2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology.

2A is an oligopeptide sequence that mediates a ribosome "skipping" effect and can mediate a co-translation cleavage of polyproteins. These sequences are widely distributed from insect to mammalian viruses and could act by accelerating adaptive capacity. These sequences have been used in many heterologous co-expression systems because they are versatile tools for cleaving proteins of biotechnological interest. In this work, we review and update the occurrence of 2A/2A-like sequences in different groups of viruses by screening the sequences available in the National Center for Biotechnology Information database. Interestingly, we reported the occurrence of 2A-like for the first time in 69 sequences. Among these, 62 corresponded to positive single-stranded RNA species, six to double stranded RNA viruses, and one to a negative-sense single-stranded RNA virus. The importance of these sequences for viral evolution and their potential in biotechnological applications are also discussed.

Design and in silico validation of polymerase chain reaction primers to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Accurate designing of polymerase chain reaction (PCR) primers targeting conserved segments in viral genomes is desirable for preventing false-negative results and decreasing the need for standardization across different PCR protocols. In this work, we designed and described a set of primers and probes targeting conserved regions identified from a multiple sequence alignment of 2341 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genomes from the Global Initiative on Sharing All Influenza Data (GISAID). We subsequently validated those primers and probes in 211,833 SARS-CoV-2 whole-genome sequences. We obtained nine systems (forward primer + reverse primer + probe) that potentially anneal to highly conserved regions of the virus genome from these analyses. In silico predictions also demonstrated that those primers do not bind to nonspecific targets for human, bacterial, fungal, apicomplexan, and other Betacoronaviruses and less pathogenic sub-strains of coronavirus. The availability of these primer and probe sequences will make it possible to validate more efficient protocols for identifying SARS-CoV-2.

Alternative primers to identify a range of apicomplexan parasites.

Here we present an alternative polymerase chain reaction (PCR) approach using 18S rDNA to identify apicomplexan parasites. A new primer set was designed and evaluated in silico and in vitro. This new PCR could detect some apicomplexan genera based on amplicon size and identify at least 45 species after sequencing.

Alternative PCR primers for genotyping of Brazilian WSSV isolates.

White spot syndrome virus (WSSV) is one of the major challenges faced by global shrimp farming in recent decades. The characterization of WSSV genetic variability has been used to determine viral dispersion and is a promising method to determine the association between genotype and virulence. The major variable regions that have been used as markers to differentiate the WSSV genomes include the VNTR loci inside ORF94, ORF75, ORF125, and insertions/deletions interspersing ORF14/15 and ORF23/24. The primers used to amplify these regions were described at least 10 years ago, but some of them do not work efficiently to identify new WSSV variants. The objective of this work was to develop improved PCR primers for WSSV genotyping based on sequence alignments that include new sequences described in recent years. We validated these new primers in a pilot study to verify the genetic variability of the WSSV in Rio Grande do Norte state (northeast Brazil), and efficiency was compared to that of other previously described primers. We confirmed that the primers we developed were more efficient for genotype Brazilian WSSV isolates, enabling us to genotype a larger number of samples. In addition, our results also introduce new data about the genetic characterization of the WSSV isolates that occur in the northeastern region of Brazil.

Evolutionary origin of 2A-like sequences in Totiviridae genomes.

In recent years there has been a significant increase in the number of new species potentially belonging to the Totiviridae family. Most of these new viruses have not yet been covered by the Committee on Taxonomy of Viruses (ICTV) official classification. In this study, a phylogenetic analysis including new sequences of Totiviridae candidates revealed a clade including Giardiavirus and a great diversity of new totiviruses, which infect arthropods, protozoa and mollusc. This expanded Giardiavirus clade comprises two monophyletic groups, one of them including Giardia lamblia virus (GLV) grouped with viruses that infect arthropods and vertebrates (GLV-like group), and the other includes the previously proposed Artivirus group (IMNV-like group). A screening of the members of the GLV-like group in search of genomic elements already described in IMNV-like group revealed the existence of sites with a high propensity to become 2 A-like oligopeptides, mainly in a specific subgroup of arthropod viruses, suggesting that these viruses preserved ancestral characteristics. The existence of these "pseudo 2 A-sites" associated to phylogenetic reconstruction indicates that these sequences appear at a decisive stage for viral evolution. If they are changed to functional 2 A-like sequences, an irreversible route to increase the genome complexity will be initiated.

A seminested PCR assay for detection and typing of human papillomavirus based on E1 gene sequences.

