Study of viral replication in HIV-1-infected CEM T-cell subclones which are reduced in their ability to form syncytia.
The derivation of ethyl-methanesulfonate (EMS) mutagenized subclones from the CEM T-cell line has been described. These clones expressed CD4 and bound soluble gp120, however, two of the generated clones were markedly reduced in their ability to form syncytia after infection with either gp160-vaccinia vector or cell-free human immunodeficiency virus type 1 (HIV-1). Here, we asked at what stage(s) viral infection is blocked in these cells. Polymerase chain reaction (PCR) analysis revealed that at 6 and 72 h postinfection with HIV-1, cells of the syncytia-deficient clones expressed markedly reduced amounts of viral-specific DNA compared with cells of the parental line or the syncytia-positive clones. Long-term cultures revealed a marked delay in the appearance of reverse transcriptase (RT) activity in the supernatants of these subclones when compared with the parental line and viral replication did not lead to massive cell death. Syncytia formation in HIV-1-infected cultures of the syncytia-deficient subclones was enhanced by tumor necrosis factor alpha (TNF alpha) when added 24 h postinfection. In contrast, pretreatment with TNF alpha for 48 h followed by washing and infection of the cells with HIV-1 augmented syncytia formation of the syncytia-positive subclones, but not of the syncytia-negative subclones. Thus, the EMS-mutagenized subclones may provide a tool to study host cell factors required for the establishment of a productive HIV-1 infection and responsiveness to TNF alpha.
Gruber
M F
MF
Division of Virology, Food and Drug Administration, Bethesda, MD 20892.
Hewlett
I K
IK
Simms
T
T
Vujcic
L
L
Manischewitz
J
J
Golding
H
H
D002999:Clone Cells; D004279:DNA, Viral / Q000032:analysis; D015726:Giant Cells / Q000166:cytology*; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000382:microbiology*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014779:Virus Replication
D004279:DNA, Viral; D014409:Tumor Necrosis Factor-alpha; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase