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Highlights • Accumulating evidence indicates that RNAi is an accessory antiviral mechanism in mammals. • Humans have at least three antiviral defense mechanisms: innate immunity, RNAi and adaptive immunity. • Invertebrates also have developed multiple layers of antiviral defense. RNA interference (RNAi) was discovered in plants where it functions as the main antiviral pathway and this antiviral role was subsequently extended to invertebrates. But it remained hotly debated whether RNAi fulfils a similar role in mammals that already have a potent innate immune system based on interferon and an elaborate adaptive immune system. On the one hand, mammalian cells do encode most of the RNAi machinery, but this could be used exclusively to control cellular gene expression via micro RNAs (miRNAs). But on the other hand, virus-derived small interfering RNAs, the hallmark of RNAi involvement, could not be readily detected upon virus infection of mammalian cells. However, recent studies have indicated that these signature molecules are generated in virus-infected embryonic cell types of mammals and that viruses actively suppress such responses by means of potent RNAi suppressor proteins. Thus, the tide seems to be changing in favor of RNAi as accessory antiviral defense mechanism in humans. Intriguingly, recent studies indicate that insects have also developed an additional innate immune system that collaborates with the RNAi response in the fight against invading viral pathogens. Thus, the presence of multiple antiviral response mechanisms seems standard outside the plant world and we will specifically discuss the interactions between these antiviral programs. [ABSTRACT FROM AUTHOR]

Summary Current drug therapies effectively suppress HIV-1 replication but do not inactivate the provirus that persists in latent reservoirs. Recent studies have found that the guide RNA (gRNA)-directed CRISPR/Cas9 system can be used for sequence-specific attack on this proviral DNA. Although potent inhibition of virus replication was reported, HIV-1 can escape from a single antiviral gRNA by mutation of the target sequence. Here, we demonstrate that combinations of two antiviral gRNAs delay viral escape, and identify two gRNA combinations that durably block virus replication. When viral escape is prevented, repeated Cas9 cleavage leads to saturation of major mutations in the conserved target sequences that encode critical proteins. This hypermutation coincides with the loss of replication-competent virus as scored in sensitive co-cultures with unprotected cells, demonstrating complete virus inactivation. These results provide a proof-of-principle that HIV-1-infected cells can be functionally cured by dual-gRNA CRISPR/Cas9 treatment. [ABSTRACT FROM AUTHOR]

Summary Background To define HBsAg-mutations correlated with different serum HBV-DNA levels in HBV chronically-infected drug-naive patients. Methods This study included 187 patients stratified into the following ranges of serum HBV-DNA:12–2000 IU/ml, 2000–100,000 IU/ml, and >100,000 IU/ml. HBsAg-mutations were associated with HBV-DNA levels by applying a Bayesian-Partitional-Model and Fisher-exact test. Mutant and wild-type HBV genotype-D genomes were expressed in Huh7 cells and HBsAg-production was determined in cell-supernatants at 3 days-post-transfection. Results Specific HBsAg-mutations (M197T,-S204N-Y206C/H-F220L) were significantly correlated with serum HBV-DNA <2000 IU/ml (posterior-probability>90%, P < 0.05). The presence of Y206C/H and/or F220L was also associated with lower median (IQR) HBsAg-levels and lower median (IQR) transaminases (for HBsAg:250[115–840] IU/ml for Y206C/H and/or F220L versus 4300[640–11,838] IU/ml for wild-type, P = 0.023; for ALT:28[21–40] IU/ml versus 53[34–90] IU/ml, P < 0.001). These mutations were localized in the HBsAg C-terminus, known to be involved in virion and/or HBsAg secretion. The co-occurrence of Y206C + F220L was found significant by cluster-analysis, ( P = 0.02). In addition, in an in-vitro model Y206C + F220L determined a 2.8–3.3 fold-reduction of HBsAg-amount released in supernatants compared to single mutants and wt (Y206C + F220L = 5,679 IU/ml; Y206H = 16,305 IU/ml; F220L = 18,368 IU/ml; Y206C = 18,680 IU/ml; wt = 14,280 IU/ml, P < 0.05). Conclusions Specific HBsAg-mutations (compartmentalized in the HBsAg C-terminus) correlated with low-serum HBV-DNA and HBsAg-levels. These findings can be important to understand mechanisms underlying low HBV replicative potential including the inactive-carrier state. [ABSTRACT FROM AUTHOR]

Eukaryotic cells and several DNA viruses encode miRNAs to regulate the expression of specific target genes. It has been controversial whether RNA viruses can encode such miRNAs as miRNA excision may lead to cleavage of the viral RNA genome. We will focus on the retrovirus family, HIV-1 in particular, and discuss the production of virus-encoded miRNAs and their putative function in the viral replication cycle. An intricate scenario of multi-layer virus–host interactions becomes apparent with small RNAs as the regulatory molecules. [ABSTRACT FROM AUTHOR]

Highlights: [•] HIV-1 latency. [•] T lymphocytes. [•] Establishment reservoir. [•] Molecular latency mechanisms. [Copyright &y& Elsevier]

