Involvement of the Matrix and Nucleocapsid Domains of the Bovine Leukemia Virus Gag Polyprotein Precursor in Viral RNA Packaging

100

The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and transacting elements within the Gag polyprotein. The packaging signal , at the 5 end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.The encapsidation of the RNA genome is essential for retrovirus replication. Because the viral genomic RNA (gRNA) constitutes only a small fraction of the total cellular mRNA, a specific Gag-RNA interaction is thought to be required for viral genome packaging (2). The determinants of virus-specific gRNA incorporation include the cis-acting element at the 5Јend of the viral gRNA, known as the packaging signal (), and the nucleocapsid (NC) domain of the Gag polyprotein (3, 14, 62). In Rous sarcoma virus (RSV), the NC domain contains basic residues that are required for the recognition of and binding to , as well as two Cys-His motifs that maintain the overall conformation of NC and are essential for RNA packaging (30, 31).Packaging of gRNA into progeny virions requires that the unspliced viral mRNA be exported from the nucleus. However, cellular proofreading mechanisms ensure that unspliced or intron-containing mRNAs are retained in the nucleus until splicing occurs. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) overcome this export block of unspliced genomes by encoding the Rev protein, which interacts with a cis-acting sequence in the viral RNA (the Revresponsive element [RRE]) to facilitate cytoplasmic accumulation of intron-containing viral mRNA (16,35). The export of the Rev-viral RNA complex is mediated through the interaction of a leucine-rich nuclear export signal (NES) in Rev with the CRM1 nuclear export factor (17,18,37,41). Simple retroviruses do no...

Section: Discussionsupporting

confidence: 52%

Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging

Garbitt-Hirst

Kenney

Parent

2009

100

“…We and others recently observed in in vitro systems that MA-bound oligonucleotides, in particular RNA, function as negative regulators of HIV-1 Gag membrane binding through the inhibition of interaction between the HBR and non-PI(4,5)P 2 acidic lipids (3,15). Notably, the MA domain of bovine leukemia virus, a deltaretrovirus, was also shown to interact specifically with viral RNA and promote RNA packaging (69). It was therefore possible that the MA domain of HTLV-1 Gag also interacts with RNA and that this interaction affects Gag-membrane binding.…”

Section: Discussionmentioning

confidence: 99%

Gag Localization and Virus-Like Particle Release Mediated by the Matrix Domain of Human T-Lymphotropic Virus Type 1 Gag Are Less Dependent on Phosphatidylinositol-(4,5)-Bisphosphate than Those Mediated by the Matrix Domain of HIV-1 Gag

Inlora

Chukkapalli

Derse

et al. 2011

104

The human immunodeficiency virus type 1 (HIV-1) Gag matrix (MA) domain facilitates Gag targeting and binding to the plasma membrane (PM) during virus assembly. Interaction with a PM phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ], plays a key role in these MA functions. Previous studies showed that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P 2 , mislocalizes HIV-1 Gag to the cytosol and greatly reduces HIV-1 release efficiency. In this study, we sought to determine the role of the MA-PI(4,5)P 2 interaction in Gag localization and membrane binding of a deltaretrovirus, human T-lymphotropic virus type 1 (HTLV-1). We compared the chimeric HIV-1 Gag (HTMA), in which MA was replaced with HTLV-1 MA, with wild-type HIV-1 and HTLV-1 Gag for PI(4,5)P 2 dependence. Our results demonstrate that, unlike HIV-1 Gag, subcellular localization of and VLP release by HTLV-1 and HTMA Gag were minimally sensitive to 5ptaseIV overexpression. These results suggest that the interaction of HTLV-1 MA with PI(4,5)P 2 is not essential for HTLV-1 particle assembly. Furthermore, liposome-binding analyses showed that both HTLV-1 and HTMA Gag can bind membrane efficiently even in the absence of PI(4,5)P 2 . Efficient HTLV-1 Gag binding to liposomes was largely driven by electrostatic interaction, unlike that of HIV-1 Gag, which required specific interaction with PI(4,5)P 2 . Furthermore, membrane binding of HTLV-1 Gag in vitro was not suppressed by RNA, in contrast to HIV-1 Gag. Altogether, our data suggest that Gag targeting and membrane binding mediated by HTLV-1 MA does not require PI(4,5)P 2 and that distinct mechanisms regulate HIV-1 and HTLV-1 Gag membrane binding.

“…MA may interact with RNA for a variety of reasons, such as to prevent premature assembly in the cytoplasm or on inappropriate membranes (4,13), to prevent premature myristoyl exposure (52,60,66), or to act along with NC in viral RNA selection and packaging (51,72). CANC and Gag⌬p6-K30,32N show elevated tRNA annealing rates, which appear to be correlated with the fact that neither protein has an MA domain capable of interacting with NA as effectively as Gag⌬p6.…”

Section: Discussionmentioning

confidence: 99%

Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding

Jones

Datta

Rein

et al. 2011

124

Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA 3 Lys serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA 3 Lys placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA 3 Lys annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing.