Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) gag-encoded proteins play key functions at almost all stages of the viral life cycle. Since these functions may require association with cellular factors, the HIV-1 matrix protein (MA) was used as bait in a yeast two-hybrid screen to identify MA-interacting proteins. MA was found to interact with elongation factor 1-alpha (EF1α), an essential component of the translation machinery that delivers aminoacyl-tRNA to ribosomes. EF1α was then shown to bind the entire HIV-1 Gag polyprotein. This interaction is mediated not only by MA, but also by the nucleocapsid domain, which provides a second, independent EF1α-binding site on the Gag polyprotein. EF1α is incorporated within HIV-1 virion membranes, where it is cleaved by the viral protease and protected from digestion by exogenously added subtilisin. The specificity of the interaction is demonstrated by the fact that EF1α does not bind to nonlentiviral MAs and does not associate with Moloney murine leukemia virus virions. The Gag-EF1α interaction appears to be mediated by RNA, in that basic residues in MA and NC are required for binding to EF1α, RNase disrupts the interaction, and a Gag mutant with undetectable EF1α-binding activity is impaired in its ability to associate with tRNA in cells. Finally, the interaction between MA and EF1α impairs translation in vitro, a result consistent with a previously proposed model in which inhibition of translation by the accumulation of Gag serves to release viral RNA from polysomes, permitting the RNA to be packaged into nascent virions.

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Tripeptide Interference with Human Immunodeficiency Virus Type 1 Morphogenesis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.11.3597-3605.2002 ◽

2002 ◽

Vol 46 (11) ◽

pp. 3597-3605 ◽

Cited By ~ 19

Author(s):

Stefan Höglund ◽

Jin Su ◽

Sara Sandin Reneby ◽

Ákos Végvári ◽

Stellan Hjertén ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Plasma Membranes ◽

Biologically Active ◽

Gag Polyprotein ◽

Immunodeficiency Virus ◽

The Matrix ◽

Hiv 1

ABSTRACT Capsid assembly during virus replication is a potential target for antiviral therapy. The Gag polyprotein is the main structural component of retroviral particles, and in human immunodeficiency virus type 1 (HIV-1), it contains the sequences for the matrix, capsid, nucleocapsid, and several small polypeptides. Here, we report that at a concentration of 100 μM, 7 of 83 tripeptide amides from the carboxyl-terminal sequence of the HIV-1 capsid protein p24 suppressed HIV-1 replication (>80%). The three most potent tripeptides, glycyl-prolyl-glycine-amide (GPG-NH2), alanyl-leucyl-glycine-amide (ALG-NH2), and arginyl-glutaminyl-glycine-amide (RQG-NH2), were found to interact with p24. With electron microscopy, disarranged core structures of HIV-1 progeny were extensively observed when the cells were treated with GPG-NH2 and ALG-NH2. Furthermore, nodular structures of approximately the same size as the broad end of HIV-1 conical capsids were observed at the plasma membranes of treated cells only, possibly indicating an arrest of the budding process. Corresponding tripeptides with nonamidated carboxyl termini were not biologically active and did not interact with p24.

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Human Immunodeficiency Virus Type 1 Virion Density Is Not Determined by Nucleocapsid Basic Residues

Journal of Virology ◽

10.1128/jvi.74.15.6734-6740.2000 ◽

2000 ◽

Vol 74 (15) ◽

pp. 6734-6740 ◽

Cited By ~ 32

Author(s):

Andrea Cimarelli ◽

Jeremy Luban

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Obvious Effect ◽

Gag Polyprotein ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Isopycnic Centrifugation ◽

Rule Out

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is sufficient for assembly and release of virion-like particles from the plasma membrane. To promote assembly, the Gag polyprotein must polymerize to form a shell that lines the inner membrane of nascent virions. Several techniques have been used to functionally map the domain required for Gag polymerization (the I domain). Among these methods, isopycnic centrifugation has been used under the assumption that changes in virion density reflect impairment in Gag-Gag interaction. If virion density is determined by efficient Gag-Gag interaction, then mutation of basic residues in the nucleocapsid (NC) domain should disrupt virion density, since these residues constitute the I domain. However, we have previously shown that simultaneous disruption of up to 10 HIV-1 NC basic residues has no obvious effect on virion density. To rule out the possibility that HIV-1 NC basic residues other than those previously mutated might be important for virion density, mutations were introduced at the remaining sites and the ability of these mutations to affect Gag-Gag interaction and virion density was analyzed. Included in our analysis is a mutant in which all NC basic residues are replaced with alanine. Our results show that disruption of HIV-1 NC basic residues has an enormous effect on Gag-Gag interaction but only a minimal effect on the density of those virions that are still produced. Therefore, the determinants of the I domain and of virion density are genetically distinguishable.

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Removal of human immunodeficiency virus type 1 (HIV-1) protease inhibitors from preparations of immature HIV-1 virions does not result in an increase in infectivity or the appearance of mature morphology.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.5.1017 ◽

1997 ◽

Vol 41 (5) ◽

pp. 1017-1023 ◽

Cited By ~ 14

Author(s):

R W Humphrey ◽

A Ohagen ◽

D A Davis ◽

T Fukazawa ◽

H Hayashi ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Protease Inhibitors ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Polyprotein Processing ◽

Gag Polyprotein ◽

Viral Particles ◽

Immunodeficiency Virus ◽

Hiv 1

The processing of gag and gag-pol polyproteins by human immunodeficiency virus type 1 (HIV-1) protease is a crucial step in the formation of infectious HIV-1 virions. In this study, we examine whether particles produced in the presence of inhibitors of HIV-1 protease can subsequently undergo gag polyprotein cleavage with restoration of infectivity following removal of the inhibitors. Viral particles produced during 7 days of culture in the presence of the protease inhibitors KNI-272 (10 microM) and saquinavir (5 microM) contained predominantly p55gag polyprotein but little or no p24gag cleavage product. Following resuspension of the particles in medium free of the inhibitor, some gag polyprotein processing was detected in particles produced from the KNI-272-treated cells, but not from the saquinavir-treated cells within the first 3 h. However, the majority of the protein remained as p55gag throughout a 48-h experimental period. The infectivity (50% tissue culture infective dose per milliliter) of the viral particles from KNI-272-treated cells was 10(6)-fold lower than that of control particles and did not significantly increase over the 48 h after the inhibitor was removed, despite the apparent return of protease function in a subset of these virions. This failure to restore infectivity was due neither to a reduction in the number of particles produced by protease inhibitor-treated cells nor to a failure of HIV RNA to be packaged in the virions. These particles also failed to express the mature phenotype by electron microscopy. Thus, while some processing of the gag polyprotein can occur in isolated HIV virions, this does not appear to be sufficient to restore infectivity in the majority of particles. This finding suggests that there may be constraints on postbudding polyprotein processing in the production of viable particles. These results should have positive implications regarding the use of protease inhibitors as anti-HIV drugs.

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