Virion instability of human immunodeficiency virus type 1 reverse transcriptase (RT) mutated in the protease cleavage site between RT p51 and the RT RNase H domain

Abstract

Each of the human immunodeficiency virus type 1 (HIV-1) pol-encoded enzymes, protease (PR), reverse transcriptase (RT), and integrase (IN), is active only as a dimer (or higher-order oligomer in the case of IN), but only RT comprises subunits of different masses. RT is a heterodimer of 66-kDa and 51-kDa subunits. The latter is formed by HIV PR-catalyzed cleavage of p66 during virion maturation, resulting in the removal of the RNase H (RNH) domain of a p66 subunit. In order to study the apparent need for RT heterodimers in the context of the virion, we introduced a variety of mutations in the RT p51-RNH protease cleavage site of an infectious HIV-1 molecular clone. Surprisingly, rather than leading to virions with increased RT p66 content, most of the mutations resulted in significantly attenuated virus that contained greatly decreased levels of RT that in many cases was primarily p51 RT. IN levels were also reduced in several mutants. However, most mutants showed normal levels of the Pr160(gag-pol) precursor polyprotein, suggesting that reduced virion RT arose from proteolytic instability rather than decreased incorporation. Mutant virion p24 Gag levels were equivalent to wild type, indicating that Gag incorporation and processing were not affected. Repeated passage of MT-2 cells exposed to mutant viruses led to the appearance of virus with improved replication capacity; these virions contained normally processed RT at near-wild-type levels. These results imply that additional proteolytic processing of RT to the p66/p51 heterodimer is essential to provide proteolytic stability of RT during HIV-1 maturation.

FIG. 1.

Western blot analysis of viral protein expression of p51-RNH cleavage site mutant viruses. Approximately 5 μg of standardized viral p24 lysate was loaded into each lane. Viral RT subunits (A1 and A2), p32 IN (B), and Gag proteins Pr55, p48, p37/41, and p24/25 (C) were detected by probing with appropriate primary and secondary antibodies followed by enhanced chemiluminescence (ECL) as indicated in Materials and Methods. A1 and A2 represent under- and overexposures of viral RT content, respectively. Contrast was enhanced in A2 to enable visualization of protein bands in certain mutants. The relative proportion of p66 RT to p51 RT (p66:p51) and the total viral content of RT and IN were determined from multiple experiments (n = 3) by densitometric scanning analysis of ECL-exposed blots under subsaturating conditions.

FIG. 2.

Western blot analysis of Pr160^gag-pol incorporation in immature p51-RNH cleavage site mutant viruses that contain an inactivated HIV-1 PR (D25A). Immature viruses, previously standardized for p55^gag content by densitometry, were ultracentrifuged, lysed, and loaded into each lane. Relative viral content of (A) Pr160^gag-pol and (B) Pr55^gag were determined by probing separate blots with anti-RT and anti-p24 monoclonal antibodies, respectively, followed by densitometry scanning analysis of ECL-exposed blots under subsaturating conditions.

FIG. 3.

Virus particle-associated RT activities. Clarified virus-containing culture supernatants (8 ng viral p24) were assayed separately for RDDP activity and DDDP activity by the incorporation of [³H]dTTP into poly(rA)-oligo(dT)_12-18 and [³H]dGTP into poly(dC)-oligo(dG)_12-18, respectively. Reaction mixtures were prepared as described in Materials and Methods, incubated at 37°C for 5 h, and quenched with 10% trichloroacetic acid-NaPP_i. Values are expressed as a percentage relative to wild-type virus. Each bar represents the average of three separate measurements.

FIG. 4.

Replication analysis of p51-RNH cleavage site mutant viruses. (A and C) Single-cycle (MAGI) viral infectivity. HeLa-CD4-LTR/β-galactosidase (MAGI) cells (4 × 10⁴) were infected separately with (A) COS-7-generated mutant virions (1 μg viral p24) or (C) MT-2 cell-generated mutant viruses derived after 35 days of culture (100 ng viral p24). MAGI cells were stained and quantitated for β-galactosidase gene expression at 48 h postinfection. Percent infectivity is expressed relative to wild type (WT), with each bar representing the average of two separate measurements. (B and D) Viral replication kinetics analysis. MT-2 lymphocytoid cells (10⁵) were (B) infected with COS-7-generated mutant virions (1 μg viral p24) or (D) reinfected with MT-2 cell-generated mutant viruses derived after 35 days of culture (50 ng viral p24). Cultures were split every 3 days to prevent overgrowth, and HIV-1-induced CPE was scored daily as percent syncytium formation. WT (•), A437I (□), V442S (▴), F440W (▿), F440V (⧫), T439S/V442G (○), Y441I/V442K (▪), F440A (▵), F440A/Y441A (▾), F440W/Y441W (⊙), E438N (⋄).

FIG. 5.

Western blot analysis of viral protein expression of p51-RNH cleavage site mutant viruses after multiple rounds of MT-2 cell infection. Approximately 100 ng of standardized viral p24 lysate was loaded into each lane. (A) RT; (B) p32 IN; (C) Gag p24-reactive protein. The relative proportion of p66 RT to p51 RT (p66:p51) and the total viral content of RT and IN were determined by imaging analysis.