Abortive herpes simplex virus infection of nonneuronal cells results in quiescent viral genomes that can reactivate.

Abortive viral infections are usually studied in populations of susceptible but nonpermissive cells. Single-cell studies of viral infections have demonstrated that even in susceptible and permissive cell populations, abortive infections can be detected in subpopulations of the infected cells. We have previously identified abortive infections in HeLa cells infected with herpes simplex virus 1 (HSV-1) at high multiplicity of infection (MOI). Here, we tested 4 additional human-derived nonneuronal cell lines (cancerous or immortalized) and found significant subpopulations that remain abortive. To characterize these abortive cells, we recovered cell populations that survived infection with HSV-1 at high MOI. The surviving cells retained proliferative potential and the ability to be reinfected. These recovered cell populations maintained the viral genomes in a quiescent state for at least 5 wk postinfection. Our results indicate that these viral genomes are maintained inside the nucleus, bound to cellular histones and occasionally reactivated to produce new progeny viruses. We conclude that abortive HSV-1 infection is a common feature during infection of nonneuronal cells and results in a latency-like state in the infected cells. Our findings question the longstanding paradigm that alphaherpesviruses can establish spontaneous latency only in neuronal cells and emphasize the stochastic nature of lytic versus latency decision of HSV-1 in nonneuronal cells.

Modulation of Cellular CpG DNA Methylation by Kaposi's Sarcoma-Associated Herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is a gammaherpesvirus associated with several human malignancies. DNA methylation at CpG dinucleotides is an epigenetic mark dysregulated in many cancer types and in KSHV-infected cells. Several previous studies have analyzed in detail the CpG methylation of the KSHV episomal genomes, but little is known about the impact of KSHV on the human genome. Our knowledge of cellular CpG methylation in the context of KSHV infection is currently limited to four hypermethylated human gene promoters. Therefore, we undertook a comprehensive CpG methylation analysis of the human methylome in KSHV-infected cells and KSHV-associated primary effusion lymphoma (PEL). We performed Infinium HumanMethylation450K and MethylationEpic BeadChip arrays and identified panels of hyper- and hypomethylated cellular promoters in KSHV-infected cells. We combined our genome-wide methylation analysis with high-throughput RNA sequencing (RNA-seq) to add functional outcomes to the virally induced methylation changes. We were able to correlate many downregulated genes with promoter hypermethylation and upregulated genes with hypomethylation. In addition, we show that treating the cells with a demethylating agent leads to reexpression of these downregulated genes, indicating that, indeed, DNA methylation plays a role in the repression of these human genes. Comparison between de novo infection and PEL suggests that the virus induces initial hypermethylation followed by a slow increase in genome-wide hypomethylation. This study extends our understanding of the relationship between epigenetic changes induced by KSHV infection and tumorigenesis.IMPORTANCE In cancer cells, certain promoters become aberrantly methylated, contributing to the phenotype of the tumor. KSHV infection seems to modify cellular CpG methylation, but only a few methylated promoters have been identified in KSHV-infected cells. Here, we investigated the CpG methylation of the human genome in KSHV-associated primary effusion lymphoma (PEL) and KSHV-infected cells. We have identified many hyper- and hypomethylated gene promoters and correlated their methylation with cellular gene expression. These differentially methylated cellular promoters can distinguish KSHV-positive cells from uninfected cells and may serve as the foundation for the use of these differentially methylated regions as potential biomarkers for KSHV-associated malignancies. Drugs that reverse these cancerous methylation patterns have the potential to inhibit tumor growth. Here, we show that treating PEL cells with a demethylating drug (5-aza-2'-deoxycytidine) led to inhibition of cell growth, raising the possibility of testing this drug for the treatment of PEL.

In Vitro Antileishmanial Activity of a Black Morel, Morchella importuna (Ascomycetes).

We studied the anti-Leishmania activity of a fractionated extract from the mushroom Morchella importuna in an in vitro system. Leishmaniasis is an important infectious disease caused by a range of Leishmania species, which are multihost protozoa parasites transmitted to humans by the sand fly and infecting macrophages. Leishmaniasis is an increasing worldwide health problem, including in the Mediterranean basin. Current chemotherapy treatments are limited by their toxic effects, the need for long-term treatment, and the increasing development of resistance by the parasite cells. Thus, alternative therapies are being considered, including herbal and mushroom products. We studied the effect of extracts from M. importuna on L. tropica promastigote cell proliferation and survival, and on their toxicity against human macrophages. The aqueous mushroom extract was compared with 3 successive extracted fractions: an 80% ethanol fraction, a water-soluble polysaccharide fraction, and a polyphenolic fraction. All 4 extracts showed anti-Leishmania activity; the aqueous extract was most active. The inhibition activity was dose dependent in killing Leishmania. No cell recovery was recorded after exposure to the mushroom extract. Microscopic observation showed morphological changes and the loss of flagella on the parasites. No cytotoxic activity was recorded against human macrophages at the same extract concentrations. The findings suggest the potential use of extracts of an edible Morchella mushroom against the Leishmania parasite in humans.