Visualization of positive and negative sense viral RNA for probing the mechanism of direct-acting antivirals against hepatitis C virus

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action

A comparative analysis of the fluorescence properties of the wild-type and active site mutants of the hepatitis C virus autoprotease NS2-3

Hepatitis C virus encodes an autoprotease, NS2-3, which is required for processing of the viral polyprotein between the non-structural NS2 and NS3 proteins. This protease activity is vital for the replication and assembly of the virus and therefore represents a target for the development of anti-viral drugs. The mechanism of this auto-processing reaction is not yet clear but the protease activity has been shown to map to the C-terminal region of NS2 and the N-terminal serine protease region of NS3. The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage. Here, intrinsic tryptophan fluorescence has been used to assess refolding in the wild-type protein and specific active site mutants. We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage. Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold

Global rescue of defects in HIV-1 envelope glycoprotein incorporation: implications for matrix structure.

The matrix (MA) domain of HIV-1 Gag plays key roles in membrane targeting of Gag, and envelope (Env) glycoprotein incorporation into virions. Although a trimeric MA structure has been available since 1996, evidence for functional MA trimers has been elusive. The mechanism of HIV-1 Env recruitment into virions likewise remains unclear. Here, we identify a point mutation in MA that rescues the Env incorporation defects imposed by an extensive panel of MA and Env mutations. Mapping the mutations onto the putative MA trimer reveals that the incorporation-defective mutations cluster at the tips of the trimer, around the perimeter of a putative gap in the MA lattice into which the cytoplasmic tail of gp41 could insert. By contrast, the rescue mutation is located at the trimer interface, suggesting that it may confer rescue of Env incorporation via modification of MA trimer interactions, a hypothesis consistent with additional mutational analysis. These data strongly support the existence of MA trimers in the immature Gag lattice and demonstrate that rescue of Env incorporation defects is mediated by modified interactions at the MA trimer interface. The data support the hypothesis that mutations in MA that block Env incorporation do so by imposing a steric clash with the gp41 cytoplasmic tail, rather than by disrupting a specific MA-gp41 interaction. The importance of the trimer interface in rescuing Env incorporation suggests that the trimeric arrangement of MA may be a critical factor in permitting incorporation of Env into the Gag lattice

Replication of S66 and T69 mutants in Jurkat cells.

<p>Jurkat cells were transfected with the indicated molecular clones. At 2-day intervals the cells were split and samples of media were assayed for RT activity. In each graph of WT pNL4-3, 12LE, 62QR or 12LE/62QR mutations are combined with (A) WT; (B) 66SA; (C) 69TA; (D) 66SR; (E) 69TR. (F) 293T cells were co-transfected with the indicated molecular clones and vectors expressing HIV-1 Env or VSV-G. At 24 h, supernatants were harvested and assayed for infectivity as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. n = 3, +/− SEM.</p

Identification of a second-site mutant capable of rescuing diverse Env-incorporation defective mutants.

<p>(A) Jurkat cells were transfected with the indicated molecular clones. At 2-day intervals the cells were split and samples of media were assayed for RT activity. Virus from the WT and 16EK peaks was normalized by RT then used to infect naïve Jurkat cells and replication of the second passage was followed as described above. Genomic DNA was extracted from cells at the time of peak replication in the 16EK samples after both first and second passage cultures, and the MA coding region was amplified by PCR and subjected to DNA sequencing, revealing the original (16EK) and second-site compensatory (62QR) mutations. (B) Jurkat cells were transfected with the indicated molecular clones and replication was monitored as in (A). (C+E) 293T cells were transfected with the indicated molecular clones. At 24 h, supernatants were filtered then virions were pelleted, lysed, and probed by western blotting for gp41 and CA. (D+F) Supernatants were harvested and assayed for infectivity as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Env incorporation was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Infectivity and Env incorporation are expressed relative to the WT value. n = 3, +/− SEM.</p

62QR is resistant to dominant-negative inhibition by defective Gag mutants in heterogeneous particles.

