Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short ...phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic ...repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Endogenous viral elements derived from nonretroviral RNA viruses have been described in various animal genomes. Whether they have a biological function, such as host immune protection against related ...viruses, is a field of intense study. Here, we investigated the repertoire of endogenous flaviviral elements (EFVEs) in
mosquitoes, the vectors of arboviruses such as dengue and chikungunya viruses. Previous studies identified three EFVEs from
cell lines and one from
cell lines. However, an in-depth characterization of EFVEs in wild-type mosquito populations and individual mosquitoes
has not been performed. We detected the full-length DNA sequence of the previously described EFVEs and their respective transcripts in several
and
populations from geographically distinct areas. However, EFVE-derived proteins were not detected by mass spectrometry. Using deep sequencing, we detected the production of PIWI-interacting RNA-like small RNAs, in an antisense orientation, targeting the EFVEs and their flanking regions
The EFVEs were integrated in repetitive regions of the mosquito genomes, and their flanking sequences varied among mosquito populations. We bioinformatically predicted several new EFVEs from a Vietnamese
population and observed variation in the occurrence of those elements among mosquitoes. Phylogenetic analysis of an
EFVE suggested that it integrated prior to the global expansion of the species and subsequently diverged among and within populations. The findings of this study together reveal the substantial structural and nucleotide diversity of flaviviral integrations in
genomes. Unraveling this diversity will help to elucidate the potential biological function of these EFVEs.
Endogenous viral elements (EVEs) are whole or partial viral sequences integrated in host genomes. Interestingly, some EVEs have important functions for host fitness and antiviral defense. Because mosquitoes also have EVEs in their genomes, characterizing these EVEs is a prerequisite for their potential use to manipulate the mosquito antiviral response. In the study described here, we focused on EVEs related to the
genus, to which dengue and Zika viruses belong, in individual
mosquitoes from geographically distinct areas. We show the existence
of flaviviral EVEs previously identified in mosquito cell lines, and we detected new ones. We show that EVEs have evolved differently in each mosquito population. They produce transcripts and small RNAs but not proteins, suggesting a function at the RNA level. Our study uncovers the diverse repertoire of flaviviral EVEs in
mosquito populations and contributes to an understanding of their role in the host antiviral system.
Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length ...has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.
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The life cycle of endogenous retroviruses (ERVs), also called long terminal repeat (LTR) retrotransposons, begins with transcription by RNA polymerase II followed by reverse transcription and ...re-integration into the host genome. While most ERVs are relics of ancient integration events, “young” proviruses competent for retrotransposition—found in many mammals, but not humans—represent an ongoing threat to host fitness. As a consequence, several restriction pathways have evolved to suppress their activity at both transcriptional and post-transcriptional stages of the viral life cycle. Nevertheless, accumulating evidence has revealed that LTR sequences derived from distantly related ERVs have been exapted as regulatory sequences for many host genes in a wide range of cell types throughout mammalian evolution. Here, we focus on emerging themes from recent studies cataloging the diversity of ERV LTRs acting as important transcriptional regulatory elements in mammals and explore the molecular features that likely account for LTR exaptation in developmental and tissue-specific gene regulation.
Solitary long terminal repeats (LTRs) have been frequently domesticated into gene regulatory elements in mammals. Here, Thompson et al. evaluate the features of LTRs that account for the prevalence of this phenomenon and highlight recent studies providing insight into the molecular mechanisms that influence the exaptation of LTRs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mechanisms of Virus Assembly Perlmutter, Jason D; Hagan, Michael F
Annual review of physical chemistry,
04/2015, Volume:
66, Issue:
1
Journal Article
Peer reviewed
Open access
Viruses are nanoscale entities containing a nucleic acid genome encased in a protein shell called a capsid and in some cases are surrounded by a lipid bilayer membrane. This review summarizes the ...physics that govern the processes by which capsids assemble within their host cells and in vitro. We describe the thermodynamics and kinetics for the assembly of protein subunits into icosahedral capsid shells and how these are modified in cases in which the capsid assembles around a nucleic acid or on a lipid bilayer. We present experimental and theoretical techniques used to characterize capsid assembly, and we highlight aspects of virus assembly that are likely to receive significant attention in the near future.
