Mark A. Wainberg

In patients with HIV/AIDS receiving antiretroviral therapy (ART), HIV-1 persistence in brain tissue is a vital and unanswered question. HIV-1 infects and replicates in resident microglia and trafficking macrophages within the brain although the impact of individual ART drugs on viral infection within these brain myeloid cells is unknown. Herein, the effects of contemporary ART drugs were investigated using in vitro and in vivo models of HIV-1 brain infection. The EC50 values for specific ART drugs in HIV-infected human microglia were significantly higher compared to bone marrow-derived macrophages and peripheral blood mononuclear cells. Intracellular ART drug concentrations in microglia were significantly lower than in human lymphocytes. In vivo brain concentrations of ART drugs in mice were 10 to 100-fold less in brain tissues compared with plasma and liver levels. In brain tissues from untreated HIV-infected BLT mice, HIV-encoded RNA, DNA and p24 were present in human leukocytes while ART eradicated viral RNA and DNA in both brain and plasma. Interruption of ART resulted in detectable viral RNA and DNA and increased human CD68 expression in brains of HIV-infected BLT mice. In aviremic HIV/AIDS patients receiving effective ART, brain tissues that were collected within hours of last ART dosing showed HIV-encoded RNA and DNA with associated neuroinflammatory responses. ART drugs show variable concentrations and efficacies in brain myeloid cells and tissues in drug-specific manner. Despite low drug concentrations in brain, experimental ART suppressed HIV-1 infection in brain although HIV/AIDS patients receiving effective ART had detectable HIV-1 in brain. These findings suggest that viral suppression in brain is feasible but new approaches to enhancing ART efficacy and concentrations in brain are required for sustained HIV-1 eradication from brain.

Flaviviruses including Zika, Dengue and Hepatitis C virus cause debilitating diseases in humans, and the former are emerging as global health concerns with no antiviral treatments. We investigated Sophora Flavecens, used in Chinese medicine, as a source for antiviral compounds. We isolated Sophoraflavenone G and found that it inhibited Hepatitis C replication, but not Sendai or Vesicular Stomatitis Virus. Pre- and post-infection treatments demonstrated anti-flaviviral activity against Dengue and Zika virus, via viral RNA polymerase inhibition. These data suggest that Sophoraflavenone G represents a promising candidate regarding anti-Flaviviridae research.

Introduction: To date, three HIV integrase strand transfer inhibitors (INSTIs), i.e. raltegravir, elvitegravir and dolutegravir, have been approved for clinical use. Recent research has focused on new integrase inhibitors including those targeting non-catalytic sites of HIV integrase. Areas covered: This paper reviews two investigational INSTIs in phase I and II clinical trials, bictegravir (BIC) and cabotegravir (CAB), as well as an investigational noncatalytic integrase inhibitor (NCINI) termed BI 224436. Expert opinion: Data from phase I and II clinical trials demonstrate that CAB has good efficacy and is well-tolerated. CAB is promising because it can be formulated both orally and as a long-acting (LA) injectable for treatment and prevention of HIV infection. Since LA-CAB formulation offers the possibility of favourable dosing, it may help individuals who struggle with adherence issues. BIC also represents a promising safe, effective and well-tolerated drug that can be administered as a single once-daily regimen in coformulation with emtricitabine and tenofovir alafenamide (FTC/TAF). Ongoing phase III trials should clarify optimal doses and reveal the potential clinical advantages of these new drugs and formulations over other current regimens. Exploration of novel HIV integrase inhibitors acting through mechanisms different from those of INSTIs is still needed.

Human immunodeficiency virus (HIV) infection persists despite decades of active antiretroviral therapy (ART), effectively preventing viral eradication. Treatment decreases plasma viral RNA, but viral DNA persists, mostly integrated within the genome of nucleated blood cells. Viral DNA blood levels correlate with comorbidities and the rapidity of viral rebound following treatment interruption. To date, no intervention aiming at decreasing HIV DNA levels below those attained through ART has been successful. This includes use of some integrase inhibitors either as part of ART or in treatment intensification studies. We have argued that using the integrase inhibitor dolutegravir (DTG) in similar studies may yield better results, but this remains to be studied. In treatment-experienced individuals, the most frequent substitution associated with failure with dolutegravir is R263K in integrase. R263K decreases integration both in cell-free and tissue culture assays. We investigated here how integrated DNA levels evolve over time during prolonged infections with R263K viruses. To investigate a potential defect in reverse transcription with R263K, the levels of reverse transcripts were measured by quantitative PCR. We measured HIV type 1 (HIV-1) integration in Jurkat cells over the course of 4-week infections using Alu-mediated quantitative PCR. The results show that R263K did not decrease reverse transcription. Prolonged infections with R263K mutant viruses led to less HIV-1 integrated DNA over time compared to wild-type viruses. These tissue culture results help to explain the absence of the R263K substitution in most individuals experiencing failure with DTG and support studies aiming at longitudinally measuring the levels of integrated DNA in individuals treated with this drug.IMPORTANCEAntiretroviral treatment decreases plasma viral RNA, but HIV DNA persists for decades within infected cells. Studies of nonhuman primates have suggested that reducing retroviral DNA levels might represent a path to eradication. The integrase inhibitor dolutegravir is less susceptible than any other anti-HIV drug to the emergence of resistance in treatment-naive individuals. In treatment-experienced individuals, in contrast, rare cases of treatment failure were commonly associated with emergence of an R263K integrase substitution that confers low-level resistance to dolutegravir. It is unclear why this substitution is not more common in individuals experiencing failure with dolutegravir. We report here that R263K progressively diminishes the levels of integrated HIV-1 DNA in tissue culture over multiple cycles of infection. Our results help to explain aspects of the clinical efficacy of dolutegravir and suggest that this drug may be able to reduce HIV DNA levels within infected individuals compared to other drugs.