HPV infection is considered one of the leading causes of cervical cancer in the world. To date, more than 180 types of HPV have been described and viral typing is critical for defining the prognosis of cancer. In this work, a seminested PCR which allow fast and inexpensively detection and typing of HPV is presented. The system is based on the amplification of a variable length region within the viral gene E1, using three primers that potentially anneal in all HPV genomes. The amplicons produced in the first step can be identified by high resolution electrophoresis or direct sequencing. The seminested step includes nine specific primers which can be used in multiplex or individual reactions to discriminate the main types of HPV by amplicon size differentiation using agarose electrophoresis, reducing the time spent and cost per analysis.

"In-House" Production of Single Stranded Oligodeoxyribonucleotides.

The most widely used technique for the production of DNA aptamers/oligonucleotides is chemical synthesis. Despite its effectiveness, this technique cannot be performed "in house", making the user fully dependent on a supplier. In this work, we present a simplified method by which it is possible to enzymatically produce DNA aptamers "in house". This new method uses the rolling circle replication followed by a unique cleavage step using the SchI endonuclease. Potentially, any oligonucleotide can be produced by the enzymatic method proposed in this study. To illustrate, we present the production of three variations of the 31-TBA aptamer, a single stranded DNA which has anticoagulant action.

An alternative nested-PCR assay for the detection of Toxoplasma gondii strains based on GRA7 gene sequences.

Toxoplasma gondii is a widespread parasite able to infect virtually any nucleated cells of warm-blooded hosts. In some cases, T. gondii detection using already developed PCR primers can be inefficient in routine laboratory tests, especially to detect atypical strains. Here we report a new nested-PCR protocol able to detect virtually all T. gondii isolates. Analyzing 685 sequences available in GenBank, we determine that GRA7 is one of the most conserved genes of T. gondii genome. Based on an alignment of 85 GRA7 sequences new primer sets that anneal in the highly conserved regions of this gene were designed. The new GRA7 nested-PCR assay providing sensitivity and specificity equal to or greater than the gold standard PCR assays for T. gondii detection, that amplify the B1 sequence or the repetitive 529bp element.

New insights about ORF1 coding regions support the proposition of a new genus comprising arthropod viruses in the family Totiviridae.

Analyzing the positions of 2A-like polypeptide cleavage sites in all available genomes of arthropod totiviruses we propose the limits of all ORF1 coding sequences and observed that two proteins previously predicted in infectious myonecrosis virus genome are unique in the arthropod totiviruses group. A putative protein cleavage site upstream the major capsid protein was also identified only in these genomes. In addition, protein models generated using ab initio and threading approaches revealed conserved structures possibly related to formation of viral protrusions and RNA packaging, clarifying the mechanisms involved in the extracellular transmission. These data appoints that the group formed by arthropod totiviruses are sufficient distinctive to be clustered in new genus belonging to the Totiviridae family, in agreement with previous phylogenetic analysis.

Analysis of new isolates reveals new genome organization and a hypervariable region in infectious myonecrosis virus (IMNV).

Infectious myonecrosis virus (IMNV) has been the cause of many losses in shrimp farming since 2002, when the first myonecrosis outbreak was reported at Brazilian's northeast coast. Two additional genomes of Brazilian IMNV isolates collected in 2009 and 2013 were sequenced and analyzed in the present study. The sequencing revealed extra 643 bp and 22 bp, at 5' and 3' ends of IMNV genome respectively, confirming that its actual size is at least 8226 bp long. Considering these additional sequences in genome extremities, ORF1 can starts at nt 470, encoding a 1708 aa polyprotein. Computational predictions reveal two stem loops and two pseudoknots in the 5' end and a putative stem loop and a slippery motif located at 3' end, indicating that these regions can be involved in the start and termination of translation. Through a careful phylogenetic analysis, a higher genetic variability among Brazilian isolates could be observed, comparing with Indonesian IMNV isolates. It was also observed that the most variable region of IMNV genome is located in the first half of ORF1, coinciding with a region which probably encodes the capsid protrusions. The results presented here are a starting point to elucidate the viral's translational regulation and the mechanisms involved in virulence.

In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae.

BACKGROUND: The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively. RESULTS: A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM. CONCLUSION: In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.

Infectious hypodermal and hematopoietic necrosis virus from Brazil: Sequencing, comparative analysis and PCR detection.

