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Retroviral Gag proteins are responsible for coordinating many aspects of virion assembly. Gag possesses two distinct nucleic acid binding domains, matrix (MA) and nucleocapsid (NC). One of the critical functions of Gag is to specifically recognize, bind, and package the retroviral genomic RNA (gRNA) into assembling virions. Gag interactions with cellular RNAs have also been shown to regulate aspects of assembly. Recent results have shed light on the role of MA and NC domain interactions with nucleic acids, and how they jointly function to ensure packaging of the retroviral gRNA. Here, we will review the literature regarding RNA interactions with NC, MA, as well as overall mechanisms employed by Gag to interact with RNA. The discussion focuses on human immunodeficiency virus type-1, but other retroviruses will also be discussed. A model is presented combining all of the available data summarizing the various factors and layers of selection Gag employs to ensure specific gRNA packaging and correct virion assembly., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Human tRNA Lys3 serves as the primer for reverse transcription in human immunodeficiency virus type-1 (HIV-1) and anneals to the complementary primer binding site (PBS) in the genome. All tRNA Lys isoacceptors interact with human lysyl-tRNA synthetase (hLysRS) and are selectively packaged into virions. tRNA Lys3 must be released from hLysRS in order to anneal to the PBS, and this process is proposed to be facilitated by the interaction of hLysRS with a tRNA-like element (TLE) first identified in the HIV-1 5'-untranslated region (5'-UTR) of the subtype B NL4-3 virus. However, a significant subset of HIV-1 strains represented by the MAL isolate possess a different secondary structure in this region of the genome. Thus, to establish the conservation of this mechanism for primer targeting and release, we investigated the subtype A-like 5'-UTR of the MAL isolate. hLysRS bound to a 229-nt MAL RNA containing the PBS domain with high affinity ( K d = 47 nM), and to a 98-nt truncated construct with ∼10-fold reduced affinity. These results resemble previous studies using analogous NL4-3-derived RNAs. However, in contrast to studies with NL4-3, no binding was observed to smaller stem-loop elements within the MAL PBS domain. The tertiary structure of the 98-nt construct was analyzed using small-angle X-ray scattering, revealing remarkable global structural similarity to the corresponding NL4-3 PBS/TLE region. These results suggest that the tRNA-like structure within the 5'-UTR is conserved across distinct HIV-1 subtypes and that hLysRS recognition of the MAL isolate is likely not conferred by specific sequence elements but by 3D structure., (© 2017 Comandur et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Background: The nucleocapsid (NC) domain of HIV-1 Gag is responsible for specific recognition and packaging of genomic RNA (gRNA) into new viral particles. This occurs through specific interactions between the Gag NC domain and the Psi packaging signal in gRNA. In addition to this critical function, NC proteins are also nucleic acid (NA) chaperone proteins that facilitate NA rearrangements during reverse transcription. Although the interaction with Psi and chaperone activity of HIV-1 NC have been well characterized in vitro, little is known about simian immunodeficiency virus (SIV) NC. Non-human primates are frequently used as a platform to study retroviral infection in vivo; thus, it is important to understand underlying mechanistic differences between HIV-1 and SIV NC., Results: Here, we characterize SIV NC chaperone activity for the first time. Only modest differences are observed in the ability of SIV NC to facilitate reactions that mimic the minus-strand annealing and transfer steps of reverse transcription relative to HIV-1 NC, with the latter displaying slightly higher strand transfer and annealing rates. Quantitative single molecule DNA stretching studies and dynamic light scattering experiments reveal that these differences are due to significantly increased DNA compaction energy and higher aggregation capability of HIV-1 NC relative to the SIV protein. Using salt-titration binding assays, we find that both proteins are strikingly similar in their ability to specifically interact with HIV-1 Psi RNA. In contrast, they do not demonstrate specific binding to an RNA derived from the putative SIV packaging signal., Conclusions: Based on these studies, we conclude that (1) HIV-1 NC is a slightly more efficient NA chaperone protein than SIV NC, (2) mechanistic differences between the NA interactions of highly similar retroviral NC proteins are revealed by quantitative single molecule DNA stretching, and (3) SIV NC demonstrates cross-species recognition of the HIV-1 Psi RNA packaging signal.

Two copies of unspliced human immunodeficiency virus (HIV)-1 genomic RNA (gRNA) are preferentially selected for packaging by the group-specific antigen (Gag) polyprotein into progeny virions as a dimer during the late stages of the viral lifecycle. Elucidating the RNA features responsible for selective recognition of the full-length gRNA in the presence of an abundance of other cellular RNAs and spliced viral RNAs remains an area of intense research. The recent nuclear magnetic resonance (NMR) structure by Keane et al. [1] expands upon previous efforts to determine the conformation of the HIV-1 RNA packaging signal. The data support a secondary structure wherein sequences that constitute the major splice donor site are sequestered through base pairing, and a tertiary structure that adopts a tandem 3-way junction motif that exposes the dimerization initiation site and unpaired guanosines for specific recognition by Gag. While it remains to be established whether this structure is conserved in the context of larger RNA constructs or in the dimer, this study serves as the basis for characterizing large RNA structures using novel NMR techniques, and as a major advance toward understanding how the HIV-1 gRNA is selectively packaged.

The field of viral molecular biology has reached a precipice for which pioneering studies on the structure of viral RNAs are beginning to bridge the gap. It has become clear that viral genomic RNAs are not simply carriers of hereditary information, but rather are active players in many critical stages during replication. Indeed, functions such as cap-independent translation initiation mechanisms are, in some cases, primarily driven by RNA structural determinants. Other stages including reverse transcription initiation in retroviruses, nuclear export and viral packaging are specifically dependent on the proper 3-dimensional folding of multiple RNA domains to recruit necessary viral and host factors required for activity. Furthermore, a large-scale conformational change within the 5'-untranslated region of HIV-1 has been proposed to regulate the temporal switch between viral protein synthesis and packaging. These RNA-dependent functions are necessary for replication of many human disease-causing viruses such as severe acute respiratory syndrome (SARS)-associated coronavirus, West Nile virus, and HIV-1. The potential for antiviral development is currently hindered by a poor understanding of RNA-driven molecular mechanisms, resulting from a lack of structural information on large RNAs and ribonucleoprotein complexes. Herein, we describe the recent progress that has been made on characterizing these large RNAs and provide brief descriptions of the techniques that will be at the forefront of future advances. Ongoing and future work will contribute to a more complete understanding of the lifecycles of retroviruses and RNA viruses and potentially lead to novel antiviral strategies., (Copyright © 2014 Elsevier B.V. All rights reserved.)