Human Transbodies to Reverse Transcriptase Connection Subdomain of HIV-1 Gag-Pol Polyprotein Reduce Infectiousness of the Virus Progeny

HIV-1 progeny are released from infected cells as immature particles that are unable to infect new cells. Gag-Pol polyprotein dimerization via the reverse transcriptase connection domain (RTCDs) is pivotal for proper activation of the virus protease (PR protein) in an early event of the progeny virus maturation process. Thus, the RTCD is a potential therapeutic target for a broadly effective anti-HIV agent through impediment of virus maturation. In this study, human single-chain antibodies (HuscFvs) that bound to HIV-1 RTCD were generated using phage display technology. Computerized simulation guided the selection of the transformed Escherichia coli-derived HuscFvs that bound to the RTCD dimer interface. The selected HuscFvs were linked molecularly to human-derived-cell-penetrating peptide (CPP) to make them cell-penetrable (i.e., become transbodies). The CPP-HuscFvs/transbodies produced by a selected transformed E. coli clone were tested for anti-HIV-1 activity. CPP-HuscFvs of transformed E. coli clone 11 (CPP-HuscFv11) that presumptively bound at the RTCD dimer interface effectively reduced reverse transcriptase activity in the newly released virus progeny. Infectiousness of the progeny viruses obtained from CPP-HuscFv11-treated cells were reduced by a similar magnitude to those obtained from protease/reverse transcriptase inhibitor-treated cells, indicating anti-HIV-1 activity of the transbodies. The CPP-HuscFv11/transbodies to HIV-1 RTCD could be an alternative, anti-retroviral agent for long-term HIV-1 treatment.

Sialidase and Sialyltransferase Inhibitors: Targeting Pathogenicity and Disease

Sialidases (SAs) and sialyltransferases (STs), the enzymes responsible for removing and adding sialic acid to other glycans, play essential roles in viruses, bacteria, parasites, and humans. Sialic acid is often the terminal sugar on glycans protruding from the cell surface in humans and is an important component for recognition and cell function. Pathogens have evolved to exploit this and use sialic acid to either “cloak” themselves, ensuring they remain undetected, or as a mechanism to enable release of virus progeny. The development of inhibitors against SAs and STs therefore provides the opportunity to target a range of diseases. Inhibitors targeting viral, bacterial, or parasitic enzymes can directly target their pathogenicity in humans. Excellent examples of this can be found with the anti-influenza drugs Zanamivir (Relenza™, GlaxoSmithKline) and Oseltamivir (Tamiflu™, Roche and Gilead), which have been used in the clinic for over two decades. However, the development of resistance against these drugs means there is an ongoing need for novel potent and specific inhibitors. Humans possess 20 STs and four SAs that play essential roles in cellular function, but have also been implicated in cancer progression, as glycans on many cancer cells are found to be hyper-sialylated. Whilst much remains unknown about how STs function in relation to disease, it is clear that specific inhibitors of them can serve both as tools to gain a better understanding of their activity and form the basis for development of anti-cancer drugs. Here we review the recent developments in the design of SA and ST inhibitors against pathogens and humans.

Patterns of virus growth across the diversity of life

Although viruses in their natural habitats add up to less than 10% of the biomass, they contribute more than 90% of the genome sequences [1]. These viral sequences or ‘viromes’ encode viruses that populate the Earth’s oceans [2, 3] and terrestrial environments [4, 5], where their infections impact life across diverse ecological niches and scales [6, 7], including humans [8–10]. Most viruses have yet to be isolated and cultured [11–13], and surprisingly few efforts have explored what analysis of available data might reveal about their nature. Here, we compiled and analyzed seven decades of one-step growth and other data for viruses from six major families, including their infections of archaeal, bacterial and eukaryotic hosts [14–191]. We found that the use of host cell biomass for virus production was highest for archaea at 10%, followed by bacteria at 1% and eukarya at 0.01%, highlighting the degree to which viruses of archaea and bacteria exploit their host cells. For individual host cells, the yield of virus progeny spanned a relatively narrow range (10–1000 infectious particles per cell) compared with the million-fold difference in size between the smallest and largest cells. Furthermore, healthy and infected host cells were remarkably similar in the time they needed to multiply themselves or their virus progeny. Specifically, the doubling time of healthy cells and the delay time for virus release from infected cells were not only correlated (r = 0.71, p < 10−10, n = 101); they also spanned the same range from tens of minutes to about a week. These results have implications for better understanding the growth, spread and persistence of viruses in complex natural habitats that abound with diverse hosts, including humans and their associated microbes.

Viral mediated tethering to SEL1L facilitates ER-associated degradation of IRE1

The unfolded protein response (UPR) and endoplasmic reticulum (ER)-associated degradation (ERAD) are two essential components of the quality control system for proteins in the secretory pathway. When unfolded proteins accumulate in the ER, UPR sensors such as IRE1 induce the expression of ERAD genes, thereby increasing protein export from the ER to the cytosol and subsequent degradation by the proteasome. Conversely, IRE1 itself is an ERAD substrate, indicating that the UPR and ERAD regulate each other. Viruses are intracellular parasites that exploit the host cell for their own benefit. Cytomegaloviruses selectively modulate the UPR to take advantage of beneficial and inhibit detrimental effects on viral replication. We have previously shown that murine and human cytomegaloviruses express homologous proteins (M50 and UL50, respectively) that dampen the UPR at late times post infection by inducing IRE1 degradation. However, the degradation mechanism has remained uncertain. Here we show that the cytomegalovirus M50 protein mediates IRE1 degradation by the proteasome. M50-dependent IRE1 degradation can be blocked by pharmacological inhibition of p97/VCP or by genetic ablation of SEL1L, both of which are components of the ERAD machinery. SEL1L acts as a cofactor of the E3 ubiquitin ligase HRD1, while p97/VCP is responsible for the extraction of ubiquitylated proteins from the ER to the cytosol. We further show that M50 facilitates the IRE1-SEL1L interaction by binding to both, IRE1 and SEL1L. These results indicate that the viral M50 protein dampens the UPR by tethering IRE1 to SEL1L, thereby promoting its degradation by the ERAD machinery. IMPORTANCE Viruses infect cells of their host and force them to produce virus progeny. This can impose stress on the host cell and activate counter-regulatory mechanisms. Protein overload in the endoplasmic reticulum (ER) leads to ER stress and triggers the unfolded protein response, which in turn upregulates protein folding and increases the degradation of proteins in the ER. Previous work has shown that cytomegaloviruses interfere with the unfolded protein response by degrading the sensor molecule IRE1. Herein we demonstrate how the cytomegalovirus M50 protein exploits the ER-associated degradation machinery to dispose of IRE1. Degradation of IRE1 curbs the unfolded protein response and helps the virus to increase the synthesis of its own proteins and the production of virus progeny.

ICTV Virus Taxonomy Profile: Ovaliviridae

Ovaliviridae is a family of enveloped viruses with a linear dsDNA genome. The virions are ellipsoidal, and contain a multi-layered spool-like capsid. The viral genome is presumably replicated through protein priming by a putative DNA polymerase encoded by the virus. Progeny virions are released through hexagonal openings resulting from the rupture of virus-associated pyramids formed on the surface of infected cells. The only known host is a hyperthermophilic archaeon of the genus Sulfolobus . This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Ovaliviridae, which is available at ictv.global/report/ovaliviridae.

Recombination in enteroviruses is a ubiquitous event independent of sequence homology and RNA structure

ABSTRACTRecombination within RNA viruses is an important evolutionary process that can significantly influence virus fitness and has been repeatedly reported to compromise vaccine effectiveness. However, its precise mechanism is poorly understood. Here, we used an established poliovirus-based in vitro assay (CRE-REP) to investigate the molecular determinants of recombination and show that neither sequence identity, nor RNA structure, have any significant effect on recombination frequency. Since the CRE-REP assay is confined by the ability to detect infectious virus progeny, we utilized deep sequencing to study the recombinant genome population that arises early in infection and before any bottleneck of selection for viable progeny. We were able to detect and analyse hundreds of recombinants containing sequence insertions or deletions, or that were of wild type genome length. While we found higher diversity in recombination events than from CRE-REP assays, the analyses demonstrate no biases towards sequence or structure, in support of the CRE-REP assay findings. The results suggest that genome functionality and fitness are of greater importance in determining the identity of recombinants. These studies provide critical information that can improve our understanding of the recombination process, and consequently allow for the production of less recombinogenic and more stable vaccines.

The folate antagonist methotrexate diminishes replication of the coronavirus SARS-CoV-2 and enhances the antiviral efficacy of remdesivir in cell culture models

ABSTRACTThe search for successful therapies of infections with the coronavirus SARS-CoV-2 is ongoing. We tested inhibition of host cell nucleotide synthesis as a promising strategy to decrease the replication of SARS-CoV-2-RNA, thus diminishing the formation of virus progeny. Methotrexate (MTX) is an established drug for cancer therapy and to induce immunosuppression. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleotides. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutic concentrations around 1 μM, leading to more than 1000-fold reductions in virus progeny in Vero C1008 (Vero E6) as well as Calu-3 cells. Virus replication was more sensitive to equivalent concentrations of MTX than of the established antiviral agent remdesivir. MTX strongly diminished the synthesis of viral structural proteins and the amount of released virus RNA. Virus replication and protein synthesis were rescued by folinic acid (leucovorin) and also by inosine, indicating that purine depletion is the principal mechanism that allows MTX to reduce virus RNA synthesis. The combination of MTX with remdesivir led to synergistic impairment of virus replication, even at 300 nM MTX. The use of MTX in treating SARS-CoV-2 infections still awaits further evaluation regarding toxicity and efficacy in infected organisms, rather than cultured cells. Within the frame of these caveats, however, our results raise the perspective of a two-fold benefit from repurposing MTX for treating COVID-19. Firstly, its previously known ability to reduce aberrant inflammatory responses might dampen respiratory distress. In addition, its direct antiviral activity described here would limit the dissemination of the virus.SIGNIFICANCEMTX is one of the earliest cancer drugs to be developed, giving rise to seven decades of clinical experience. It is on the World Health Organization’s List of Essential Medicines, can be administered orally or parenterally, and its costs are at single digit € or $ amounts/day for standard treatment. In case of its successful further preclinical evaluation for treating SARS-CoV-2 infections, its repurposing to treat COVID-19 would thus be feasible, especially under low-resource conditions.Additional drugs exist to interfere with the synthesis of nucleotides, e.g. additional folate antagonists, inhibitors of GMP synthetase, or inhibitors of dihydroorotate dehydrogenase (DHODH). Such inhibitors have been approved as drugs for different purposes and might represent further therapeutic options against infections with SARS-CoV-2Remdesivir is currently the most established drug for treating COVID-19. Our results argue that MTX and remdesivir, even at moderate concentrations, can act in a synergistic fashion to repress virus replication to a considerably greater extent than either drug alone.COVID-19, in its severe forms, is characterized by pneumonia and acute respiratory distress syndrome, and additional organ involvements. These manifestations are not necessarily a direct consequence of virus replication and cytopathic effects, but rather a result of an uncontrolled inflammatory and immune response. Anti-inflammatory drugs such as glucocorticoids are thus being evaluated for treating COVID-19. However, this bears the risk of re-activating virus spread by suppressing a sufficient and specific immune response. In this situation, it is tempting to speculate that MTX might suppress both excessive inflammation as well as virus replication at the same time, thus limiting both the pathogenesis of pneumonia and also the spread of virus within a patient.

