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# Retroviral integration site selection: a running ?

Paul Lesbats, Vincent Parissi

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Paul Lesbats, Vincent Parissi. Retroviral integration site selection: a running ?. Microbial Cell, 2018, 5 (12), pp.569-571. 10.15698/mic2018.12.663. hal-04274654

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# Retroviral integration site selection: a running Gag?

Paul Lesbats<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,* and Vincent Parissi<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

The ability of retroviruses to integrate their genomes into host chromatin is a key step for the completion of their replication cycle. Selection of a suitable chromosomal integration site has been described as a hierarchical mechanism involving both cellular and viral proteins but the exact molecular determinants are still unclear. We recently showed that the spumaretrovirus prototype foamy virus (PFV) Gag protein is acting as a chromatin tether by interacting with the nucleosome acidic patch (Lesbats et al. PNAS 114(21)). Disruption of the nucleosome binding leads to a dramatic delocalization of both the viral particles and the integration sites accompanied with a reduction of integrated genes expression. These data show for the first time a direct interaction between retroviral structural proteins with the host chromosomes, and highlight their importance in the integration sites selection.

The retroviral Gag proteins encoded by the *gag* (group antigen) gene compose the main structural components of the viral particle, with the exception of the envelope. Their involvement during retroviral cycle is pleiotropic, ranging from intracellular viral trafficking to viral assembly and integration. Retroviral integration site selection of a suitable chromatin environment is crucial for the fate of both the virus and the cell. Several studies on the lentivirus Human Immunodeficiency Virus-1 (HIV-1) and gammaretrovirus Murine Leukemia Virus (MLV) showed that their integration site selection is partly dictated by the interaction of their integrases (IN) with host cell chromatin-bound proteins, like LEDGF/p75 and BRD2-4, respectively. The chromatin-bound host factors act as tethers or receptors for the cognate pre-integration complexes. In addition, an increasing body of recent evidence demonstrates that the

HIV-1 capsid protein, a Gag product, is involved in integration site selection via binding to the cellular cleavage and polyadenylation specificity factor 6 (CPSF6) protein. This interaction induces a directing of the bulk of integration into gene dense regions of the nucleus. For the Spumaretrovirus family, previous data showed a tight association of the Gag proteins with mitotic chromosomes. The functional significance as well as the molecular determinants of this interaction were unclear. By using biochemistry and x-ray crystallography we could obtain the high-resolution structure of the chromatin-binding sequence (CBS) domain of the PFV Gag protein bound to a nucleosome. The CBS peptide adopts an elongated conformation along the face of the nucleosome core. The CBS-nucleosome complex involves one H2A-H2B heterodimer and both H3 histones chains with an array of hydrogen bounds and hydrophobic contacts between the two partners. We noted that PFV Gag Arg540 projects into the H2A-H2B acidic patch to interact with H2A carboxylates Glu61, Asp90, and Glu92. Interestingly, most of the chromatin factors-nucleosomes structures solved to date show the use of a conserved arginine anchor residue for the interaction of the H2A-H2B acidic patch carboxylates. Consistently, substitution of Gag Arg540 as well as conserved Tyr537 and Leu539 abolish *in vitro* interaction with the nucleosome. In agreement with published results, immunofluorescence of incoming viral particles showed a colocalization with centrosomes in interphase cells and a chromatin association during mitosis. The R540Q mutant viruses still localize around centrosomes during interphase, but lost the capacity to associate with mitotic chromosomes, phenocopying the *in vitro* substitutions results. Quantification of viral infectivity in the context of viruses harboring the R540Q substitution showed a modest two-fold defect compared to wild type (WT) viruses. Interestingly, the integration step was found

**MICROREVIEW** on: Lesbats P, Serrao E, Maskell DP, Pye VE, O'Reilly N, Lindemann D, Engelman AN, Cherepanov P (2017). Structural basis for spumavirus GAG tethering to chromatin. Proc Natl Acad Sci U S A 114(21):5509-5514. doi: 10.1073/pnas.1621159114

doi: 10.15698/mic2018.12.663

Received 31.10.2018, Accepted 09.11.2018, Published 19.11.2018.

Keywords: retrovirus, integration, nucleosome, chromatin-binding, Gag.

<footnote n="1">¹ Fundamental Microbiology and Pathogenicity Laboratory, UMR 5234 CNRS, SFR TransBioMed. Bordeaux, France.</footnote>

<footnote n="2">² International Associated Laboratory (LIA) of Microbiology and Immunology, CNRS / Heinrich Pette Institute-Leibniz Institute for Experimental Virology, Bordeaux, France.</footnote>

<footnote n="3">³ Viral DNA Integration and Chromatin Dynamics Network (DyNAVir), Bordeaux, France.</footnote>

<footnote>* Corresponding Author:</footnote>

<footnote>Paul Lesbats, Fundamental Microbiology and Pathogenicity Laboratory, UMR 5234 CNRS, SFR TransBioMed. Bordeaux, France;</footnote>

