Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral chromatin tether

Abstract

By tethering their circular genomes (episomes) to host chromatin, DNA tumor viruses ensure retention and segregation of their genetic material during cell divisions. Despite functional genetic and crystallographic studies, there is little information addressing the 3D structure of these tethers in cells, issues critical for understanding persistent infection by these viruses. Here, we have applied direct stochastic optical reconstruction microscopy (dSTORM) to establish the nanoarchitecture of tethers within cells latently infected with the oncogenic human pathogen, Kaposi's sarcoma-associated herpesvirus (KSHV). Each KSHV tether comprises a series of homodimers of the latency-associated nuclear antigen (LANA) that bind with their C termini to the tandem array of episomal terminal repeats (TRs) and with their N termini to host chromatin. Superresolution imaging revealed that individual KSHV tethers possess similar overall dimensions and, in aggregate, fold to occupy the volume of a prolate ellipsoid. Using plasmids with increasing numbers of TRs, we found that tethers display polymer power law scaling behavior with a scaling exponent characteristic of active chromatin. For plasmids containing a two-TR tether, we determined the size, separation, and relative orientation of two distinct clusters of bound LANA, each corresponding to a single TR. From these data, we have generated a 3D model of the episomal half of the tether that integrates and extends previously established findings from epifluorescent, crystallographic, and epigenetic approaches. Our findings also validate the use of dSTORM in establishing novel structural insights into the physical basis of molecular connections linking host and pathogen genomes.

Epstein–Barr virus enhances genome maintenance of Kaposi sarcoma-associated herpesvirus

“…Cell. The number of "LANA dots" in an interphase nucleus correlates with the number of KSHV genomes (48,49). We used 3D immunofluorescence coupled to image reconstruction to count the number of distinct LANA + foci ( Fig.…”

Rainbow Kaposi's Sarcoma-Associated Herpesvirus Revealed Heterogenic Replication with Dynamic Gene Expression

“…LANA directly binds to the terminal repeat (TR) region of the viral genome and associates with nucleosomal proteins to tether to the host cell chromatin (35,36). Tight binding to the 35 copies of the TR sequences concentrates LANA proteins on the KSHV genome in the nucleus of infected cells, which makes them visible as "LANA dots" under fluorescence microscopy (37). Accordingly, a LANA dot represents a single viral episome in the nucleus; therefore, the fluorescence signal can be used to measure the viral DNA copy number in an infected cell nucleus (38).…”

Nanoscale organization of rotavirus replication machineries

“…VPs have been previously studied using electron and fluorescence microscopy, however, due to the limited resolution of classic fluorescence microscopy techniques, and the difficulty of analysis of immunoelectron microscopy, the existence of any complex structural organization of the viral elements inside the VPs has not been reported. In recent years, the development of SRM has facilitated research into the nanoscale organization of a diverse range of cellular structures [26,27], however, until now SRM had not been applied to study the replication cycle of rotavirus. In this work, for the first time, we visualized and determined quantitatively the location of several protein and nucleic acid components of VPs, what led us to propose a detailed model of the VP that should be of great value for understanding virus morphogenesis.…”