Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (VSV) containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.

A genome-wide RNA interference screen using Caenorhabditis elegans LRP-1/megalin as a model for LDLR transport was employed to identify factors critical to LDLR uptake. We provide evidence that epsin1 promotes LDLR internalization via a FxNPxY-independent pathway. We complement C. elegans in vivo approaches with loss-of-function and biochemical analyses, using mammalian cell culture systems to evaluate epsin1’s mode of action in LDLR endocytosis.

Human non-small cell lung cancer (NSCLC) cells were transfected with recombinant prodrug herpes simplex virus type I thymidine kinase (HSV-tk) cDNA, and the selected clones underwent apoptosis in response to induction by antiviral ganciclovir (GCV). The efficiency of GCV-induced growth inhibition and the extent of the bystander effect were associated with the expression level of HSV-TK in stable transfectants. Development in the HSV-tk/GCV system toward cell death was initiated with cell-cycle accumulation at S and G 2 /M phases, immediately followed by the appearance of sub-G 0 /G 1 cells after drug exposure. To investigate the regulation of cell-cycle modulators during drug treatment, we analyzed release of the apoptosis initiator cytochrome c and activation of the downstream effectors caspase-9, caspase-3 and poly(ADP-ribose)polymerase 16 hr after GCV sensitization, followed by transient escalation of tumor-suppressor p53 and cell-cycle modulators cyclin A and B 1 before committing to programmed cell death. Furthermore, tumor regression was proportional to the degree of ectopic expression of the transferred HSV-tk gene. Our results demonstrate that the HSV-tk/GCV system effectively inhibits the proliferation of NSCLC cells in vitro and in vivo through potent induction of apoptosis, thus providing a rationale for further development. Key words: non-small cell lung cancer; herpes simplex virus-thymidine kinase; apoptosis; cell cycleLung cancer is the leading malignancy in the global population and the second most prominent overall cause of cancer deaths in men. Being the major fatality among lung cancer victims, nonsmall cell lung cancer (NSCLC) remains lethal in approximately 90% of all diagnosed cases because of failure of sensitive diagnosis, failure of effective therapy and resistance to various therapies. In restraining such malignant cells, new therapeutic paradigms are constantly pursued and improved, specifically against solid tumors such as lung cancer. One promising approach takes advantage of the normal physiologic response to suicide/prodrug therapy leading to terminal cellular apoptosis. The strategy has gradually become a promising alternative to the conventional therapeutic modalities. 1 It makes use of the transfer and expression of exogenous genes encoding enzymes that convert nontoxic prodrugs into cellular toxins.One of the promising gene therapeutic approaches utilizes the herpes simplex virus type I thymidine kinase (HSV-tk) gene, which demonstrates sensitivity to exogenous ganciclovir [9-(1,3-dihydroxy-2-proproxy-methyl)-guanine, GCV]. An important aspect of this enzyme-prodrug antitumor therapy is the ability to kill cells that do not carry the transgene, a course termed the bystander effect, in which not only cells with transferred HSV-tk but also nontransfected cells nearby are killed. Expression of the HSV-tk gene in the minority of tumor cells in animal models accounts for the complete tumor regression after GCV treatment. The efficacy of HSV-tk gene transfer and GCV treatment (HSV-t...

The homeobox gene superfamily has been highly conserved throughout evolution. These genes act as transcription factors during several important developmental processes. To explore the functional roles of homeobox genes in spermatogenesis, we performed a degenerate oligonucleotide polymerase chain reaction (PCR) screening of a testis cDNA library and isolated a novel mouse homeobox gene. This gene, which we named Tox, encodes a homeodomain protein distantly related to members of the Paired/Pax (Prd/Pax) family. A phylogenetic analysis revealed Tox to be a member of the recently defined PEPP subfamily of Paired-like homeobox genes. Tox was mapped to chromosome X, with its homeodomain organized into three exons. A special feature of Tox is that the encoded protein sequence contains two poly-glutamic acid (poly E) stretches, which make Tox highly acidic. Tox transcripts were detected predominately in the testis and ovary of mice. Tox expression in testes was initiated soon after birth, mainly in Sertoli cells and spermatogonia; however, in adult mice, Tox expression shifts to the spermatids and spermatozoa. Tox expression in ovaries was detected in somatic cells of follicles, early on in theca cells, and in both granulosa and theca cells at the later stages of follicular development. Based on these results, Tox may play an important role during gametogenesis.