Cloning and characterization of human immunodeficiency virus type 1 variants diminished in the ability to induce syncytium-independent cytolysis.
The phenomenon of interference was exploited to isolate low-abundance noncytopathic human immunodeficiency virus type 1 (HIV-1) variants from a primary HIV-1 isolate from an asymptomatic HIV-1-seropositive hemophiliac. Successive rounds of virus infection of a cytolysis-susceptible CD4+ cell line and isolation of surviving cells resulted in selective amplification of an HIV-1 variant reduced in the ability to induce cytolysis. The presence of a PvuII polymorphism facilitated subsequent amplification and cloning of cytopathic and noncytopathic HIV-1 variants from the primary isolate. Cloned virus stocks from cytopathic and noncytopathic variants exhibited similar replication kinetics, infectivity, and syncytium induction in susceptible host cells. The noncytopathic HIV-1 variant was unable, however, to induce single-cell killing in susceptible host cells. Construction of viral hybrids in which regions of cytopathic and noncytopathic variants were exchanged indicated that determinants for the noncytopathic phenotype map to the envelope glycoprotein. Sequence analysis of the envelope coding regions indicated the absence of two highly conserved N-linked glycosylation sites in the noncytopathic HIV-1 variant, which accompanied differences in processing of precursor gp160 envelope glycoprotein. These results demonstrate that determinants for syncytium-independent single-cell killing are located within the envelope glycoprotein and suggest that single-cell killing is profoundly influenced by alterations in envelope sequence which affect posttranslational processing of HIV-1 envelope glycoprotein within the infected cell.
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198-5120.
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M
Stevenson
S
S
Haggerty
C
C
Lamonica
A M
AM
Mann
C
C
Meier
A
A
Wasiak
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D014644:Genetic Variation; D015726:Giant Cells / Q000502:physiology*; D006031:Glycosylation; D015497:HIV-1 / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D006367:HeLa Cells / Q000201:enzymology; D015368:Human T-lymphotropic virus 1 / Q000235:genetics; D006801:Humans; D007256:Information Systems; D007700:Kinetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014759:Viral Envelope Proteins / Q000235:genetics*; D014779:Virus Replication
D004279:DNA, Viral; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D012194:RNA-Directed DNA Polymerase