Evolution of the HIV-1 Rev Response Element during Natural Infection Reveals Nucleotide Changes That Correlate with Altered Structure and Increased Activity over Time.

The HIV-1 Rev response element (RRE) is a cis-acting RNA element characterized by multiple stem-loops. Binding and multimerization of the HIV Rev protein on the RRE promote the nucleocytoplasmic export of incompletely spliced mRNAs, an essential step in HIV replication. Most of our understanding of the Rev-RRE regulatory axis comes from studies of lab-adapted HIV clones. However, in human infection, HIV evolves rapidly, and mechanistic studies of naturally occurring Rev and RRE sequences are essential to understanding this system. We previously described the functional activity of two RREs found in circulating viruses in a patient followed during the course of HIV infection. The early RRE was less functionally active than the late RRE, despite differing in sequence by only 4 nucleotides. In this study, we describe the sequence, function, and structural evolution of circulating RREs in this patient using plasma samples collected over 6 years of untreated infection. RRE sequence diversity varied over the course of infection, with evidence of selection pressure that led to sequence convergence as disease progressed being found. An increase in RRE functional activity was observed over time, and a key mutation was identified that correlates with a major conformational change in the RRE and increased functional activity. Additional mutations were found that may have contributed to increased activity as a result of greater Shannon entropy in RRE stem-loop II, which is key to primary Rev binding.IMPORTANCE HIV-1 replication requires interaction of the viral Rev protein with a cis-acting regulatory RNA, the Rev response element (RRE), whose sequence changes over time during infection within a single host. In this study, we show that the RRE is subject to selection pressure and that RREs from later time points in infection tend to have higher functional activity. Differences in RRE functional activity are attributable to specific changes in RNA structure. Our results suggest that RRE evolution during infection may be important for HIV pathogenesis and that efforts to develop therapies acting on this viral pathway should take this into account.

A new HIV-1 Rev structure optimizes interaction with target RNA (RRE) for nuclear export.

HIV-1 Rev mediates the nuclear export of unspliced and partially-spliced viral transcripts for the production of progeny genomes and structural proteins. In this process, four (or more) copies of Rev assemble onto a highly-structured 351-nt region in such viral transcripts, the Rev response element (RRE). How this occurs is not known. The Rev assembly domain has a helical-hairpin structure which associates through three (A-A, B-B and C-C) interfaces. The RRE has the topology of an upper-case letter A, with the two known Rev binding sites mapping onto the legs of the A. We have determined a crystal structure for the Rev assembly domain at 2.25 Šresolution, without resort to either mutations or chaperones. It shows that B-B dimers adopt an arrangement reversed relative to that previously reported, and join through a C-C interface to form tetramers. The new subunit arrangement shows how four Rev molecules can assemble on the two sites on the RRE to form the specificity checkpoint, and how further copies add through A-A interactions. Residues at the C-C interface, specifically the Pro31-Trp45 axis, are a potential target for intervention.

Diverse activities of viral cis-acting RNA regulatory elements revealed using multicolor, long-term, single-cell imaging.

Cis-acting RNA structural elements govern crucial aspects of viral gene expression. How these structures and other posttranscriptional signals affect RNA trafficking and translation in the context of single cells is poorly understood. Herein we describe a multicolor, long-term (>24 h) imaging strategy for measuring integrated aspects of viral RNA regulatory control in individual cells. We apply this strategy to demonstrate differential mRNA trafficking behaviors governed by RNA elements derived from three retroviruses (HIV-1, murine leukemia virus, and Mason-Pfizer monkey virus), two hepadnaviruses (hepatitis B virus and woodchuck hepatitis virus), and an intron-retaining transcript encoded by the cellular NXF1 gene. Striking behaviors include "burst" RNA nuclear export dynamics regulated by HIV-1's Rev response element and the viral Rev protein; transient aggregations of RNAs into discrete foci at or near the nuclear membrane triggered by multiple elements; and a novel, pulsiform RNA export activity regulated by the hepadnaviral posttranscriptional regulatory element. We incorporate single-cell tracking and a data-mining algorithm into our approach to obtain RNA element-specific, high-resolution gene expression signatures. Together these imaging assays constitute a tractable, systems-based platform for studying otherwise difficult to access spatiotemporal features of viral and cellular gene regulation.

