Dominant Negative MA-CA Fusion Protein Is Incorporated into HIV-1 Cores and Inhibits Nuclear Entry of Viral Preintegration Complexes

Abstract

Particle maturation is a critical step in the HIV-1 replication cycle that requires proteolytic cleavage of the Gag polyprotein into its constitutive proteins: the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins. The accurate and efficient cleavage of Gag is essential for virion infectivity; inhibitors of the viral protease are potent antivirals, and substitutions in Gag that prevent its cleavage result in reduced HIV-1 infectivity. In a previous study, a mutation inhibiting cleavage at the MA-CA junction was observed to potently inhibit virus infection: incorporation of small amounts of uncleaved MA-CA protein into HIV-1 particles inhibited infectivity by ∼95%, and the resulting viral particles exhibited aberrant capsids. Here we report a detailed mechanistic analysis of HIV-1 particles bearing uncleaved MA-CA protein. We show that the particles contain stable cores and can efficiently saturate host restriction by TRIMCyp in target cells. We further show that MA-CA associates with CA in particles without detectably affecting the formation of intermolecular CA interfaces. Incorporation of MA-CA did not markedly affect reverse transcription in infected cells, but nuclear entry was impaired and integration targeting was altered. Additionally, results from mutational analysis of Gag revealed that membrane-binding elements of MA contribute to the antiviral activity of uncleaved MA-CA protein. Our results suggest that small amounts of partially processed Gag subunits coassemble with CA during virion maturation, resulting in impaired capsid functions. IMPORTANCE To become infectious, newly formed HIV-1 particles undergo a process of maturation in which the viral polyproteins are cleaved into smaller components. A previous study demonstrated that inclusion of even small quantities of an uncleavable mutant Gag polyprotein results in a strong reduction in virus infectivity. Here we show that the mechanism of transdominant inhibition by uncleavable Gag involves inhibition of nuclear entry and alteration of viral integration sites. Additionally, the results of mutational analysis suggest that the membrane-binding activity of Gag is a major requirement for the antiviral activity. These results further define the antiviral mechanism of uncleavable Gag, which may be useful for exploiting this effect to develop new antivirals.

Inositol phosphates promote HIV-1 assembly and maturation to facilitate viral spread in human CD4+ T cells

“…Impairments in Gag processing can alter particle morphology and inhibit HIV-1 replication [32][33][34][35][36]. To test for evidence of budding defects and alterations to particle morphology upon depletion of IP5 and/or IP6, we examined infected MT-4 cells by thin section transmission electron microscopy at 3 days following infection.…”

CPSF6-Dependent Targeting of Speckle-Associated Domains Distinguishes Primate from Nonprimate Lentiviral Integration

“…****, P < 0.0001. (E) Mapped HIV NLX.Luc.R-U3-Tag -Luc integration sites in indicated cell lines; the EIAV U3-tagged virus affords integration site determinations in cells that harbor preexisting integrated LentiCRISPRv2 ( 66 ). See Table S1 for associated statistical analyses.…”

“…Plasmid psPAX2 was obtained from the NIH AIDS Reagent Program while plasmids pIRES2-eGFP ( 13 ), pNLX.LucR-U3-tag ( 66 ), and pCG-VSV-G ( 67 ) were previously described. Other plasmids that encoded single-round HIV-1 ( 68 ), HIV-2 ( 69 ), SIV agm Sab, SIV agm Tan ( 70 ), SIV mac ( 71 ), BIV ( 72 ), EIAV ( 73 ), FIV ( 74 ), and MLV ( 75 ) GFP constructs as well as HIV-1 (WT, N74D) ( 76 , 77 ), SIV mac ( 78 ), MLV ( 13 ), and FIV ( 79 ) Luc reporter viruses were also previously described.…”

“…Illumina reads were processed and integration sites were mapped essentially as previously described ( 17 , 66 , 82 ). Briefly, read 1 of the paired-end sequences contained viral information linked to host DNA whereas the second read contained the linker and opposing end of the captured host fragment.…”

HIV-1 Maturation: Lessons Learned from Inhibitors

“…However, blocking other cleavage events may impede virus replication by different mechanisms. For example, previous studies have shown that the uncleaved MA-CA Gag fragment is a very strong dominant negative HIV inhibitor with the ability to induce aberrant and eccentric core morphology, block reverse transcription in target cells, and disrupt proper nuclear entry and integration [ 191 , 192 ]. However, the MA-CA junction is unstructured [ 193 ] and, therefore, may not be readily inhibited by small molecules.…”