A 3739 nucleotide fragment of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) from Brazil was amplified and sequenced. This fragment contains the entire coding sequences of viral proteins, the full 3' untranslated region (3'UTR) and a partial sequence of 5' untranslated region (5'UTR). The genome organization of IHHNV revealed the three typical major coding domains: a left ORF1 of 2001 bp that codes NS1, a left ORF2 (NS2) of 1091 bp that codes NS2 and a right ORF3 of 990 bp that codes VP. Nucleotide and amino acid sequences of the three viral proteins were compared with putative amino acid sequences of viruses reported from different regions. Comparisons among genomes from different geographic locations reveal 31 nucleotide regions that are 100% similar, distributed throughout the genome. An analysis of secondary structure of UTR regions, revealed regions with high probability to form hairpins, that may be involved in mechanisms of viral replication. Additionally, a maximum likelihood analysis indicates that Brazilian IHHNV belongs to lineage III, in the infectious IHHNV group, and is clustered with IHHNV isolates from Hawaii, China, Taiwan, Vietnam and South Korea. A new nested PCR targeting conserved nucleotide regions is proposed to detect IHHNV.

FEZ1 interacts with CLASP2 and NEK1 through coiled-coil regions and their cellular colocalization suggests centrosomal functions and regulation by PKC.

FEZ1 was initially described as a neuronal protein that influences axonal development and cell polarization. CLASP2 and NEK1 proteins are present in a centrosomal complex and participate in cell cycle and cell division mechanisms, but their functions were always described individually. Here, we report that NEK1 and CLASP2 colocalize with FEZ1 in a perinuclear region in mammalian cells, and observed that coiled-coil interactions occur between FEZ1/CLASP2 and FEZ1/NEK1 in vitro. These three proteins colocalize and interact with endogenous gamma-tubulin. Furthermore, we found that CLASP2 is phosphorylated and interacts with active PKC isoforms, and that FEZ1/CLASP2 colocalization is inhibited by PMA treatment. Our results provide evidence that these three proteins cooperate in centrosomal functions and open new directions for future studies.

Human FEZ1 has characteristics of a natively unfolded protein and dimerizes in solution.

The fasciculation and elongation protein Zeta 1 (FEZ1) is the mammalian orthologue of the Caenorhabditis elegans protein UNC-76, which is necessary for axon growth. Human FEZ1 interacts with Protein Kinase C (PKC) and several regulatory proteins involved in functions ranging from microtubule associated transport to transcriptional regulation. Theoretical prediction, circular dichroism, fluorescence spectroscopy, and limited proteolysis of recombinant FEZ1 suggest that it contains disordered regions, especially in its N-terminal region, and that it may belong to the group of natively unfolded proteins. Small angle X-ray scattering experiments indicated a mainly disordered conformation, proved that FEZ1 is a dimer of elongated shape and provided overall dimensional parameters for the protein. In vitro pull down experiments confirmed these results and demonstrated that dimerization involves the N-terminus. Ab-initio 3D low resolution models of the full-length conformation of the dimeric constructs 6xHis-FEZ1(1-392) and 6xHis-FEZ1(1-227) were obtained. Furthermore, we performed in vitro phosphorylation assays of FEZ1 with PKC. The phosphorylation occurred mainly in its C-terminal region, and does not cause any significant conformational changes, but nonetheless inhibited its interaction with the FEZ1 interacting domain of the protein CLASP2 in vitro. The C terminus of FEZ1 has been reported to bind to several interacting proteins. This suggests that FEZ1 binding and transport function of interacting proteins may be subject to regulation by phosphorylation.

Over-expression of GFP-FEZ1 causes generation of multi-lobulated nuclei mediated by microtubules in HEK293 cells.

FEZ1 (Fasciculation and elongation protein zeta 1) is an ortholog of the Caenorhabditis elegans protein UNC-76, involved in neuronal development and axon outgrowth, in that worm. Mammalian FEZ1 has already been reported to cooperate with PKC-zeta in the differentiation and polarization of PC12 neuronal cells. Furthermore, FEZ1 is associated with kinesin 1 and JIP1 to form a cargo-complex responsible for microtubule based transport of mitochondria along axons. FEZ1 can also be classified as a hub protein, since it was reported to interact with over 40 different proteins in yeast two-hybrid screens, including at least nine nuclear proteins. Here, we transiently over-expressed GFP-FEZ1full in human HEK293 and HeLa cells in order to study the sub-cellular localization of GFP-FEZ1. We observed that over 40% of transiently transfected cells at 3 days post-transfection develop multi-lobulated nuclei, which are also called flower-like nuclei. We further demonstrated that GFP-FEZ1 localizes either to the cytoplasm or the nuclear fraction, and that the appearance of the flower-like nuclei depends on intact microtubule function. Finally, we show that FEZ1 co-localizes with both, alpha- and especially with gamma-tubulin, which localizes as a centrosome like structure at the center of the multiple lobules. In summary, our data suggest that FEZ1 has an important centrosomal function and supply new mechanistic insights to the formation of flower-like nuclei, which are a phenotypical hallmark of human leukemia cells.