Refrex-1, a soluble restriction factor against feline endogenous and exogenous retroviruses.

The host defense against viral infection is acquired during the coevolution or symbiosis of the host and pathogen. Several cellular factors that restrict retroviral infection have been identified in the hosts. Feline leukemia virus (FeLV) is a gammaretrovirus that is classified into several receptor interference groups, including a novel FeLV-subgroup D (FeLV-D) that we recently identified. FeLV-D is generated by transduction of the env gene of feline endogenous gammaretrovirus of the domestic cat (ERV-DCs) into FeLV. Some ERV-DCs are replication competent viruses which are present and hereditary in cats. We report here the determination of new viral receptor interference groups and the discovery of a soluble antiretroviral factor, termed Refrex-1. Detailed analysis of FeLV-D strains and ERV-DCs showed two receptor interference groups that are distinct from other FeLV subgroups, and Refrex-1 specifically inhibited one of them. Refrex-1 is characterized as a truncated envelope protein of ERV-DC and includes the N-terminal region of surface unit, which is a putative receptor-binding domain, but lacks the transmembrane region. Refrex-1 is efficiently secreted from the cells and appears to cause receptor interference extracellularly. Two variants of Refrex-1 encoded by provirus loci, ERV-DC7 and DC16, are expressed in a broad range of feline tissues. The host retains Refrex-1 as an antiretroviral factor, which may potentially prevent reemergence of the ERVs and the emergence of novel ERV-related viruses in cats. Refrex-1 may have been acquired during endogenization of ERV-DCs and may play an important role in retroviral restriction and antiviral defense in cats.

First molecular characterization of feline immunodeficiency virus in Turkey.

In this study, strains of feline immunodeficiency virus (FIV), designated TR-D, TR-Mo and TR-Mi, isolated from three cats in Turkey, were characterized. PCR products (859 bp) from the envelope (env) gene region were amplified and sequenced, and possible geographical differences in the env gene region of Turkish FIV strains are discussed. Phylogenetic analysis of two strains showed that FIV subtype B was present in Turkey. Phylogenetic analysis showed that one new Turkish FIV strain occupies a separate branch from known clusters (subtypes A to E) from the USA, Canada, Europe and Japan.

Domain- and nucleotide-specific Rev response element regulation of feline immunodeficiency virus production.

Computational analysis of feline immunodeficiency virus (FIV) RNA sequences indicated that common FIV strains contain a rev response element (RRE) defined by a long unbranched hairpin with 6 stem-loop sub-domains, termed stem-loop A (SLA). To examine the role of the RNA secondary structure of the RRE, mutational analyses were performed in both an infectious FIV molecular clone and a FIV CAT-RRE reporter system. These studies disclosed that the stems within SLA (SA1, 2, 3, 4, and 5) of the RRE were critical but SA6 was not essential for FIV replication and CAT expression. These studies also revealed that the secondary structure rather than an antisense protein (ASP) mediates virus expression and replication in vitro. In addition, a single synonymous mutation within the FIV-RRE, SA3/45, reduced viral reverse transcriptase activity and p24 expression after transfection but in addition also showed a marked reduction in viral expression and production following infection.

Molecular and biological characterization of equine infectious anemia virus Rev.

