Eytan Herzig

MicroRNA‐mediated regulation of p21 and TASK1 cellular restriction factors enhances HIV‐1 infection

MicroRNAs (miRNAs) are short non‐coding RNAs that play a central role in the regulation of gene expression by binding to target mRNAs. Several studies have revealed alterations in cellular miRNA profiles following HIV‐1 infection, mostly for miRNAs involved in inhibiting viral infection. These miRNA expression modifications might also serve to block the innate HIV‐1 inhibition mechanism. As a result, it is expected that during HIV‐1 infection miRNAs target genes that hinder or prevent the progression of the HIV‐1 replication cycle. One of the major sets of genes known to inhibit the progression of HIV‐1 infection are cellular restriction factors. In this study, we identified a direct miRNA target gene that modulates viral spread in T‐lymphocytes and HeLa‐CCR5 cell lines. Following infection, let‐7c, miR‐34a or miR‐124a were upregulated, and they targeted and downregulated p21 and TASK1 (also known as CDKN1A and KCNK3, respectively) cellular proteins. This eventually led to increased virion release and higher copy number of viral genome transcripts in infected cells. Conversely, by downregulating these miRNAs, we could suppress viral replication and spread. Our data suggest that HIV‐1 exploits the host miRNA cellular systems in order to block the innate inhibition mechanism, allowing a more efficient infection process.

dUTPase: the frequently overlooked enzyme encoded by many retroviruses

Retroviruses are among the best studied viruses in last decades due to their pivotal involvement in cellular processes and, most importantly, in causing human diseases, most notably—acquired immunodeficiency syndrome (AIDS) that is triggered by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively). Numerous studied were conducted to understand the involvement of the three cardinal retroviral enzymes, reverse transcriptase, integrase and protease, in the life cycle of the viruses. These studies have led to the development of many inhibitors of these enzymes as anti-retroviral specific drugs that are used for routine treatments of HIV/AIDS patients. Interestingly, a fourth virus-encoded enzyme, the deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is also found in several major retroviral groups. The presence and the importance of this enzyme to the life cycle of retroviruses were usually overlooked by most retrovirologists, although the occurrence of dUTPases, particularly in beta-retroviruses and in non-primate retroviruses, is known for more than 20 years. Only more recently, retroviral dUTPases were brought into the limelight and were shown in several cases to be essential for viral replication. Therefore, it is likely that future studies on this enzyme will advance our knowledge to a level that will allow designing novel, specific and potent anti-dUTPase drugs that are effective in combating retroviral diseases. The aim of this review is to give concise background information on dUTPases in general and to summarize the most relevant data on retroviral dUTPases and their involvement in the replication processes and pathogenicity of the viruses, as well as in possibly-associated human diseases.

The dUTPase-related gene of bovine immunodeficiency virus is critical for viral replication, despite the lack of dUTPase activity of the encoded protein

BackgroundDeoxyuridine 5′-triphosphate nucleotide-hydrolases (dUTPases) are essential for maintaining low intra-cellular dUTP/dTTP ratios. Therefore, many viruses encode this enzyme to prevent dUTP incorporation into their genomes instead of dTTP. Among the lentiviruses, the non-primate viruses express dUTPases. In bovine immunodeficiency virus (BIV), the putative dUTPase protein is only 74 residues-long, compared to ~130 residues in other lentiviruses.ResultsIn this study, the recombinant BIV dUTPase, as well as infectious wild-type (WT) BIV virions, were shown to lack any detectable dUTPase activity. Controls of recombinant dUTPase from equine infectious anemia virus (EIAV) or of EIAV virions showed substantial dUTPase activities. To assess the importance of the dUTPase to BIV replication, we have generated virions of WT BIV or BIV with mutations in the dUTPase gene. The two mutant viral dUTPases were the double mutant D48E/N57S (in the putative enzyme active site and its vicinity) and a deletion of 36 residues. In dividing Cf2Th cells and under conditions where the WT virus was infectious and generated progeny virions, both mutant viruses were defective, as no progeny viruses were generated. Analyses of the integrated viral cDNA showed that cells infected with the mutant virions carry in their genomic DNA levels of integrated BIV DNA that are comparable to those in WT BIV-infected cells.ConclusionsThe herby presented results show that the two BIV mutants with the modified dUTPase gene could infect cells, as viral cDNA was synthesized and integrated into the host cell DNA. However, no virions were generated by cells infected by these mutants. The most likely explanation is that either the integrated cDNA of the mutants is defective (due to potential multiple mutations, introduced during reverse-transcription) or that the original dUTPase mutations have led to severe blocks in viral replication at steps post integration. These results emphasize the importance of the dUTPase-related sequence to BIV replication, despite the lack of any detectable catalytic activity.

The removal of RNA primers from DNA synthesized by the reverse transcriptase of the retrotransposon Tf1 is stimulated by Tf1 integrase

ABSTRACT The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RTs RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RTs activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposons reverse transcription process.

