The present disclosure provides interfering, conditionally replicating human immunodeficiency virus (HIV) constructs; infectious particles comprising the constructs; and compositions comprising the constructs or the particles. The constructs, particles, and compositions are useful in methods of reducing HIV viral load in an individual, which methods are also provided.

Provided are methods and compositions for generating a deletion library, and methods and compositions for generating and identifying a defective interfering particle (DIP). Also provided are transposon cassettes. A subject method can include: inserting a transposon cassette comprising a target sequence for a sequence specific DNA endonuclease into a population of circular target DNAs to generate a population of transposon-inserted circular target DNAs; contacting the population of transposon-inserted circular target DNAs with the sequence specific DNA endonuclease to generate a population of cleaved linear target DNAs; contacting the population of cleaved linear target DNAs with one or more exonucleases to generate a population of deletion DNAs; and circularizing the deletion DNAs to generate a library of circularized deletion DNAs. The population of circular target DNAs can include viral genomic DNA. Also provided are human immunodeficiency virus (HIV) deletion mutants, e.g., interfering, conditionally replicating, HIV deletion mutants, and related constructs.

The invention pertains to an inducible CRISPR system for controlling expression of a CRISPR complex with an inducible fusion promoter. One embodiment of the invention provides HIV LTR-minimal Drosophila hsp70 fusion promoter that can be used for inducible co-expression of gRNA and Cas9 in HIV-infected cells to target cellular cofactors such as Cyclin T1. A single introduction of such embodiment leads to sustained suppression of HIV replication in stringent, chronically infected HeLa-CD4 cell lines as well as in T-cell lines. In another embodiment, the invention further relates to enhancement of HIV suppression by incorporating cis-acting ribozymes immediately upstream of the gRNA in the inducible CRISPR system construct. The inducible fusion promoter is adaptable for other tissue- or cell-type specific expression of the inducible CRISPR system.

The invention pertains to an inducible CRISPR system for controlling expression of a CRISPR complex with an inducible fusion promoter. One embodiment of the invention provides HIV LTR-minimal Drosophila hsp70 fusion promoter that can be used for inducible co-expression of gRNA and Cas9 in HIV-infected cells to target cellular cofactors such as Cyclin T1. A single introduction of such embodiment leads to sustained suppression of HIV replication in stringent, chronically infected HeLa-CD4 cell lines as well as in T-cell lines. In another embodiment, the invention further relates to enhancement of HIV suppression by incorporating cis-acting ribozymes immediately upstream of the gRNA in the inducible CRISPR system construct. The inducible fusion promoter is adaptable for other tissue- or cell-type specific expression of the inducible CRISPR system.

Provided here are certain recombinant HIV compositions and animal models to evaluate prophylactic and therapeutic antiviral compositions.

The present disclosure provides an interfering, conditionally replicating human immunodeficiency virus (HIV) construct; infectious particles comprising the constructs; and compositions comprising the construct or the particle. The constructs, particles, and compositions are useful in methods of reducing HIV viral load in an individual, which methods are also provided.

The invention pertains to an inducible CRISPR system for controlling expression of a CRISPR complex with an inducible fusion promoter. One embodiment of the invention provides HIV LTR-minimal Drosophila hsp70 fusion promoter that can be used for inducible co-expression of gRNA and Cas9 in HIV-infected cells to target cellular cofactors such as Cyclin T1. A single introduction of such embodiment leads to sustained suppression of HIV replication in stringent, chronically infected HeLa-CD4 cell lines as well as in T-cell lines. In another embodiment, the invention further relates to enhancement of HIV suppression by incorporating cis-acting ribozymes immediately upstream of the gRNA in the inducible CRISPR system construct. The inducible fusion promoter is adaptable for other tissue- or cell-type specific expression of the inducible CRISPR system.

The present invention provides an expression vector for preventing or inhibiting HIV entry, fusion or replication in mammalian cells. In particular, the invention provides a recombinant retroviral vector that encodes an inhibitor of a HIV co-receptor, such as CCR5 or CXCR4, and a protein that inhibits HIV fusion to target cells and/or HIV replication. Pharmaceutical compositions comprising such constructs and methods of use thereof to prevent or treat HIV infection in a patient are also disclosed.

The invention provides a method for determining whether a human immunodeficiency virus is resistant to a viral entry inhibitor. The methods are particularly useful for determining resistance to inhibitors that act by a non-competitive mechanism. In certain aspects, the methods comprise determining whether an HIV population is resistant to an HIV entry inhibitor, comprising determining a log-sigmoid inhibition curve comprising data points for entry of the HIV population in the presence of varying concentrations of the HIV entry inhibitor, wherein if the entry of the HIV population cannot be completely inhibited by the HIV entry inhibitor, the HIV population is resistant to the HIV entry inhibitor.

The present invention provides a novel method for detecting replication competent virus in a test sample. The method comprises culturing and diluting a plurality of individual cell culture aliquots comprising virus-permissive cells and a portion of the test sample, followed by testing for the presence of replication competent virus. The method may be used in parallel with a positive control, which is also provided herein.