Antiretroviral Therapy: New Mechanistic and Therapeutic Insights for HIV Single-Entity and Combination Drug Products by

The infectivity of cell-free HIV-1 is consistently reported to be less than 0.1% and the mechanisms influencing this low infectivity are not yet fully understood. Some hypothesize that this observed low infectivity results from the presence of defective HIV-1 particles, formed from mutations introduced in the reverse transcription step of virus replication. Using molecularly cloned HIV-1 that is capable of only a single round of infection, we can bypass the reverse transcription step during virus production in order to investigate this hypothesis and the extent to which infectivity can be influenced by other factors during virus production. Herein we show that optimal infectivity is obtained by harvesting virions from culture media 18 hours after transfection, with complete media changes 4-6 hours post-transfection. This optimal infectivity appears to primarily result from the emergence of “noninfectious” virus particles at later time points. Our data demonstrate that HIV-1 infectivity increases in the presence of 18 greater amounts of viral envelope glycoproteins. Thus, the larger proportion of “noninfectious” virus particles apparent in harvested virus cultures at later time points may be attributable to the production of virus lacking sufficient amounts of envelope glycoproteins. However, although we demonstrate ways by which virus infectivity can be optimized by varying parameters to enhance culture and production conditions, the overall infectivity of HIV-1 remains low. This suggests that the observed low infectivity of the virus is principally attributable to other, potentially-related, mechanism(s). Introduction Infectivity, a unitless number that quantitates the proportion of virus particles that are infectious, is a critical parameter for characterizing the human immunodeficiency virus type 1 (HIV-1). The infectivity of HIV-1 is consistently reported to be less than 0.1% [22] [23] [24]. There is not yet a clear, confirmatory understanding of why HIV-1 possesses such low infectivity, but there is a great deal of literature suggesting that one of the primary limitations in the establishment of a productive HIV infection is the target cell engagement and/or entry step. In fact, several engineered/artificial methods for increasing interactions between virions and target cells have been documented to result in greater infection efficiency. Spinoculation, a method of inoculating cells with virions using centrifugation, for example, results in greater amounts of cellassociated virus and a proportional increase in virus replication [27]. Furthermore, inoculating virions in the presence of positively-charged molecules 19 (e.g., polybrene) has been shown to enhance adsorption of virus particles onto target cells and result in greater infectivity [24] [28] [29]. And the overexpression adhesion molecules on virion-producer cells has also been shown to result in virions that possess more adhesion molecules (acquired from budding out of producer cells) and enhanced ability to interact with and infect target cells [30] [31] [32]. Therefore, it is plausible that HIV-1 infectivity is primarily limited by the ability to efficiently engage target cells. On the other hand, however, some have hypothesized that this observed low infectivity results from the presence of defective HIV-1 particles, formed from mutations introduced in the reverse transcription step of virus replication [25]. This theory is supported by the well-documented, naturally high error rate of HIV reverse transcriptase [49] [50] [51]. Furthermore, the identification of defense mechanisms, such as APOBEC3 cytidine deaminases, in the host cell that can influence the rate of mutation during the reverse transcription step of virus replication also strongly point to the possibility that the low infectivity of HIV-1 might be due to the presence of large numbers of defective virions resulting from mutations introduced to the proviral DNA during reverse transcription [52] [53]. Using molecularly cloned HIV-1 that is capable of only a single round of infection, we can bypass the reverse transcription step during virus production and essentially eliminate the influence of reverse transcriptase errors and APOBEC3 in order to investigate this hypothesis and determine the extent to which infectivity can be influenced by other factors during virus production. 