<p>(A) 293T cells were co-transfected with molecular clones expressing WT or 62QR Gag with the 12LE molecular clone in the ratios indicated (µg∶µg of DNA). At 24 h, supernatants were harvested and assayed for infectivity as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Infectivity is expressed relative to the WT value. n = 4, +/− SEM. (B–E) 293T cells were co-transfected with molecular clones expressing WT or 62QR Gag with Env-incorporation-defective Gag in the ratios indicated (µg∶µg of DNA). Infectivity relative to WT was determined as described for (A). n = 3, +/− SEM. (F) 293T cells were co-transfected with molecular clones expressing WT or 62QR Gag with d8 gp41 in the ratios indicated (µg∶µg of DNA). Infectivity relative to WT was determined as described for (A) n = 4, +/− SEM.</p

Drug Interactions in Lenacapavir-Based Long-Acting Antiviral Combinations

Long-acting (LA) anti-HIV regimens show promise for increasing dosing intervals and consequently, improving the patients&rsquo; quality of life. The first FDA-approved LA therapy is Cabenuva, which comprises rilpivirine (a non-nucleoside reverse transcriptase inhibitor) and cabotegravir (integrase strand transfer inhibitor). Novel promising LA anti-HIV agents such as lenacapavir (a capsid-targeting antiviral) and islatravir (EFdA, a nucleoside reverse transcriptase translocation inhibitor) need to be explored as combination therapies. Therefore, we sought to determine whether combination of lenacapavir with islatravir, rilpivirine, or cabotegravir displayed synergy, additivity, or antagonism. We performed dose-response matrices of these drug combinations in an HIV-1 reporter cell line and subsequently analyzed the data with SynergyFinder Plus, which employs four major drug interaction models: highest single agent, Bliss independence, Loewe additivity, and zero interaction potency. Most of these models predict additive inhibition by the studied drug combinations This work highlights the importance of effective drug combinations in LA-regimens

Potential for intersubunit interactions in the MA trimer.

<p>MA trimer as described in Hill <i>et al.</i> PNAS (1996), showing (A) a top-down view and (B) a side-on view <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#ppat.1003739-Hill1" target="_blank">[32]</a>. Env incorporation defects, red; Q62, green; Ser66 and Thr69, cyan. (C) Close-up view of boxed area from (A), showing Q62 side chain (green), and the side chains of S66 and T69 (cyan) of a second MA monomer. Chain a, black; chain b, gray. Distances between the oxygen atoms of Q62 carbonyl group and the S66 and T69 hydroxyl groups are indicated. Modeled configurations for R62 (D) K62 (E), R66 (F), R66, in combination with R62 (G) and R69 (H). Mutagenesis and rendering performed using MacPymol <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#ppat.1003739-Pymol1" target="_blank">[70]</a>.</p

Vertical scanning of MA residue 62 to determine effects on Env incorporation and ability to rescue Env-incorporation-defective mutants.

<p>(A) HeLa cells were transfected with the molecular clones indicated. Virus release efficiency was determined by metabolic labeling with ³⁵S[Met/Cys] as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. n = 3, +/− SEM. (B) 293T cells were transfected with the indicated molecular clones. At 24 h, supernatants were filtered then virions pelleted, lysed, and probed by western blotting for gp41 and CA. (C) Supernatants from (B) were harvested and assayed for infectivity as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Env incorporation was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Infectivity and Env incorporation are expressed relative to the WT value. n = 6, +/− SEM. (D) Jurkat cells were transfected with the indicated molecular clones. At 2-day intervals the cells were split and samples of media were assayed for RT activity.</p

The effect of mutations at the trimer interface on rescue of Env incorporation.

<p>293T cells were transfected with the indicated molecular clones. At 24<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Infectivity is expressed relative to the WT value. Supernatants were also filtered and virions pelleted, lysed, and probed by western blotting for gp41 and CA. Env incorporation was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a> and is indicated relative to WT. Representative blots are shown below each graph. n = 5–7, +/− SEM. (A) Ala mutants of S66 and T69. (B) Arg mutants of S66 and T69.</p