HIV can spread by both cell-free and cell-to-cell transmission. Here, we show that many of the amino acid changes in Env that are close to the CD4 binding pocket can affect HIV replication. We generated a number of mutant viruses that were unable to infect T cells as cell-free viruses but were nevertheless able to infect certain T cell lines as cell-associated viruses, which was followed by reversion to the wild type. However, the activation of JAK-STAT signaling pathways caused the inhibition of such cell-to-cell infection as well as the reversion of multiple HIV Env mutants that displayed differences in their abilities to bind to the CD4 receptor. Specifically, two T cell activators, interleukin-2 (IL-2) and phorbol 12-myristate 13-acetate (PMA), both capable of activation of JAK-STAT pathways, were able to inhibit cell-to-cell viral transmission. In contrast, but consistent with the above result, a number of JAK-STAT and mTOR inhibitors actually promoted HIV-1 transmission and reversion. Hence, JAK-STAT signaling pathways may differentially affect the replication of a variety of HIV Env mutants in ways that differ from the role that these pathways play in the replication of wild-type viruses. IMPORTANCE Specific alterations in HIV Env close to the CD4 binding site can differentially change the ability of HIV to mediate infection for cell-free and cell-associated viruses. However, such differences are dependent to some extent on the types of target cells used. JAK-STAT signaling pathways are able to play major roles in these processes. This work sheds new light on factors that can govern HIV infection of target cells.

Codon usage bias has been described for various organisms and is thought to contribute to the regulation of numerous biological processes including viral infections. HIV-1 codon usage has been previously shown to be different from that of other viruses and man. It is evident that the antiretroviral drugs used to restrict HIV-1 replication also select for resistance variants. We wanted to test whether codon frequencies in HIV-1 sequences from treatment-experienced patients differ from those of treatment-naive individuals due to drug pressure affecting codon usage bias. We developed a JavaScript to determine the codon frequencies of aligned nucleotide sequences. Irrespective of subtypes, using HIV-1 pol sequences from 532 treatment-naive and 52 treatment-experienced individuals, we found that pol sequences from treatment-experienced patients had significantly increased AGA (arginine; p = 0.0002***) and GGU (glycine; p = 0.0001***), and decreased AGG (arginine; p = 0.0001***) codon frequencies. The same pattern was not observed when subtypes B and C sequences were analyzed separately. Additionally, irrespective of subtypes, using HIV-1 gag sequences from 524 treatment-naive and 54 treatment-experienced individuals, gag sequences from treatment-experienced patients had significantly increased CUA (leucine; p < 0.0001***), CAG (glutamine; p = 0.0006***), AUC (isoleucine; p < 0.0001***) and UCU (serine; p = 0.0005***), and decreased AUA (isoleucine; p = 0.0003***) and CAA (glutamine; p = 0.0006***) codon frequencies. Using pol and gag genes derived from the same HIV-1 genome, we show that antiretroviral therapy changed certain HIV-1 codon frequencies in a subtype specific way.

Background: The viral RNA-dependent RNA polymerase (RdRp) enzymes of the Flaviviridae family are essential for viral replication and are logically important targets for development of antiviral therapeutic agents. Zika virus (ZIKV) is a rapidly re-emerging human pathogen for which no vaccine or antiviral agent is currently available. Methods: To facilitate development of ZIKV RdRp inhibitors, we have established an RdRp assay using purified recombinant ZIKV NS5 polymerase. Results: We have shown that both the hepatitis C virus (HCV) nucleoside inhibitor sofosbuvir triphosphate and a pyridoxine-derived non-nucleoside small-molecule inhibitor, DMB213, can act against ZIKV RdRp activity at IC₅₀s of 7.3 and 5.2 μM, respectively, in RNA synthesis reactions catalysed by recombinant ZIKV NS5 polymerase. Cell-based assays confirmed the anti-ZIKV activity of sofosbuvir and DMB213 with 50% effective concentrations (EC₅₀s) of 8.3 and 4.6 μM, respectively. Control studies showed that DMB213 did not inhibit recombinant HIV-1 reverse transcriptase and showed only very weak inhibition of HIV-1 integrase strand-transfer activity. The S604T substitution in motif B of the ZIKV RdRp, which corresponds to the S282T substitution in motif B of HCV RdRp, which confers resistance to nucleotide inhibitors, also conferred resistance to sofosbuvir triphosphate, but not to DMB213. Enzyme assays showed that DMB213 appears to be competitive with natural nucleoside triphosphate (NTP) substrates. Conclusions: Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.