Anti-Neuraminidase Bioactives from Manggis Hutan (Garcinia celebica L.) Leaves: Partial Purification and Molecular Characterization

The neuraminidase enzyme (NA) from the influenza virus is responsible for the proliferation and infections of the virus progeny, prompting several efforts to discover and optimize effective neuraminidase inhibitors. The main aim of this study is to discover a new potential neuraminidase inhibitor that comes from Garcinia celebica leaves (GCL). The bioassay-guided isolation method was performed to obtain lead compounds. The binding interaction of the isolated compounds was predicted by using molecular docking studies. Friedeline (GC1, logP > 5.0), two lanastone derivatives (methyl-3α,23-dihydroxy-17,14-friedolanstan-8,14,24-trien-26-oat (GC2) and 24E-3a,9,23-trihydroxy-17,14-friedolanostan-14,24-dien-26-oate (GC3) with LogP > 5.0) and catechin (GC4, LogP = 1.4) were identified. The inhibitory potency of these four compounds on NA from C. perfringens and H1N1 was found to be as follows: GC4 > GC2 > GC3 > GC1. All compounds exhibited higher inhibitory activity towards C. perfringens NA compared to H1N1 NA. From the molecular docking results, GC4 favorably docked and interacted with Arg118, Arg371, Arg292, Glu276 and Trp178 residues, whilst GC2 interacted with Arg118, Arg371, Arg292, Ile222, Arg224 and Ser246. GC3 interacted with Tyr406 only. GC4 had potent NA inhibition with free energy of binding of −12 kcal/mol. In the enzyme inhibition study, GC4 showed the highest activity with an IC50 of 60.3 µM and 91.0 µM for C. perfringens NA and H1N1 NA—respectively.

Use of an Infectious cDNA Clone of Pepper Veinal Mottle Virus to Confirm the Etiology of a Disease in Capsicum chinense

The pepper cultivar Yellow Lantern, one of the spiciest pepper varieties, is a local germplasm of Capsicum chinense, cultivated exclusively on Hainan Island, China. However, this variety is susceptible to viral diseases that severely affect its production. In this study, we report that pepper veinal mottle virus (PVMV) is associated with foliar chlorosis and rugosity symptoms in Yellow Lantern. To verify this correlation, we constructed a full-length cDNA clone of a PVMV isolate named HNu. The virus progeny derived from the cDNA clone replicated and moved systemically in the pepper, inducing the same symptoms as those induced by PVMV-HNu in Yellow Lantern peppers in the field. The results support that PVMV-HNu is the causal agent of foliar chlorosis and rugosity disease in Yellow Lantern. This knowledge will help in the diagnosis and prevention of disease caused by PVMV. Furthermore, the cDNA clone serves as a reverse genetic tool to study the molecular pathogenesis of PVMV.

ADAPTATION OF HUMAN ROTAVIRUS STRAINS OF GROUP A TO THE REPRODUCTION IN PASSAGED CELL CULTURES

The incidence of rotavirus gastroenteritis in the world still has no tendency to reduction. The development of an effective vaccine would reduce or, in the future, even defeat this highly contagious dangerous disease. However, both in Russia and abroad there is still no developed technique for adapting and cultivating strains of the human rotavirus A that would stably produce a high "yield" of virus progeny in transplanted culture cells. The phenomenon of gene exchange for the segmented genome of rotavirus was used by foreign researchers to create the rotavirus vaccine using reassortant strains which are the result of joint cultivation of low-titer (1-2·106 virions per ml) human rotavirus strains and rotavirus strains of animals, such as monkey rotavirus SA-11 or Nebraska calf rotavirus diarrhea providing a relatively high "yield" of virus progeny (1·107-1·108). It is clear that such vaccine compositions will not be able to replace a full-fledged vaccine of human rotavirus strains of different serotypes, but they can be used for the time being as a solution to the problem. Ideally, a rotavirus vaccine is needed that includes the full set of G and P serotypes of rotaviruses circulating in the territory of their application. The paper describes an original technique for adaptation and cultivation of human rotaviruses of group A on the culture of transplantable cells developed by the authors. This technique allows 5·108 virions to be obtained per 1 ml of culture fluid. High-titer cultivated strains of human rotavirus that can be used as vaccine strains were obtained, as well as highly-active antigens for the construction of diagnostic test-systems.