Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Apr;78(7):3429-35.
doi: 10.1128/jvi.78.7.3429-3435.2004.

Coupling of human immunodeficiency virus type 1 fusion to virion maturation: a novel role of the gp41 cytoplasmic tail

Donald J Wyma  1 , Jiyang Jiang, Jiong Shi, Jing Zhou, Janet E Lineberger, Michael D Miller, Christopher Aiken
Affiliations

Coupling of human immunodeficiency virus type 1 fusion to virion maturation: a novel role of the gp41 cytoplasmic tail

Donald J Wyma et al. J Virol. 2004 Apr.

Abstract

Retrovirus particles are not infectious until they undergo proteolytic maturation to form a functional core. Here we report a link between human immunodeficiency virus type 1 (HIV-1) core maturation and the ability of the virus to fuse with target cells. Using a recently developed reporter assay of HIV-1 virus-cell fusion, we show that immature HIV-1 particles are 5- to 10-fold less active for fusion with target cells than are mature virions. The fusion of mature and immature virions was rendered equivalent by truncating the gp41 cytoplasmic domain or by pseudotyping viruses with the glycoprotein of vesicular stomatitis virus. An analysis of a panel of mutants containing mutated cleavage sites indicated that HIV-1 fusion competence is activated by the cleavage of Gag at any site between the MA and NC segments and not as an indirect consequence of an altered core structure. These results suggest a mechanism by which binding of the gp41 cytoplasmic tail to Gag within immature HIV-1 particles inhibits Env conformational changes on the surface of the virion that are required for membrane fusion. This "inside-out" regulation of HIV-1 fusion could play an important role in the virus life cycle by preventing the entry of immature, noninfectious particles.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
gp41 CT inhibits the fusion of immature HIV-1 particles with target cells. (a) Wild-type (NL4-3) and immature (MA/p6) HIV-1 reporter particles and corresponding mutants lacking the gp41 CT (ΔCT) were assayed for fusion with SupT1 target cells in the BlaM-Vpr reporter assay. Data shown are the mean values of duplicate determinations as a function of the quantity of virus added to the cells. The results are representative of at least three independent experiments. (b) Immunoblot analysis of viral lysates. Pelleted virions were lysed and subjected to SDS-polyacrylamide gel electrophoresis. After being transferred to nitrocellulose membranes, blots were probed sequentially with antisera that were specific for gp120, gp41, BlaM, and CA. (c) Restored fusion ability of immature HIV-1 particles lacking the gp41 CT is not due to enhanced levels of Env proteins on the virions. HIV-1 particles containing reduced levels of gp41 with the CT deleted were produced by cotransfection of the NL4-3ΔCT or MA/p6ΔCT proviral construct with various quantities of the respective Env-defective proviruses. The total quantity of proviral DNA was kept constant, at 20 μg. Values shown on the abscissa represent the amounts of Env-expressing proviral DNA used in the cotransfections. Viruses were assayed for fusion with target cells, and the fusion efficiency was expressed as a function of the fluorescence ratio per nanogram of p24 input virus. (d) Fusion activity of VSV-pseudotyped HIV-1. Reporter particles were produced by the cotransfection of a pNL4-3Env− or pNL-MA/p6Env− proviral clone with pHCMV-G (34) and the BlaM-Vpr expression construct pMM310. Threefold dilutions of the viruses were tested to verify the dose dependence of the assay for pseudotyped particles. The virus dilutions were reassayed by p24 ELISAs to confirm the accuracy of the dilutions. Results shown are the averages of duplicate determinations from one of two independent experiments.
FIG. 2.
FIG. 2.
Cleavage of Gag between MA and NC is required for efficient HIV-1 fusion. (a) Schematic of Gag cleavage site mutants. The CA6 mutant was previously described (32). PCR overlap extension was used to replace the P1 amino acid of the remaining Pr55Gag cleavage sites with an Ile, which prevents cleavage by the HIV-1 PR. (b) SupT1 cells were inoculated with wild-type (NL4-3) reporter particles or Gag cleavage mutants, and fusion was assayed as described for Fig. 1. Results shown are the averages of duplicate determinations (which typically varied by <10%) from one of two independent experiments. (c) Immunoblot analysis of Pr55Gag cleavage site mutants for gp120, gp41, BlaM, and CA.
FIG. 3.
FIG. 3.
Transmission electron microscopy of mutant HIV-1 particles. Wild-type (WT) and Gag cleavage site mutant (MA/CA, MA/p2, MA/NC, and MA/p6) proviruses were transfected into 293T cells; the cells were cultured for 1 day and then harvested, fixed, sectioned, and stained; and the samples were analyzed by electron microscopy. Scale bars are shown in micrometers. All of the transfections yielded large quantities of HIV-1 particles of similar sizes, but with distinct internal morphologies.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adachi, A., H. E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, and M. A. Martin. 1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59:284-291. - PMC - PubMed
    1. Aguilar, H. C., W. F. Anderson, and P. M. Cannon. 2003. Cytoplasmic tail of Moloney murine leukemia virus envelope protein influences the conformation of the extracellular domain: implications for mechanism of action of the R peptide. J. Virol. 77:1281-1291. - PMC - PubMed
    1. Aiken, C. 1997. Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A. J. Virol. 71:5871-5877. - PMC - PubMed
    1. Brody, B. A., S. S. Rhee, and E. Hunter. 1994. Postassembly cleavage of a retroviral glycoprotein cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. J. Virol. 68:4620-4627. - PMC - PubMed
    1. Cavrois, M., C. De Noronha, and W. C. Greene. 2002. A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat. Biotechnol. 20:1151-1154. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources