Nelfinavir inhibition of Kaposi's sarcoma-associated herpesvirus protein expression and capsid assembly.

BACKGROUND: Antiviral therapies that target herpesviruses are clinically important. Nelfinavir is a protease inhibitor that targets the human immunodeficiency virus (HIV) aspartyl protease. Previous studies demonstrated that this drug could also inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) production. Our laboratory demonstrated nelfinavir can effectively inhibit herpes simplex virus type 1 (HSV-1) replication. For HSV-1 we were able to determine that virus capsids were assembled and exited the nucleus but did not mature in the cytoplasm indicating the drug inhibited secondary envelopment of virions. METHODS: For KSHV, we recently derived a tractable cell culture system that allowed us to analyze the virus replication cycle in greater detail. We used this system to further define the stage at which nelfinavir inhibits KSHV replication. RESULTS: We discovered that nelfinavir inhibits KSHV extracellular virus production. This was seen when the drug was incubated with the cells for 3 days and when we pulsed the cells with the drug for 1-5 min. When KSHV infected cells exposed to the drug were examined using ultrastructural methods there was an absence of mature capsids in the nucleus indicating a defect in capsid assembly. Because nelfinavir influences the integrated stress response (ISR), we examined the expression of viral proteins in the presence of the drug. We observed that the expression of many were significantly changed in the presence of drug. The accumulation of the capsid triplex protein, ORF26, was markedly reduced. This is an essential protein required for herpesvirus capsid assembly. CONCLUSIONS: Our studies confirm that nelfinavir inhibits KSHV virion production by disrupting virus assembly and maturation. This is likely because of the effect of nelfinavir on the ISR and thus protein synthesis and accumulation of the essential triplex capsid protein, ORF26. Of interest is that inhibition requires only a short exposure to drug. The source of infectious virus in saliva has not been defined in detail but may well be lymphocytes or other cells in the oral mucosa. Thus, it might be that a "swish and spit" exposure rather than systemic administration would prevent virion production.

Activation of NIX-mediated mitophagy by an interferon regulatory factor homologue of human herpesvirus.

Viral control of mitochondrial quality and content has emerged as an important mechanism for counteracting the host response to virus infection. Despite the knowledge of this crucial function of some viruses, little is known about how herpesviruses regulate mitochondrial homeostasis during infection. Human herpesvirus 8 (HHV-8) is an oncogenic virus causally related to AIDS-associated malignancies. Here, we show that HHV-8-encoded viral interferon regulatory factor 1 (vIRF-1) promotes mitochondrial clearance by activating mitophagy to support virus replication. Genetic interference with vIRF-1 expression or targeting to the mitochondria inhibits HHV-8 replication-induced mitophagy and leads to an accumulation of mitochondria. Moreover, vIRF-1 binds directly to a mitophagy receptor, NIX, on the mitochondria and activates NIX-mediated mitophagy to promote mitochondrial clearance. Genetic and pharmacological interruption of vIRF-1/NIX-activated mitophagy inhibits HHV-8 productive replication. Our findings uncover an essential role of vIRF-1 in mitophagy activation and promotion of HHV-8 lytic replication via this mechanism.

Rapid identification of germline mutations in retinoblastoma by protein truncation testing.

OBJECTIVE: To demonstrate the utility of protein truncation testing (PTT) for rapid detection and sequencing of germline mutations in the retinoblastoma tumor suppressor gene (RB1). METHODS: We performed PTT, a technique based on the in vitro synthesis of protein from amplified RNA, on 27 probands from 27 kindreds with hereditary retinoblastoma. In 4 kindreds, PTT was also performed on 1 additional affected relative. Ten unrelated patients without retinoblastoma were included as negative control subjects. All PTT-detected mutations were further analyzed by focused sequencing of genomic DNA. When no mutation was detected by PTT, we performed exon-by-exon sequencing, as well as cytogenetic analysis by Giemsa-trypsin-Giemsa banding and by fluorescent in situ hybridization for RB1. The results of proband testing were used for direct genetic testing by polymerase chain reaction and sequencing in 11 relatives from 7 of the 27 kindreds. RESULTS: Of the probands tested, 19 (70%) of 27 tested positive for germline mutations by PTT. In 1 kindred, the proband had negative PTT results but an additional affected relative had positive PTT results. Focused DNA sequencing of 1 patient with positive PTT results from each of the 20 kindreds with positive PTT results revealed truncating mutations in 19 kindreds. Four demonstrated frameshift deletions, 6 had splice site mutations, and 9 showed nonsense mutations. Further analysis by genomic exon-by-exon sequencing and karyotype analysis of the 8 probands who tested negative for germline mutations by PTT revealed 1 splice site mutation, 2 missense mutations, and 1 chromosomal deletion. Focused sequencing based on positive PTT results was successfully used to confirm shared truncating mutations in additional affected family members in 2 kindreds. Using a multitiered approach to genetic testing, 23 (85%) of 27 kindreds had mutations identified and those detected by PTT received a positive result in as few as 7 days. In control subjects, PTT produced no false-positive results. CONCLUSIONS: Protein truncation testing is an effective, rapid single-modality screen for germline mutations in patients with retinoblastoma. When used as an initial screen, PTT can increase the yield of additional testing modalities, such as sequencing and chromosomal analysis, providing a timely and cost-effective approach for the diagnosis of heritable germline mutations in patients with retinoblastoma.Clinical Relevance The clinical application of PTT in retinoblastoma will improve detection of germline retinoblastoma mutations, which will supply critical information for prognosis, treatment planning, follow-up care, and genetic counseling.