatin immunoprecipitation to investigate the nature of unintegrated HIV-1 DNAs and discovered that core histones, the histone variant H3.

Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout.

The recently discovered CRISPR/Cas9 system is widely used in basic research and is a useful tool for disease modeling and gene editing therapies. However, long-term expression of DNA-modifying enzymes can be associated with cytotoxicity and is particularly unwanted in clinical gene editing strategies. Because current transient expression methods may still suffer from cytotoxicity and/or low efficiency, we developed non-integrating retrovirus-based CRISPR/Cas9 all-in-one particles for targeted gene knockout.

Correction to: Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

The authors wish to apologize for an error within the scale bar of the microarray heatmap in Additional File 5 of the supplementary information. Two values were incorrectly displayed on the scale bar (11 instead of 10 and 13 instead of 12).

Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

Background: Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers.