Mutational analysis of the C-terminal gag cleavage sites in human immunodeficiency virus type 1

Abstract

Human immunodeficiency virus type 1 (HIV-1) Gag is expressed as a polyprotein that is cleaved into six proteins by the viral protease in a maturation process that begins during assembly and budding. While processing of the N terminus of Gag is strictly required for virion maturation and infectivity, the necessity for the C-terminal cleavages of Gag is less well defined. To examine the importance of this process, we introduced a series of mutations into the C terminus of Gag that interrupted the cleavage sites that normally produce in the nucleocapsid (NC), spacer 2 (SP2), or p6(Gag) proteins. Protein analysis showed that all of the mutant constructs produced virions efficiently upon transfection of cells and appropriately processed Gag polyprotein at the nonmutated sites. Mutants that produced a p9(NC/SP2) protein exhibited only minor effects on HIV-1 infectivity and replication. In contrast, mutants that produced only the p8(SP2/p6) or p15(NC/SP2/p6) protein had severe defects in infectivity and replication. To identify the key defective step, we quantified reverse transcription and integration products isolated from infected cells by PCR. All mutants tested produced levels of reverse transcription products either similar to or only somewhat lower than that of wild type. In contrast, mutants that failed to cleave the SP2-p6(Gag) site produced drastically less provirus than the wild type. Together, our results show that processing of the SP2-p6(Gag) and not the NC-SP2 cleavage site is important for efficient viral DNA integration during infection in vitro. In turn, this finding suggests an important role for the p9(NC/SP2) species in some aspect of integration.

FIG. 1.

Proviral constructs. Diagrams of the Gag regions of the various pNL4-3 constructs used in this study are presented. The mature proteins within Gag and the minor cleavage sites (denoted by dotted lines in SP2 and p6^Gag) are indicated on the NL4-3 wild-type construct map. The region mutated is presented for each mutant with the expected product highlighted in black. A dotted line indicates the C-terminal partial protease cleavage site (after position 36) in p6^Gag.

FIG. 2.

Immunoblots of mutant virion preparations. p24^CA, RT, p7^NC, and p6^Gag immunoblots of equal volumes of virion preparations are presented. Samples are identified above the respective lanes. sssDNA, a virus preparation produced from transfected, sheared salmon sperm DNA as a mock control. Molecular masses, as calculated by relative mobilities, and identities of bands are indicated at the margins of the blots. Due to the poor retention of wild-type p6^Gag, a 10-fold-longer exposure is provided to the right of the second p6^Gag immunoblot to show the migration of wild-type p6^Gag.

FIG. 3.

HPLC analysis of mutants. The complete HPLC chromatogram for wild-type NL4-3 virions is shown at the top of the figure with the region that is presented for the mutants indicated by arrows. The identities of the Gag proteins, determined by Coomassie brilliant blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, protein sequencing, and mass spectrometry, are identified above each respective peak. The mutant proteins as identified by MALDI-TOF mass spectrometry are shaded in black. The p6^Gag-containing species that are missing the C-terminal 16 amino acids due to cleavage after residue 36 are indicated by a “-cf” notation, e.g., p6^-cf, in the identifying superscript. The C-terminal fragment itself was labeled p6^cf when observed.

FIG. 4.

Reverse transcription and integration products assayed by real-time PCR. Representative results of a quantitative real-time PCR analysis are presented as percent conversion between two representative steps in the infection process. Results for VSV-G-mediated infection are presented as black bars, and data from HIV-1 Env-mediated infections are in white. Parameters presented are indicated next to each graph. Typical samples analyzed contained extract from about 1.1 × 10⁴ cells. For the wild type, the target quantities detected were 3.2 × 10⁵ copies of R-U5, 1.33 × 10⁵ copies of R-5′UTR, 3.3 × 10⁴ copies of provirus, and 4.8 × 10³ copies of 2-LTR circles. The amounts of provirus in H9 cells infected with HIV-1 Env-containing virus were below the level of detection and thus were omitted. Full data sets of experiments are presented in Tables S1 to S6 in the supplemental material.