Equine infectious anemia virus (EIAV) is one of the most divergent members of the lentivirus subfamily of retroviruses and is considered a useful comparative model for molecular studies of lentivirus replication. The Rev protein of EIAV is functionally homologous with other lentiviral Revs and facilitates export of incompletely spliced viral mRNAs through a Crm1-dependent pathway. The trans- and cis-acting elements that mediate EIAV Rev function are similar to, but distinct from, the well-characterized elements in human immunodeficiency virus (HIV-1), the prototypical Rev protein. In addition, the EIAV rev sequence is highly variable in vivo, and changes in Rev phenotype correlate with changes in clinical stages of EIAV infection. This review summarizes the molecular biology of EIAV Rev-RRE interactions and the consequences of Rev variation in vivo. A comparative perspective of Rev activity may enhance understanding of an essential lentiviral protein and stimulate new strategies for treatment and prevention of lentivirus infections in vivo.

SIVagm containing the SHIV89.6P Envelope gene replicates poorly and is non-pathogenic.

SIVagm does not induce disease in its African green monkey (AGM) host. In comparison, the hybrid simian-human immunodeficiency virus SHIV89.6P that carries the HIV env gene induces disease in rhesus macaques more rapidly than the SIVmac parent virus. To address the possibility that this enhancement of disease by HIV env would also occur when present in SIVagm, a full-length SIVagm/89.6Penv chimeric lentivirus genome (termed SHIV-MP) was constructed. SHIV-MP replicated similarly to SIVagm in simian peripheral blood mononuclear cells (PBMCs). In inoculated AGMs, rhesus macaques and pig-tailed (PT) macaques the absolute number of CD4(+) T lymphocytes remained at normal levels. The peak levels of productively infected cells in SHIV-MP-infected monkeys ranged from 10(1) to 10(2) per 10(6) PBMCs, while in SIVagm infected macaques the levels were 10-100-fold higher. The env gene of SHIV89.6P therefore appears insufficient to confer acute pathogenicity to a non-pathogenic primate lentivirus due to poor in vivo replication.

Bacterial RNase P RNA is a drug target for aminoglycoside-arginine conjugates.

The ribonuclease P (RNase P) holoenzymes are RNPs composed of RNase P RNA (PRNA) and a variable number of P protein subunits. Primary differences in structure and function between bacterial and eukaryotic RNase P and its indispensability for cell viability make the bacterial enzyme an attractive drug target. On the basis of our previous studies, aminoglycoside-arginine conjugates (AACs) bind to HIV-1 TAR and Rev responsive element (RRE) RNAs significantly more efficiently than neomycin B. Their specific inhibition of bacterial rRNA as well as the findings that the hexa-arginine neomycin derivative (NeoR6) is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, led us to explore the structure-function relationships of AACs in comparison to a new set of aminoglycoside-polyarginine conjugates (APACs). We here present predicted binding modes of AACs and APACs to PRNA. We used a multistep docking approach comprising rigid docking full scans and final refinement of the obtained complexes. Our docking results suggest three possible mechanisms of RNase P inhibition by AACs and APACs: competition with the P protein and pre-tRNA on binding to P1-P4 multihelix junction and to J19/4 region (probably including displacement of Mg2+ ions from the P4 helix) of PRNA; competition with Mg2+ ions near the P15 loop; and competition with the P protein and/or pre-tRNA near the P15 helix and interfering with interactions between the P protein and pre-tRNA at this region. The APACs revealed about 10-fold lower intermolecular energy than AACs, indicating stronger interactions of APACs than AACs with PRNA.

Heterocyclic compounds that inhibit Rev-RRE function and human immunodeficiency virus type 1 replication.

A cell-based screening assay was performed to identify compounds that inhibited the postintegration stage of the human immunodeficiency virus (HIV) life cycle. This assay utilized a cell line that contains the HIV gag and pol genes expressed in a Rev-dependent fashion. The cell line produces about 10 to 15 ng of p24 per milliliter of medium over a 24-h period in the form of viruslike particles. Any compound that inhibits a postintegration step in the HIV life cycle scores in this assay by decreasing particle production. Forty thousand compounds were screened, and 192 compounds were selected from the original screen because they showed more than 50% inhibition at a 10 muM concentration. The cumulative evidence presented in this study strongly suggests that 2 of the 192 compounds work as inhibitors of HIV Rev function. This was determined by a variety of cell-based assays, although the compounds do not interfere with Rev-RRE (Rev response element) binding in vitro. Both compounds inhibit replication of the lab isolate NL4-3 as well as an HIV primary isolate from Brazil (93BR021) and thus are promising leads as therapeutic candidates that target HIV replication through inhibition of Rev function.