The viral reservoir represents a critical challenge for human immunodeficiency virus type 1 (HIV-1) eradication strategies. However, it remains unclear when and where the viral reservoir is seeded ...during acute infection and the extent to which it is susceptible to early antiretroviral therapy (ART). Here we show that the viral reservoir is seeded rapidly after mucosal simian immunodeficiency virus (SIV) infection of rhesus monkeys and before systemic viraemia. We initiated suppressive ART in groups of monkeys on days 3, 7, 10 and 14 after intrarectal SIVMAC251 infection. Treatment with ART on day 3 blocked the emergence of viral RNA and proviral DNA in peripheral blood and also substantially reduced levels of proviral DNA in lymph nodes and gastrointestinal mucosa as compared with treatment at later time points. In addition, treatment on day 3 abrogated the induction of SIV-specific humoral and cellular immune responses. Nevertheless, after discontinuation of ART following 24 weeks of fully suppressive therapy, virus rebounded in all animals, although the monkeys that were treated on day 3 exhibited a delayed viral rebound as compared with those treated on days 7, 10 and 14. The time to viral rebound correlated with total viraemia during acute infection and with proviral DNA at the time of ART discontinuation. These data demonstrate that the viral reservoir is seeded rapidly after intrarectal SIV infection of rhesus monkeys, during the 'eclipse' phase, and before detectable viraemia. This strikingly early seeding of the refractory viral reservoir raises important new challenges for HIV-1 eradication strategies.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV ...DNA-containing virus particles with a mean antiviral 50% effective concentration (EC
) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs
resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited
infection and viral replication in primary human hepatocytes with EC
values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies
The overall preclinical profile of NVR 3-778 predicts antiviral activity
and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.
Over the past few decades, a large number of studies have identified herpesvirus sequences from many mammalian species around the world. Among the different nonhuman primate species tested so far for ...cytomegaloviruses (CMVs), only a few were from the New World. Seeking to identify CMV homologues in New World monkeys (NWMs), we carried out molecular screening of 244 blood DNA samples from 20 NWM species from Central and South America. Our aim was to reach a better understanding of their evolutionary processes within the Platyrrhini parvorder. Using PCR amplification with degenerate consensus primers targeting highly conserved amino acid motifs encoded by the herpesvirus DNA polymerase gene, we characterized novel viral sequences from 12 species belonging to seven genera representative of the three NWM families. BLAST searches, pairwise nucleotide and amino acid sequence comparisons, and phylogenetic analyses confirmed that they all belonged to the
genus. Previously determined host taxa allowed us to demonstrate a good correlation between the distinct monophyletic clades of viruses and those of the infected primates at the genus level. In addition, the evolutionary branching points that separate NWM CMVs were congruent with the divergence dates of their hosts at the genus level. These results significantly expand our knowledge of the host range of this viral genus and strongly support the occurrence of cospeciation between these viruses and their hosts. In this respect, we propose that NWM CMV DNA polymerase gene sequences may serve as reliable molecular markers with which to infer Platyrrhini phylogenetics.
Investigating evolutionary processes between viruses and nonhuman primates has led to the discovery of a large number of herpesviruses. No study published so far on primate cytomegaloviruses has extensively studied New World monkeys (NWMs) at the subspecies, species, genus, and family levels. The present study sought to identify cytomegalovirus homologues in NWMs and to decipher their evolutionary relationships. This led us to characterize novel viruses from 12 of the 20 primate species tested, which are representative of the three NWM families. The identification of distinct viruses in these primates not only significantly expands our knowledge of the host range of this viral genus but also sheds light on its evolutionary history. Phylogenetic analyses and molecular dating of the sequences obtained support a virus-host coevolution.
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