Relationships between Infectious Titer, Capsid Protein Levels, and Reverse Transcriptase Activities of Diverse Human Immunodeficiency Virus Type 1 Isolates

Abstract

Most studies on human immunodeficiency virus type 1 (HIV-1) replication kinetics or fitness must rely on a particular assay to initially standardize inocula from virus stocks. The most accurate measure of infectious HIV-1 titers involves a limiting dilution-infection assay and a calculation of the dose required for 50% infectivity of susceptible cells in tissue culture (TCID 50 ). Surrogate assays are now commonly used to measure the amount of p24 capsid, the endogenous reverse transcriptase (RT) activity, or the amount of viral genomic RNA in virus particles. However, a direct comparison of these surrogate assays and actual infectious HIV-1 titers from TCID 50 assays has not been performed with even the most conserved laboratory strains, let alone the highly divergent primary HIV-1 isolates of different subtypes. This study indicates that endogenous RT activity, not p24 content or viral RNA load, is the best surrogate measure of infectious HIV-1 titer in both cell-free supernatants and viruses purified on sucrose cushions. Sequence variation between HIV-1 subtypes did not appear to affect the function or activity of the RT enzyme in this endogenous assay but did affect the detection of p24 capsid by both enzyme immunoassays and Western blots. Clear groupings of non-syncytiuminducing (NSI), CCR5-tropic (R5), and SI/CXCR4-tropic (X4) HIV-1 isolates were observed when we compared the slopes derived from correlations of RT activity with infectious titers. Finally, the replication efficiency or fitness of both the NSI/R5 and SI/X4 HIV-1 isolates was not linked to the titers of the virus stocks.

Recapitulating Cross-Species Transmission of Simian Immunodeficiency Virus SIVcpz to Humans by Using Humanized BLT Mice

“…We first sought to normalize the inoculum of the 4 SIVcpz strains and HIV-1 SUMA . Since a dose based on infectious units or 50% tissue culture infective dose (TCID 50 ) may be biased due to the humanorigin indicator cells, we used reverse transcriptase activity to normalize the dose of inoculum (19). We then reduced the inoculation dose to 0.52 RT units, which is significantly lower (9.42-fold reduction of IU on average) than the high-dose inoculum that we used in initial infections of hu-BLT mice.…”

“…To test human susceptibility to SIVcpz, 25 female hu-BLT mice with high immune reconstitution were randomly divided into 5 groups ( Cross-species transmission barrier of SIVcpz. To quantify the crossspecies transmission barrier of SIVcpz to infect humans, each of the SIVcpz strains and HIV-1 SUMA was titrated based on viral reverse transcriptase (RT) activity, which is the best available method (19). The RT was measured in triplicate using the EnzChek reverse transcriptase assay kit (Invitrogen, Eugene, OR, USA).…”

A Quantitative Affinity-Profiling System That Reveals Distinct CD4/CCR5 Usage Patterns among Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus Strains

“…Envelope-pseudotyped luciferase reporter viruses were generated by cotransfection of 293T cells with 1 g of the luciferase-encoding pseudotyping vector pNL-Luc.AM (37) and 1 g of envelope expression vector. Cells were washed after 24 h, and pseudoviruses were collected after a subsequent 48 h. The relative numbers of particles were determined by limiting-dilution reverse transcriptase (RT) assay (25). All pseudotyped reporter viruses were used within the linear range of the assay.…”

Rapid, Fully Automated Digital Immunoassay for p24 Protein with the Sensitivity of Nucleic Acid Amplification for Detecting Acute HIV Infection

“…Third-generation antibody assays (6 ) detect the earliest stage of the immune response (immunoglobulin M), reducing the serological window to approximately 3 weeks (7 ). Viral proteins are present at the start of infection, and each HIV particle is composed of approximately 2000 copies of p24 capsid protein and 2 viral RNA copies (8 ). A titer of 30 viruses per mL equates to 60 000 p24 molecules, or a concentration of 3 femtograms per milliliter, which is well below the detection limit of 11 000 -70 000 fg/mL with contemporary p24 and fourth-generation HIV combination immunoassays (9,10 ).…”