Molecular characterization of the HERV-W env gene in humans and primates: expression, FISH, phylogeny, and evolution.

We characterized the human endogenous retrovirus (HERV-W) family in humans and primates. In silico expression data indicated that 22 complete HERV-W families from human chromosomes 1-3, 5-8, 10-12, 15, 19, and X are randomly expressed in various tissues. Quantitative real-time RT-PCR analysis of the HERV-W env gene derived from human chromosome 7q21.2 indicated predominant expression in the human placenta. Several copies of repeat sequences (SINE, LINE, LTR, simple repeat) were detected within the complete or processed pseudo HERV-W of the human, chimpanzee, and rhesus monkey. Compared to other regions (5'LTR, Gag, Gag-Pol, Env, 3'LTR), the repeat family has been mainly integrated into the region spanning the 5'LTRs of Gag (1398 bp) and Pol (3242 bp). FISH detected the HERV-W probe (fosWE1) derived from a gorilla fosmid library in the metaphase chromosomes of all primates (five hominoids, three Old World monkeys, two New World monkeys, and one prosimian), but not in Tupaia. This finding was supported by molecular clock and phylogeny data using the divergence values of the complete HERV-W LTR elements. The data suggested that the HERV-W family was integrated into the primate genome approximately 63 million years (Myr) ago, and evolved independently during the course of primate radiation.

Rev-dependent indicator T cell line.

Measuring virion infectivity is critical for studying and monitoring the process of HIV-1 infection. The easiest and the most common method utilizes reporter cell lines based on the HIV LTR promoter. The early HIV gene product Tat amplifies expression from the LTR; however, there is a background transcriptional activity that is independent of Tat. Furthermore, LTR activity can be influenced by cellular activation states. We have recently constructed a Rev-dependent expression vector, and as a test of this construct's functionality, we have integrated this vector into a continuous T cell line. This novel indicator cell has no measurable background signal, is not affected by elevated metabolic states, and yet responds robustly to the presence of HIV. The line is able to complete TCID50 assays in 3-5 days, and appears sensitive to both CCR5- and CXCR4-utilizing viruses.

HIV-l and the microRNA-guided silencing pathway: an intricate and multifaceted encounter.

MicroRNAs (miRNAs) are approximately 21-24 nucleotide RNAs that mediate repression of messenger RNA (mRNA) translation through recognition of specific miRNA binding sites usually located in the 3' non-translated region. Designed to simulate miRNAs, small interfering RNAs represent a powerful genetic approach to potently inhibit gene expression by mediating cleavage of the intended mRNA target. This strategy has been applied successfully to suppress replication of several viruses, including human immunodeficiency virus type 1 (HIV-1). However, recent evidences indicate that viral RNAs may themselves be processed, to some extent, by the endogenous miRNA biosynthetic machinery in mammalian cells, extending previous observations in plants. The resulting viral miRNAs may exert regulatory effects towards host and/or viral genes that may influence viral replication and modulate the course of infection. Viral miRNA generation and/or action may be limited by counteraction through inhibitory viral RNAs and/or proteins. This review article will focus on the relationship between HIV-1 and miRNA-guided RNA silencing, and discuss the different aspects of their interaction. As we learn more about the mechanism and importance of small RNA-based antiviral systems, a more intricate picture of the interaction between HIV-1 and a proven antiviral defense mechanism in lower eukaryotes is emerging.

