Replication of Herpes Simplex Virus Type 1 within Trigeminal Ganglia Is Required for High Frequency but Not High Viral Genome Copy Number Latency

Abstract

The replication properties of a thymidine kinase-negative (TK ؊ ) mutant of herpes simplex virus type 1 (HSV-1) were exploited to examine the relative contributions of replication at the body surface and within trigeminal ganglia (TG) on the establishment of latent infections. ؊ in the absence of significant replication within the TG resulted in a modest increase in the number of latent sites. Importantly, these latently infected neurons displayed a wild-type latent-genome copy number profile, with some neurons containing hundreds of copies of the TK ؊ mutant genome. As expected, the replication of the TK ؊ mutant appeared to be blocked prior to DNA replication in most ganglionic neurons in that (i) virus replication was severely restricted in ganglia, (ii) the number of neurons expressing HSV proteins was reduced 30-fold compared to the rescued isolate, (iii) cell-to-cell spread of virus was not detected within ganglia, and (iv) the proportion of infected neurons expressing late proteins was reduced by 89% compared to the rescued strain. These results demonstrate that the viral TK gene is required for the efficient establishment of latency. This requirement appears to be primarily for efficient replication within the ganglion, which leads to a sixfold increase in the number of latent sites established. Further, latent sites with high genome copy number can be established in the absence of significant virus genome replication in neurons. This suggests that neurons can be infected by many HSV virions and still enter the latent state.

Analyses of herpes simplex virus type 1 latency and reactivation at the single cell level using fluorescent reporter mice

“…We report here that HSV-1 expressing Cre recombinase under control of the human cytomegalovirus (HCMV) major immediate early promoter (MIEP) marks the latent reservoir with high efficiency in Ai6 fluorescent reporter mice, and that HSV-1 DNA copy numbers can vary by at least three orders of magnitude amongst infected neurons, consistent with previous studies using contextual analyses (Sawtell, 1997;Thompson & Sawtell, 2000).…”

“…The biological significance of these marked latent populations is currently unknown. Several studies have described a small population of latently infected cells containing w1000 HSV DNA copies (Chen et al, 2002;Ma et al, 2014;Sawtell, 1997;Thompson & Sawtell, 2000;Wang et al, 2005), and indeed our own data describe populations of neurons containing several hundred genome copies (Fig. 2).…”

“…Much has been learnt from small animal models of herpes simplex virus (HSV) latency both at the molecular and immunological level (Efstathiou & Preston, 2005;Nicoll et al, 2012b;Wagner & Bloom, 1997) but our understanding of latency within single neurons in vivo has been hampered by the lack of amenable systems to identify and isolate live cells from the infected host. For example, careful analyses of HSV-1 DNA copy numbers within individual human or mouse neurons using contextual analysis of DNA and laser-capture microdissection methodologies have shown that virus genome copies can vary over three orders of magnitude (Chen et al, 2002;Sawtell, 1997;Thompson & Sawtell, 2000;Wang et al, 2005), but studying the potential biological effect of this phenomenon has been limited to indirect examination (Sawtell et al, 1998). Reporter mice encoding conditionally expressed LacZ or yellow fluorescent protein (YFP) from the endogenous ROSA locus have proved useful for the stable visual marking of latently infected sensory neurons following infection with Cre recombinase-expressing HSV-1 recombinants, and have also revealed that lytic gene promoter activation and low level lytic gene transcription is compatible with latency in at least a subset of latently infected neurons (Ma et al, 2014;Nicoll et al, 2012a;Proença et al, 2008Proença et al, , 2011Wakim et al, 2008).…”

Anatomical mapping of human herpesvirus reservoirs of infection

“…Primary HSV-1 infection of immunocompetent persons is characterized by lesions of the oral mucosa, followed by latent persistence within neurons of cervical and cranial nerve sensory ganglia. [3][4][5][6][7][8][9][10][11] In some cases, HSV-1 and HSV-2 can be found in brain tissue. 12 Viral reactivation can lead to recurrent mucocutaneous disease, whereas dissemination may lead to widespread organ involvement, including esophagus, lower gastrointestinal tract, trachea, lungs, brain, eyes, and skin.…”

Latence et réactivation du virus de l’herpès simplex de type 1 (HSV-1)

“…L'utilisation de certains mutants viraux incapables de se multiplier dans les neurones montre que l'établissement de la latence ne nécessite pas de multiplication du virus dans les ganglions sensitifs [28]. Cependant, elle est beaucoup nombre de génomes viraux qui peuvent être visualisés par hybridation in situ (FISH) sous la forme d'un seul point fluorescent par neurone.…”