20 Herein we specifically demonstrate that the culture conditions commonly used to produce cell-free HIV-1 in cultured media significantly influence the resulting infectivity of virions and can result in the production of a greater proportion of defective virus over time. These results emphasize the optimal conditions for producing cell-free virus and point to a potential molecular mechanism, related to the alternate theory of inefficient virus-cell interactions, which may more significantly impede the infectivity of HIV-1 in plasma or cultured media. Materials and Methods Production of single-cycle HIV-1 virions. Virions were produced by transfecting HEK 293T/17 cells (ATCC, Manassas, VA) as previously described [54]. Briefly, 293T were cultured at 37C with 5% CO2 in DMEM supplemented with 10% FBS (HyClone Laboratories, Logan, UT) and seeded overnight in culture media. Using the TransIT LT-1 transfection reagent (MirusBio, Madison, WI), HIV virions carrying free EGFP were generated by transfection with variable amounts of pNL4-3Eplasmid, pNL4-3E-MA-EGFP-CA plasmid and pcDNA3.1REC. In certain experiments, after a specified number of hours of incubation at 37C, the culture media (with transfection reagents) was removed and replaced with fresh culture media. The 37C incubation continued for an additional period. At the experimentallydetermined time point post transfection, the culture media, containing EGFPlabeled single-cycle virions was collected and filtered through a 0.45-mm syringe filter (Millex-HV PVDF, Millipore). The filtrate was then aliquoted on ice, flash21 frozen in a dry ice/ ethanol bath and stored in a -80C freezer. Subsequent analysis using p24 ELISA (HIV-1 p24 Antigen Capture Kit, Advanced Bioscience Laboratories, Rockville, MD) was conducted to determine the number of virus particles in a specified volume, assuming 2,500 molecules of p24 per virion. Calculating the Infectivity of free-EGFP HIV-1. Infection assay in TZM-bl cell line. The infectivity of virions was calculated by normalizing the virus titer (i.e., number of infectious units in a certain volume) to the physical number of virus particles present in the specified volume. The titer was be determined by using an established β-galactosidasebased infection assay. This assay relies upon the TZM-bl indicator cell line a genetically engineered HeLa-derived cell line that expresses CD4, CXCR4, and CCR5 [55] [56]. TZM-bl cells function as indicators of HIV infection because they also possess Luciferase and bacterial β-galactosidase reporter genes, which are driven by an HIV LTR promoter. This LTR promoter is induced to express the reporter enzymes following HIV infection when the viral protein Tat (Transactivator of transcription) is produced [57]. Thus, in theory, only infected cells will express the reporter genes. These infected cells can be detected by providing a chromogenic substrate for either of the reporter enzymes. For these studies, infected cells will be quantitated by using 5-bromo-4-chloro-3-indolyl-βD-galactopyranoside (X-gal) as a substrate for β-galactosidase. βgalactosidase catalyzes the hydrolysis of X-gal and produces a blue-colored byproduct, 5,5'-dibromo-4,4'-dichloro-indigo. 22 The infectious titer of HIV-1 virions can therefore be determined by enumerating the number of blue TZM-bl cells following inoculation with various dilutions of the virus stock in accordance with the following equation: mL Units Infectious (mL) Volume Inoculum mL 1 Factor Dilution Cells TZMbl Blue # Titer    TZM-bl cells (cat#8129, NIH AIDS Research and Reference Reagent Program) were cultured at 37C with 5% CO2 in DMEM supplemented with 10% FBS (HyClone Laboratories, Logan, UT). Prior to tenth passage, TZM-bl cells were trypsinized, counted, sedimented by centrifugation at 1,000g for 5 minutes and resuspended in DMEM with 10% FBS. 8X10 TZM-bl cells in a 1-ml culture volume were seeded in each well of a 12-well plate one day prior to infection. EGFP-labeled HIV-1 particles (obtained from stocks stored at -80C) were added to these pre-seeded aliquots of TZM-bl cells in 100 μl of DMEM with 10% FBS and DEAE dextran (final concentration 20 mg/ml). The virion and TZM-bl cell mixture was incubated at 37C for 2hrs with gentle rocking every 30 min. At the end of two hours, 1 ml of complete media was added to each well and the incubation was continued at 37C for 48 hours with 5% CO2. After 2 days of incubation, cells were fixed in 2% gluteraldehyde at room temperature for five minutes. Cells were then washed three times with PBS, and stained for 50 min at 37C using cell staining solution provided in the beta-galactosidase staining kit (Mirus Bio, Madison, WI). Cells were washed three times with milliQ water and