A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein–protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives’ action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C₇₀ derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C₇₀ derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription—without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives’ oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.

The early detection of upcoming disease outbreaks is essential to avoid both health and economic damage. The last four years of COVID-19 pandemic have proven wastewater-based epidemiology is a reliable system for monitoring the spread of SARS-CoV-2, a causative agent of COVID-19, in an urban population. As this monitoring enables the identification of the prevalence of spreading variants of SARS-CoV-2, it could provide a critical tool in the fight against this viral disease. In this study, we evaluated the presence of variants and subvariants of SARS-CoV-2 in Prague wastewater using nanopore-based sequencing. During August 2021, the data clearly showed that the number of identified SARS-CoV-2 RNA copies increased in the wastewater earlier than in clinical samples indicating the upcoming wave of the Delta variant. New SARS-CoV-2 variants consistently prevailed in wastewater samples around a month after they already prevailed in clinical samples. We also analyzed wastewater samples from smaller sub-sewersheds of Prague and detected significant differences in SARS-CoV-2 lineage progression dynamics among individual localities studied, e.g., suggesting faster prevalence of new variants among the sites with highest population density and mobility.

Many reports have documented that the presence of SARS-CoV-2 RNA in the influents of municipal wastewater treatment plants (WWTP) correlates with the actual epidemic situation in a given city. However, few data have been reported thus far on measurements upstream of WWTPs, i.e. throughout the sewer network. In this study, the monitoring of the presence of SARS-CoV-2 RNA in Prague wastewater was carried out at selected locations of the Prague sewer network from August 2020 through May 2021. Various locations such as residential areas of various sizes, hospitals, city center areas, student dormitories, transportation hubs (airport, bus terminal), and commercial areas were monitored together with four of the main Prague sewers. The presence of SARS-CoV-2 RNA was determined by reverse transcription – multiplex quantitative polymerase chain reaction (RT-mqPCR) after the precipitation of nucleic acids with PEG8000 and RNA isolation with TRIzol™ Reagent. The number of copies of the gene encoding SARS-CoV-2 nucleocapsid (N1) per liter of wastewater was compared with the number of officially registered COVID-19 cases in Prague. Although the data obtained by sampling wastewater from the major Prague sewers were more consistent than those obtained from the small sewers, the correlation between wastewater-based and clinical-testing data was also good for the residential areas with more than 1 000 registered inhabitants. It was shown that monitoring SARS-CoV-2 RNA in wastewater sampled from small sewers could identify isolated occurrences of COVID-19-positive cases in local neighborhoods. This can be very valuable while tracking COVID-19 hotspots within large cities. Highlights SARS-CoV-2 RNA presence was measured at 24 locations in the Prague sewer network Residential areas (100–13 000 inhab.), transport hubs, hospitals etc. were included Consistent wastewater monitoring by RT-mqPCR took place from August 2020 – May 2021 The sampling of major Prague sewers correlated well with clinical-based data Grab samples can identify COVID-19 hotspots in local neighborhoods