Summary: Introduction: The identification of novel reverse-transcriptase (RT) drug-resistance mutations is critical in predicting the probability of success to anti-HBV treatment. Furthermore, due to HBV-RT/HBsAg gene-overlap, they can have an impact on HBsAg-detection and quantification. Methods: 356 full-length HBV-RT sequences from 197 drug-naive patients and 159 patients experiencing virological-breakthrough to nucleoside/nucleotide-analogs (NUCs) were analyzed. Mutants and wild-type HBs-antigens were expressed in HuH7-hepatocytes and quantified in cell-supernatants and cell-lysates by Architect HBsAg-assay. Results: Ten novel RT-mutations (rtN53T-rtS78T-rtS85F-rtS135T-rtA181I-rtA200V-rtK212Q-rtL229V/F-rtM309K) correlated with specific NUC-treatments and classical drug-resistance mutations on divergent evolutionary pathways. Some of them reduced RT-binding affinity for anti-HBV drugs and altered S-antigen structure. Indeed, rtS78T (prevalence: 1.1% in drug-naïve and 12.2% in adefovir-failing patients) decreased the RT-affinity for adefovir more than the classical adefovir-resistance mutations rtA181 T/V (WT:−9.63 kcal/mol, rtA181T:−9.30 kcal/mol, rtA181V:−7.96 kcal/mol, rtS78T:−7.37 kcal/mol). Moreover, rtS78T introduced a stop-codon at HBsAg-position 69, and completely abrogated HBsAg-quantification in both supernatants and cell-lysates, indicating an impaired HBsAg-secretion/production. Furthermore, the HBsAg-mutation sP217L, silent in RT, significantly correlated with M204V/I-related virological-breakthrough and increased HBsAg-quantification in cell-lysate. Conclusions: Mutations beyond those classically known can affect drug-binding affinity of mutated HBV-RT, and may have potential effects on HBsAg. Their cumulative effect on resistance and HBV-pathogenicity indicates the importance of preventing therapeutic failures. [Copyright &y& Elsevier]

Cancer and human immunodeficiency virus (HIV) are both scary things to have in your body, but a new treatment is successfully using the latter against the former. Recent news reports, among others in the New York Times, talked about this new cure for leukemia by using HIV. This mini-review puts this news in perspective and provides a broader view as there appear to be several areas where clinical research on HIV and leukemia seem to connect. The topics covered range from antiviral gene therapy approaches using HIV-based lentiviral vectors to the risk of leukemia induction by these integrating vectors, and from an anti-leukemia transplantation strategy that turned out to provide a functional cure for HIV, to novel vaccination approaches. [ABSTRACT FROM AUTHOR]

Entry of human immunodeficiency virus type 1 (HIV-1) into cells is mediated by attachment of the envelope glycoproteins, gp120 and gp41, to the CD4 receptor and a chemokine receptor (CCR5 or CXCR4) and subsequent fusion of viral and cellular membranes. Several steps of the entry process can be targeted by drugs. Receptor antagonists prevent virus attachment and fusion inhibitors block conformational changes that are required for membrane fusion. The T20 peptide (Enfuvirtide, Fuzeon), which is homologous to part of the gp41-encoded fusion machinery, is the only clinically approved fusion inhibitor, but over the last decade new generations of T20-like peptides have been developed with improved potency and stability, as well as fusion inhibitors that target alternative gp41 domains. Here, we will review the field of HIV-1 fusion inhibitors. [Copyright &y& Elsevier]

Abstract: The discovery of RNA interference (RNAi), an evolutionary conserved gene silencing mechanism that is triggered by double stranded RNA, has led to tremendous efforts to use this technology for basic research and new RNA therapeutics. RNAi can be induced via transfection of synthetic small interfering RNAs (siRNAs), which results in a transient knockdown of the targeted mRNA. For stable gene silencing, short hairpin RNA (shRNA) or microRNA (miRNA) constructs have been developed. In mammals and humans, the natural RNAi pathway is triggered via endogenously expressed miRNAs. The use of modified miRNA expression cassettes to elucidate fundamental biological questions or to develop therapeutic strategies has received much attention. Viral vectors are particularly useful for the delivery of miRNA genes to specific target cells. To date, many viral vectors have been developed, each with distinct characteristics that make one vector more suitable for a certain purpose than others. This review covers the recent progress in miRNA-based gene-silencing approaches that use viral vectors, with a focus on their unique properties, respective limitations and possible solutions. Furthermore, we discuss a related topic that involves the insertion of miRNA-target sequences in viral vector systems to restrict their cellular range of gene expression. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation. [Copyright &y& Elsevier]

Abstract: RNA silencing or RNAi interference (RNAi) serves as an innate antiviral mechanism in plants, fungi and animals. Human viruses, like plant viruses, encode suppressor proteins or RNAs that block or modulate the RNAi pathway. This review summarizes the mechanisms by which pathogenic human viruses affect the RNAi pathway. Furthermore, some applications of the viral RNAi suppressor functions and the consequences for antiviral therapeutic strategies that are based on RNAi are discussed. [Copyright &y& Elsevier]