<footnote>E-mail: Paul.lesbats@u-bordeaux.fr</footnote>
unaffected suggesting an expression alteration of the inte-
grated genes. To get deeper insights on the role of the
Gag-chromatin interaction, we mapped the integration
sites of WT and R540Q PFV in various cell types. In agree-
ment with previously published results and in contrast to
HIV-1 and MLV, PFV avoids integration into genes. Surpris-
ingly, the virus showed varied preference for heterochro-
matic or promoters regions depending on the cell line used
for infection (with HT1080 and HepG2 showing the most
divergences). Indeed, while heterochromatin regions like
constitutive lamina associated domains (cLADs) or giemsa
positive cytobands are favored in HT1080 cells, they are
disfavored in HepG2. Conversely, compared to HT1080, the
virus integrates more often near transcription start sites
and CpG islands in HepG2 cells. Nevertheless, analysis of
integration sites of the R540Q virus revealed a massive
redistribution of integration toward centromeres in all cell
lines studied. These data demonstrate that Gag tethering
to host chromatin is critical for the virus to select an opti-
mal integration site.

In light of the discordances observed in integration
sites selection of the wt virus in the different cell lines, it is
possible that the conditions of infections can widely affect
the outcome of the integration targeting observed. Indeed,
in contrast to HepG2, HT1080 cells are highly permissive
to foamy virus (FV) infection. Since deep sequencing anal-
yses require many unique integration sites, infection ex-
periments are performed under high multiplicity of infec-
tion (MOI). We can then speculate that in HT1080 cells, a
massive amount of viral particles can enter each cell, pos-
sibly titrating specific cellular proteins or pathways that
will impact the resulting integration site selection. In
HepG2 such “viral flooding” will be limited, potentially
revealing the original FV integration preference. This hy-
pothesis would require more analyses, specifically under
natural zoonotic infections.

In contrast to foamy viruses, orthoretroviral Gag pro-
teins are matured into separate polypeptides. Recently, it

has been described that gammaretrovirus MLV Gag prod-
uct p12 associates directly to host nucleosomes. Whether
this p12 protein is involved in gammaretroviral integration
site selection is currently unclear, but the correct tether-
ing to chromatin is critical, since abolishment of p12-
nucleosome binding abrogates viral infectivity.

The role of Gag in chromatin targeting in the other ret-
roviral family is currently obscure. Lentiviruses rely on the
capsid-binding partners like CPSF6 or nucleoporin NUP153
to gain access to gene dense chromosomal regions via an
active nuclear import process. In the case of spumaretro-
virus and gammaretrovirus this function is achieved by
direct binding of Gag products to host chromatin during
mitosis.

Chromatin binding by viral proteins is not restricted to
retroviruses as it was also described for Kaposi's sarcoma
herpes virus (KSHV) latency-associated nuclear antigen
(LANA) and cytomegalovirus (CMV) immediate early 1
protein (IE1). As described for PFV Gag, structural analyses
showed a conserved interaction mode using an arginine
anchor motif poking to the acidic patch. Interestingly, bio-
physical approaches revealed an interesting feature about
these acidic patch binders viral proteins. As the acidic
patch constitutes the binding pocket for the neighboring
histone H4 tail during higher order chromatin assembly, it
was shown that competition for the acidic patch using the
viral proteins LANA and IE1 could induce an alteration of
the chromatin compaction in vitro. Whether this feature is
also expressed during viral replication is currently not
known. As foamy viruses favor integration into deep het-
erochromatic regions of the chromosomes, it is tempting
to speculate that the binding of Gag to compacted nucleo-
somes might locally remodel the chromatin architecture in
order to create a replication/gene-expression permissive
environment (Figure 1). Such a hypothesis is currently
under investigation.

Virus-host interaction is an arms race, which has
shaped the biology we know today during the course of

<figure><img src="image_3.png" /></figure>

**FIGURE 1:** Hypothesis model for viral protein mediated chromatin remodeling. The ground state chromatin structure is mediated by H4 tail binding to the acidic patch. Alteration of the chromatin state can be achieved by competition of viral proteins with H4 tail for binding to the acidic patch leading to a viral replication-permissive chromatin structure. Abbreviations: H1, histone H1. H4, histone H4. See text for details.
evolution. The retrovirus family originated more than 500
millions years ago and nowadays infects a broad range of
hosts. From the oldest Foamy viruses to the most recent
pandemic causative HIV-1, latest researches showed a new
mechanism of regulation of integration site selection by
the viral capsid/Gag. Further experiments investigating the
potential involvement of Gag products from other retrovi-
ral genera on the targeting of integration is of great inter-
est and will shed light on the replicative strategy evolved
by retroviruses.

ACKNOWLEDGMENTS

We thank all the authors who contributed to the original
article.

CONFLICT OF INTEREST

No conflicts.

COPYRIGHT

© 2018 Lesbats and Parissi. This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged.

Please cite this article as: Paul Lesbats and Vincent Parissi (2018). Retroviral integration site selection: a running *Gag*? **Microbial Cell** 5(12): 569-571. doi: 10.15698/mic2018.12.663