Substrate variations that affect the nucleic acid clamp activity of reverse transcriptases

We have recently shown that reverse transcriptases (RTs) perform template switches when there is a very short (two‐nucleotide) complementarity between the 3′ ends of the primer (donor) strand and the DNA or RNA template acceptor strands [Oz‐Gleenberg et al. (2011) Nucleic Acids Res39, 1042–1053]. These dinucleotide pairs are stabilized by RTs that are capable of ‘clamping’ together the otherwise unstable duplexes. This RT‐driven stabilization of the micro‐homology sequence promotes efficient DNA synthesis. In the present study, we have examined several factors associated with the sequence and structure of the DNA substrate that are critical for the clamp activity of RTs from human immunodeficiency virus type 1 (HIV‐1), murine leukemia virus (MLV), bovine immunodeficiency virus (BIV) and the long terminal repeat retrotransposon Tf1. The parameters studied were the minimal complementarity length between the primer and functional template termini that sustains stable clamps, the effects of gaps between the two template strands on the clamp activity of the tested RTs, the effects of template end phosphorylations on the RT‐associated clamp activities, and clamp activity with a long ‘hairpin’ double‐stranded primer comprising both the primer and the complementary non‐functional template strands. The results show that the substrate conditions for clamp activity of HIV‐1 and MLV RTs are more stringent, while Tf1 and BIV RTs show clamp activity under less rigorous substrate conditions. These differences shed light on the dissimilarities in catalytic activities of RTs, and suggest that clamp activity may be a potential new target for anti‐retroviral drugs.

Template-independent DNA synthesis activity associated with the reverse transcriptase of the long terminal repeat retrotransposon Tf1

Reverse transcriptases (RTs) possess a non‐templated addition (NTA) activity while synthesizing DNA with blunt‐ended DNA primer/templates. Interestingly, the RT of the long terminal repeat retrotransposon Tf1 has an NTA activity that is substantially higher than that of HIV‐1 or murine leukemia virus RTs. By performing steady state kinetics, we found that the differences between the NTA activities of Tf1 and HIV‐1 RTs can be explained by the substantially lower KM value for the incoming dNTP of Tf1 RT (while the differences between the apparent kcat values of these two RTs are relatively small). Furthermore, the KM values, calculated for both RTs with the same dNTP, are much lower for the template‐dependent synthesis (TDS) than those of NTA. However, TDS of HIV‐1 RT is higher than that of Tf1 RT. The overall relative order of the apparent kcat/KM values for dATP is: HIV‐1 RT (TDS) > Tf1 RT (TDS) >> Tf1 RT (NTA) > HIV‐1 RT (NTA). Under the employed conditions, Tf1 RT can add up to seven nucleotides to the blunt‐ended substrate, while the other RTs add mostly a single nucleotide. The NTA activity of Tf1 RT is restricted to DNA primers. Furthermore, the NTA activity of Tf1 and HIV‐1 RTs is suppressed by ATP, as it competes with the incoming dATP (although ATP is not incorporated by the NTA activity of the RTs). The unusually high NTA activity of Tf1 RT can explain why, after completing cDNA synthesis, the in vivo generated Tf1 cDNA has relatively long extra sequences beyond the highly conserved CA at its 3′‐ends.

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

ABSTRACT The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RTs strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RTs L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. IMPORTANCE Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzymes DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

The importance of glutamine 294 that affects the ribonuclease H activity of the reverse transcriptase of HIV-2 to viral replication.

Most currently-used antiretroviral drugs inhibit the reverse-transcriptase (RT) of HIV. The differences between HIV-1 and HIV-2 RTs explain why some of the anti-HIV-1 drugs are not effective against HIV-2. One major difference between the two HIV RTs is the low ribonuclease H (RNase H) activity of HIV-2 RT relative to HIV-1 RT. Our previous studies showed that residue Gln294 in HIV-2 RT accounts for this RNase H reduction (the comparable residue in HIV-1 RT is Pro294), as the Q294P mutant of HIV-2 RT has ~10-fold higher RNase H. Here, we show that infectious HIV-2 cannot bear the replacement of the RTs Gln294 by the HIV-1 RT Pro counterpart, as it results in substantially reduced HIV-2 replication and fast reversions to the wild-type Gln294 virus. These findings prove the critical role of maintaining low RT-associated RNase H activity in HIV-2. In contrast, HIV-1 can tolerate an about 10-fold higher RNase H.

Insights into the molecular and biological features of the dUTPase-related gene of bovine immunodeficiency virus

This study was stimulated by our previous research of the dUTPase-related protein from bovine immunodeficiency virus (BIV) (Voronin et al., 2014). Despite the lack of detectable enzymatic BIV dUTPase activity (both of the recombinant protein and in virions), mutating the dUTPase gene was deleterious to viral production. However, cDNA synthesis and integration were apparently unaffected. Consequently, we have studied here two important issues. First, we showed that in cDNA produced by the dUTPase-mutated virions, the incidence of mutations was not higher than that found in wild-type BIV-infected cells. Second, single mutations, introduced in preserved dUTPase residues Asp48 and Asn57 (in the putative dUTPase active site or close to it), have led to abortive BIV infections (except for the conservative Asp48Glu mutation). Therefore, we postulate that the BIV dUTPase-related protein has a critical role in retroviral replication at steps that take place after viral cDNA synthesis and integration.

Erratum to: dUTPase: the frequently overlooked enzyme encoded by many retroviruses

© 2015 Hizi and Herzig. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Erratum to: Retrovirology (2015) 12:70 DOI 10.1186/s12977‐015‐0198‐9 The original version of this article unfortunately contained a typographical mistake. The spelling of ‘retroviruses’ was incorrect. The corrected text is given below.