Compositions and Methods for Determining Resistance to Inhibitors of Virus Enter Using Recombinant Virus Assays

A method for detecting mutations of a virus that are correlated with reduced susceptibility to a virus entry inhibitor, comprising:

• (a) obtaining individual biological samples from a subject at a plurality of different time points during infection, the samples each comprising virus nucleic acid molecules;
• (b) cloning a plurality of nucleic acid molecules, each comprising a gene that encodes an envelope protein for one of the virus molecules in the sample of step (a);
• (c) transfecting into a first cell:
• (i) a nucleic acid comprising one of the cloned envelope protein genes of step (b) or a population thereof;
• and (ii) a viral expression vector which lacks the viral envelope protein gene, such that a population of identical pseudoviruses or a population of representative pseudoviruses are produced by the first cell, and optionally, contacting the pseudoviruses produced by the first cell with a virus entry inhibitor;
• (d) contacting the pseudoviruses of step (c) with a second cell, wherein the second cell expresses at least one cell surface receptor to which the virus binds;
• (e) measuring the ability of the pseudoviruses to infect the second cell;
• (f) comparing the infectivity of the pseudoviruses of step (e) when the pseudoviruses have been exposed to the virus entry inhibitor as compared to when the pseudoviruses have not been exposed to the virus entry inhibitor, wherein a partial decrease, no change, or an increase in infectivity for the pseudoviruses exposed to the virus entry inhibitor indicates that the pseudoviruses have reduced susceptibility to the virus entry inhibitor;
• and
• (g) determining the nucleic acid sequence of at least some of the pseudoviruses of step (f) to determine whether a mutation or mutations in the gene encoding the envelope protein is correlated with the reduced susceptibility to the virus entry inhibitor.