Furin cleavage potentiates the membrane fusion-controlling intersubunit disulfide bond isomerization activity of leukemia virus Env.

The membrane fusion protein of murine leukemia virus is a trimer of a disulfide-linked peripheral-transmembrane (SU-TM) subunit complex. The intersubunit disulfide bond is in SU linked to a disulfide bond isomerization motif, CXXC, with which the virus controls its fusion reaction (M. Wallin, M. Ekström, and H. Garoff, EMBO J. 23:54-65, 2004). Upon receptor binding the isomerase rearranges the intersubunit disulfide bond into a disulfide bond isomer within the motif. This facilitates SU dissociation and fusion activation in the TM subunit. In the present study we have asked whether furin cleavage of the Env precursor potentiates the isomerase to be triggered. To this end we accumulated the late form of the precursor, gp90, in the cell by incubation in the presence of a furin-inhibiting peptide. The isomerization was done by NP-40 incubation or by a heat pulse under alkylation-free conditions. The cells were lysed in the presence of alkylator, and the precursor was immunoprecipitated, gel isolated, deglycosylated, and subjected to complete trypsin digestion. Disulfide-linked peptide complexes were separated by sodium dodecyl sulfate-tricine-polyacrylamide gel electrophoresis under nonreducing conditions. This assay revealed the size of the characteristic major disulfide-linked peptide complex that differentiates the two isomers of the disulfide bond between Cys336 (or Cys339) and Cys563, i.e., the bond corresponding to the intersubunit disulfide bond. The analyses showed that the isomerase was five- to eightfold more resistant to triggering in the precursor than in the mature, cleaved form. This suggests that the isomerase becomes potentiated for triggering by a structural change in Env that is induced by furin cleavage in the cell.

Functional variability of Rev response element in HIV-1 primary isolates.

We have previously studied sequence heterogeneity of HIV-1 Rev response element (RRE), and showed uneven variations in different stem-loops of both primary sequence and secondary structure. Here we studied the functional variation of RRE clones from a set of 10 primary isolates, and demonstrated a variation in the function of these RRE clones on the expression of Gag proteins from a truncated HIV-1 genome. The difference in Gag level was, in part, if not exclusively, resulted from the differential efficiency of RNA transport and enhancing of translation. These data suggested that variation of HIV-1 RRE may play a role in regulation of viral replication rate in HIV-1 primary isolates.

Functional variation of HIV-1 Rev Response Element in a longitudinally studied cohort.

We showed previously that HIV-1 Rev Response Element (RRE) contains a certain degree of structural variation, and in a set of limited samples, RRE from HIV-1 natural isolates were found to have functional variability. The significance of the RRE heterogeneity is addressed further by analyzing the functional variation of RREs in a longitudinal cohort. While the RRE activity at early time points was not a good predictor of disease outcome, the RRE activity at late time points was correlated with rates of CD4+ count decline. These data suggest that RRE heterogeneity may be important in viral pathogenesis and disease progression.

Mutations conferring resistance to human immunodeficiency virus type 1 fusion inhibitors are restricted by gp41 and Rev-responsive element functions.

One of the human immunodeficiency virus (HIV) envelope proteins, gp41, plays a key role in HIV fusion. A gp41-derived peptide, T-20, efficiently inhibits HIV fusion and is currently approved for treatment of HIV-infected individuals. Although resistant variants have been reported, the mechanism of the resistance remains to be defined. To elucidate the mechanism in detail, we generated variants resistant to C34, a peptide derived from the gp41 carboxyl terminus heptad repeat (C-HR) in vitro. The resistant variants had a 5-amino-acid deletion in gp120 and a total of seven amino acid substitutions in gp41. Binding assays revealed that an I37K substitution in the N-terminal heptad repeat (N-HR) impaired the binding of C34, whereas an N126K substitution in the C-HR enhanced the binding to mutated N-HR, indicating that both mutations were directly involved in resistance. On the other hand, substitutions for A30 and D36 seemed to be secondary mutations, located complementary to each other in the Rev-responsive element (RRE), and were mutated simultaneously to maintain the secondary structure of the RRE that was impaired by the mutations at I37. Thus, HIV acquired resistance to C34 by mutations in N-HR, which directly interacted with C34. However, since this region also encoded the RRE, additional mutations were required to maintain viral replication. These results suggest that HIV fusion is one of the attractive targets for HIV chemotherapy.