Abstract: Reverse transcription is an essential step in the retroviral life cycle, as it converts the genomic RNA into DNA. In this review, we describe recent developments concerning the initiation step of this complex, multi-step reaction. During initiation of reverse transcription, a cellular tRNA primer is placed onto a complementary sequence in the viral genome, called the primer binding site or PBS. The viral enzyme reverse transcriptase (RT) recognizes this RNA–RNA complex, and catalyzes the extension of the 3′ end of the tRNA primer, with the viral RNA (vRNA) acting as template. The initiation step is highly specific and most retroviruses are restricted to the use of the cognate, self-tRNA primer. Human immunodeficiency virus type 1 (HIV-1) uses the cellular tRNALys,3 molecule as primer for reverse transcription. No spontaneous switches in tRNA usage by HIV-1 or other retroviruses have been described and attempts to change the identity of the tRNA primer were unsuccessful in the past. These observations indicate that the virus strongly prefers the self-primer, suggesting that a very specific mechanism for primer selection must exist. Indeed, tRNA primers are selectively packaged into virus particles, are specifically recognized by RT and are placed onto the viral RNA genome via base pairing to the PBS and other sequence motifs, thus rendering a specific initiation complex. Analysis of this critical step in the viral life cycle may result in the discovery of novel antiviral drugs in the battle against HIV/AIDS. [Copyright &y& Elsevier]

The article reports on the discovery that plants and animals have thousands of genes that encode non-protein-coding (nc)RNAs opening a new vista on RNA-mediated biology. NcRNAs include rRNA, tRNA, small nuclear (sn)RNA, small nucleolar (sno)RNAs, micro-(mi)RNAs and some of lesser known RNAs such as vault RNAs, Y RNAs, repeat-associated small interfering (rasi)-RNAs and PIWI-interacting (pi)RNAs.

Presents evidence that the full-length 5'-untranslated region of the HIV-1 RNA genome also adopts the long distance interaction and branched multiple hairpin conformations. Identification of the U5-AUG long distance interaction; Structure probing; Influence of U5-AUG duplex on the translation of the Gag protein.

In a search for natural proteins with anti-HIV activity, we screened a large set of purified proteins from bovine milk and peptide fragments thereof. Because several charged proteins and peptides are known to inhibit the process of virus entry, we selected proteins with an unusual charge composition or hydrophobicity profile. In contrast with some chemically modified (strongly negative) milk proteins, unmodified αs2-, β- and κ-casein, as well as several negatively and positively charged fragments thereof, did not show significant inhibition of virus replication. In fact, HIV-1 replication was elevated in the presence of β-casein or amphiphilic fragments thereof. Bovine lactoferrin (bLF), a milk protein of 80 kDa, showed considerable inhibitory activity against HIV-1 with an IC50 of 0.4 μM. Modest inhibition was obtained with lactoferricin, a highly positively charged loop domain of bLF, indicating that other domains within the native bLF protein may also be required for inhibition. bLF blocked HIV-1 variants that use either the CXCR4 or the CCR5 coreceptor. In order to obtain further insight into the mechanism of action of this antiviral protein, we selected a bLF-resistant HIV-1 variant. The bLF-resistance phenotype is mediated by the viral envelope protein, which contains two interesting mutations that have previously been associated with an altered virus-host interaction and a modified receptor–coreceptor interaction. These results demonstrate that bLF targets the HIV-1 entry process. [Copyright &y& Elsevier]

The HIV latent reservoir forms the major hurdle to an HIV cure. The discovery of CD32 as marker of this reservoir has aroused much interest, but subsequent reports have challenged this finding. Here, we observe a positive correlation between the percentages of CD32+ cells among CD4+ T cells of aviremic cART-treated, HIV-infected individuals and their HIV DNA loads in peripheral blood. Moreover, optimization of the CD32+CD4+ T cell purification protocol reveals prominent enrichment for HIV DNA (mean, 292-fold) in these cells. However, no enrichment for HIV RNA is observed in CD32+CD4+ cells, yielding significantly reduced HIV RNA/DNA ratios. Furthermore, HIV proviruses in CD32+CD4+ cells can be reactivated ex vivo to produce virus, strongly suggesting that these cells support HIV transcriptional latency. Our results underscore the importance of isolating pure, bona fide CD32+CD4+ T cells for future studies and indicate that CD32 remains a promising candidate marker of the HIV reservoir. • Percentages of CD32+ cells among CD4+ T cells correlate with HIV DNA loads in PBMC • High enrichment for HIV DNA in purified CD32+CD4+ cells from cART-treated patients • HIV transcription level per provirus is reduced in patient-derived CD32+CD4+ cells • HIV proviruses in purified CD32+CD4+ cells can be reactivated ex vivo to produce virus CD32a was recently proposed to mark the HIV reservoir, but this finding was subsequently challenged. By using a sequential cell-sorting protocol to purify bona fide CD32+CD4+ cells, Darcis et al. demonstrate HIV DNA enrichment and ex vivo reactivation-mediated virus production in these cells, reinforcing CD32 as an HIV reservoir marker. [ABSTRACT FROM AUTHOR]