HIV-1 reverse transcription initiation: A potential target for novel antivirals?

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

A novel long distance base-pairing interaction in human immunodeficiency virus type 1 rna occludes the gag start codon

The 5′-untranslated region (5′-UTR) is the most conserved part of the HIV-1 RNA genome, and it contains regulatory motifs that mediate various steps in the viral life cycle. Previous work showed that the 5′-terminal 290 nucleotides of HIV-1 RNA adopt two mutually exclusive secondary structures, long distance interaction (LDI) and branched multiple hairpin (BMH). BMH has multiple hairpins, including the dimer initiation signal (DIS) hairpin that mediates RNA dimerization. LDI contains a long distance base-pairing interaction that occludes the DIS region. Consequently, the two conformations differ in their ability to form RNA dimers. In this study, we have presented evidence that the full-length 5′-UTR also adopts the LDI and BMH conformations. The downstream 290-352 region, including the Gag start codon, folds differently in the context of the LDI and BMH structures. These nucleotides form an extended hairpin structure in the LDI conformation, but the same sequences create a novel long distance interaction with upstream U5 sequences in the BMH conformation. The presence of this U5-AUG duplex was confirmed by computer-assisted RNA structure prediction, biochemical analyses, and a phylogenetic survey of different virus isolates. The U5-AUG duplex may influence translation of the Gag protein because it occludes the start codon of the Gag open reading frame

Cellular factors involved in HIV-1 RNA transport

HIV-1 assembly and genomic RNA encapsidation have been intensively studied for many years. Many details of the interaction between the RNA packaging signal and the nucleocapsid (NC) domain of the structural Gag protein are now understood. However, there are still many unknowns regarding the spatial and temporal control of the RNA packaging process. It is generally assumed that cellular mRNAs are complexed with RNA-binding proteins during or shortly after transcription. These ribonucleoprotein complexes (RNPs) are subsequently trafficked out of the nucleus into the cytoplasm, where they can be translated at various locations, stored in stress granules during stress responses or destroyed by various RNA degradation machineries. It is assumed that the HIV-1 genomic RNA (gRNA) is also subject to these processes. The composition of the genomic RNP may thus determine the fate of the viral RNA. Indeed, it has been shown that altering the expression level of certain host proteins can affect HIV-1 translation, gRNA localization or particle assembly, implying that these proteins are important for efficient viral replication. Consequently, there is an increasing interest in targeting these host factors as an additional approach to antiviral treatment

The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation

The untranslated leader RNA is the most conserved part of the human immunodeficiency virus type I (HIV-1) genome. It contains many regulatory motifs that mediate a variety of steps in the viral life cycle. Previous work showed that the full-length leader RNA can adopt two alternative structures: a long distance interaction (LDI) and a branched multiple-hairpin (BMH) structure. The BMH structure exposes the dimer initiation site (DIS) hairpin, whereas this motif is occluded in the LDI structure. Consequently, these structures differ in their capacity to form RNA dimers in vitro. The BMH structure is dimerization-competent, due to DIS hairpin formation, but also presents the splice donor (SD) and RNA packaging (Ψ) hairpins. In the LDI structure, an extended RNA packaging (ΨE) hairpin is folded, which includes the splice donor site and gag coding sequences. The gag initiation codon is engaged in a long distance base pairing interaction with sequences in the upstream U5 region in the BMH structure, thus forming the evolutionarily conserved U5-AUG duplex. Therefore, the LDI-BMH equilibrium may affect not only the process of RNA dimer formation but also translation initiation. In this study, we designed mutations in the 3′-terminal region of the leader RNA that alter the equilibrium of the LDI-BMH structures. The mutant leader RNAs are affected in RNA dimer formation, but not in their translation efficiency. These results indicate that the LDI-BMH status does not regulate HIV-1 RNA translation, despite the differential presentation of the gag initiation codon in both leader RNA structures

Tobacco mosaic virus helicase domain induces necrosis in N gene-carrying tobacco in the absence of virus replication

Tobacco mosaic virus (TMV) elicits a hypersensitive response (HR) in tobacco plants that carry the N gene. To identify the elicitor of this HR, Agrobacteriumn tumefaciens was used as a vector for the transient expression of TMV replicase proteins, movement protein, and coat protein in NN and nn tobacco. Transient expression of the 126K protein and fragments thereof containing the helicase motifs induced necrosis and systemic expression of the pathogenesis-related PR-1a gene in NN plants but not in nn plants. The results confirm previous evidence that the TMV helicase sequence is the elicitor of the HR (H. S. Padgett, Y. Watanabe, and R. N. Beachy, Mol. Plant-Microbe Interact. 10:709-715, 1997) and demonstrate that this helicase sequence acts as an elicitor of HR in the absence of other viral proteins or RNA replication

Neuron-specific translational control shift ensures proteostatic resilience during ER stress

Proteostasis is essential for cellular survival and particularly important for highly specialised post-mitotic cells such as neurons. Transient reduction in protein synthesis by protein kinase R-like endoplasmic reticulum (ER) kinase (PERK)-mediated phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α) is a major proteostatic survival response during ER stress. Paradoxically, neurons are remarkably tolerant to PERK dysfunction, which suggests the existence of cell type-specific mechanisms that secure proteostatic stress resilience. Here, we demonstrate that PERK-deficient neurons, unlike other cell types, fully retain the capacity to control translation during ER stress. We observe rescaling of the ATF4 response, while the reduction in protein synthesis is fully retained. We identify two molecular pathways that jointly drive translational control in PERK-deficient neurons. Haem-regulated inhibitor (HRI) mediates p-eIF2α and the ATF4 response and is complemented by the tRNA cleaving RNase angiogenin (ANG) to reduce protein synthesis. Overall, our study elucidates an intricate back-up mechanism to ascertain translational control during ER stress in neurons that provides a mechanistic explanation for the thus far unresolved observation of neuronal resilience to proteostatic stress

Silencing of a gene encoding a protein component of the oxygen-evolving complex of photosystem ii enhances virus replication in plants

It has been suggested that, in addition to viral proteins, host proteins are involved in RNA virus replication. In this study the RNA helicase domain of the Tobacco mosaic virus (TMV) replicase proteins was used as bait in the yeast two-hybrid system to identify tobacco proteins with a putative role in TMV replication. Two host proteins were characterized. One protein (designated #3) belongs to a protein family of ATPases associated with various activities (AAA), while the second host protein (designated #13) is the 33K subunit of the oxygen-evolving complex of photosystem II. Using Tobacco rattle virus vectors, genes #3 and #13 were silenced in Nicotiana benthamiana, after which the plants were challenged by TMV infection. Silencing of gene #13 resulted in a 10-fold increase of TMV accumulation, whereas silencing of gene #3 caused a twofold reduction of TMV accumulation. Additionally, silencing of genes #3 and #13 decreased and increased, respectively, the accumulation of two other viruses. Similar to silencing of gene #13, inhibition of photosystem II by application of an herbicide increased TMV accumulation several fold. Infection of N. benthamiana with TMV resulted in a decrease of #13 mRNA levels. Silencing of gene #13 may reflect a novel strategy of TMV to suppress basal host defense mechanisms. The two-hybrid screenings did not identify tobacco proteins involved in helicase domain-induced N-mediated resistance