Construction and expression of a Rev-dependent TNF-R1 expressing HIV-infected-cell injurious vectors.

BACKGROUND: Rev is necessary for exporting unspliced and incompletely spliced intron containing HIV mRNAs and for HIV replication. The aim of this study is to develop a kind of selective suicide construct that can specifically and directly induce HIV infected cells into apoptosis based on the high affinity of Rev and Rev response element (RRE). METHODS: Molecular-cloning technique was used to synthesis Rev dependent TNF-R1 expression construct pDM128-TNF-R1 (pT128) that contains RRE and TNFR1 gene. Restriction digestion, Polymerase Chain Reaction (PCR) and DNA sequencing were processed and the exactness and correctness of the inserted TNF-R1 gene in pT128 were confirmed repeatedly. The expression of pT128 co-transfected with different combination of other plasmids by calcium phosphate-DNA co-precipitation in Helas and by gene gun transfection in keratinocytes was further tested by flow-cytometry and cell counted under microscope. RESULTS: The new plasmid specifically expressed TNF-R1 in Helas when co-transfected with pRev but did not when without pRev. Indirect expression of TNF-R1 from pT128 was slower than the direct expression of that from Hu p60 TNFR1 in pDC302 (pT60), but all those pT60 or pT128 transfected cells showed apoptosis at last while TNF-R1 was sufficiently expressed. Other kinds of Rev expression construct such as pAD8 and a chimeric HIV vaccine also can switched on the selective expression of pT128. Not only Rev-dependent expression in Helas, pT128 also normally expressed its TNF-R1 in keratinocytes. Co-transfected with pRev or pAD8 that expressed Rev, pT128 expressed TNF-R1 and induced apoptosis of green fluorescent keratinocytes in skin explant. The number of green fluorescent keratinocytes co-transfected by pT128 plus pRev or pAD8 was gradually outnumbered by that co-transfected by pT128 only. The difference was more significant after culturing for 72 hours. CONCLUSIONS: Rev dependent pT128 is able to selectively induce apoptosis of HIV-infected or Rev-expressed target cells by expression of TNF-R1. The new strategy based on manipulation of the regulatory protein of HIV may be valuable in design of new HIV vaccine.

A DEAD box protein facilitates HIV-1 replication as a cellular co-factor of Rev.

HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions. We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as "bait". DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev- cells rescued by exogenous Rev. Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production. DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis. Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance. These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein. Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics.

Requirement of DDX3 DEAD box RNA helicase for HIV-1 Rev-RRE export function.

A single transcript in its unspliced and spliced forms directs the synthesis of all HIV-1 proteins. Although nuclear export of intron-containing cellular transcripts is restricted in mammalian cells, HIV-1 has evolved the viral Rev protein to overcome this restriction for viral transcripts. Previously, CRM1 was identified as a cellular cofactor for Rev-dependent export of intron-containing HIV-1 RNA. Here, we present evidence that Rev/CRM1 activity utilizes the ATP-dependent DEAD box RNA helicase, DDX3. We show that DDX3 is a nucleo-cytoplasmic shuttling protein, which binds CRM1 and localizes to nuclear membrane pores. Knockdown of DDX3 using either antisense vector or dominant-negative mutants suppressed Rev-RRE-function in the export of incompletely spliced HIV-1 RNAs. Plausibly, DDX3 is the human RNA helicase which functions in the CRM1 RNA export pathway analogously to the postulated role for Dbp5p in yeast mRNA export.