Matrix-Induced Inhibition of Membrane Binding Contributes to Human Immunodeficiency Virus Type 1 Particle Assembly Defects in Murine Cells

2017

Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing fulllength viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans. In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNAdependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5= packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM.IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy.KEYWORDS Gag, MS2, packaging, Psi, RNA trafficking, RRE, genomes, human immunodeficiency virus, translation, virus assembly T he spatial distribution of mRNAs within the cytoplasm is a core determinant of mRNA turnover, cytoplasmic utilization, and the formation of functional macromolecular complexes (1-3). Viruses face severe challenges in this regard during the p...

Section: Resultssupporting

confidence: 79%

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

Becker

2017

“…Deletion of the MA globular-head domain, resulting in constitutive exposure of the myristoyl group, can significantly improve Gag membrane targeting and virion assembly at low intracellular Gag levels, demonstrating that the globular-head domain plays an autoinhibitory function in this context and suggesting that myristoyl exposure is driven by cooperative Gag-Gag interactions (39). Consistent with these ideas, similar deletions of the MA globular head that retain the native myristoylation signal (or, alternatively, the addition of the myristoylated membranetargeting signal from the Src kinase to the N terminus of intact Gag) substantially improves HIV-1 assembly in murine cells (23). Additionally, replacements of HIV-1 MA with other retroviral MA regions (e.g., from murine leukemia virus) also improve assembly in murine cells (8,9,42).…”

supporting

confidence: 61%

“…The 4؋CTE-dependent rescue of assembly requires an intact MA domain. MA-dependent autoinhibition is at least partially responsible for Gag assembly defects under conditions of low Gag expression (23,39). To address assembly efficiency (i.e., levels of VLP production relative to intracellular Gag levels) in the context of varying intracellular Gag levels, we performed a twofold vector dilution series comparing GP-4ϫCTE or ⌬8-126-RRE/Rev to the GP-RRE/Rev context.…”

Section: Vol 83 2009mentioning

confidence: 99%

“…Many experimental vector systems in murine cells yield low levels of Gag expression, and low intracellular Gag levels have been correlated with poor Gag plasma membrane targeting, even in some human cell lines (e.g., HOS and 293T cells) (23,39). This defect may be explained, at least in part, by a requirement for a threshold concentration of intracellular Gag to activate membrane targeting through a "myristoyl switch" mechanism (23,39,51). Gag is cotranslationally modified with a hydrophobic myristoyl group at its N terminus, a modification required for efficient membrane targeting and virus particle production (6,20).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Matrix Mediates the Functional Link between Human Immunodeficiency Virus Type 1 RNA Nuclear Export Elements and the Assembly Competency of Gag in Murine Cells

Swanson

Papaioannou

et al. 2009

Human immunodeficiency virus type 1 (HIV-1) assembles poorly in murine cells, reflecting inefficient targeting of the Gag structural polyprotein to the plasma membrane. Virus particle production can be restored by replacing the cis-acting Rev response element (RRE) in Gag-Pol mRNAs with multiple copies of the CTE (4؋CTE), suggesting a mechanistic link between HIV-1 RNA trafficking and productive Gag assembly. In this report, we demonstrate that Gag molecules generated from RRE-dependent transcripts are intrinsically defective for assembly in murine 3T3 cells. When controlled for the intracellular Gag level, modulations of the Gag matrix (MA) domain that enhance Gag membrane association (e.g., deletion of the MA globular head) substantially improve assembly for Gag derived from RRE-but not 4؋CTE-dependent transcripts. Gag mutants carrying a leucine zipper replacement of the nucleocapsid (NC) domain remain largely assembly defective when derived from RRE-dependent transcripts, indicating that the defect does not reflect aberrant NC/RNA-driven Gag multimerization. We further demonstrate that single changes in uncharged amino acids implicated in Gag/MA myristoyl switch regulation, most notably replacing the leucine at position 21 with serine, improve assembly for Gag derived from RRE-dependent transcripts. In sum, we provide genetic evidence to suggest that HIV-1 RNA metabolism specifically modulates the activation of MA-dependent membrane targeting.The nuclear export of full-length genomic RNAs (gRNAs) initiates the late stages of retrovirus replication, including virus particle assembly and egress (19). In the cytosol, gRNAs serve as the mRNAs encoding the Gag (and Gag-Pol [GP]) structural polyproteins. Although Gag is sufficient to drive assembly in the absence of other viral factors, gRNAs not only bind to Gag to ensure their packaging into viral cores, but may also play a structural role in promoting Gag self-association (16). As with all mRNAs, gRNA metabolism and transport are highly regulated processes, and the cellular mechanisms underlying the interplay between the Gag protein and gRNA during assembly are not well defined (11,49).In the nucleus, all primary retroviral RNA transcripts undergo pre-mRNA splicing, so that gRNAs, containing functional introns, require specialized machineries to ensure their stabilization and nuclear export (14,22). Human immunodeficiency virus type 1 (HIV-1) encodes its own viral accessory factor, Rev, that regulates nuclear export by binding to a structured cis-acting RNA signal, the Rev response element (RRE), found only in full-length gRNAs and partially spliced viral transcripts (40). Rev contains a leucine-rich nuclear export signal (NES) that recruits the cellular factor CRM1 (also known as exportin 1) to direct nuclear export of a gRNA/Rev ribonucleoprotein (RNP) complex. Of retroviruses that do not encode Rev equivalents, the best understood is Mason-Pfizer monkey virus, which carries a distinct RNA export element, the constitutive transport element (CTE), in all viral ...

“…Gag is myristylated at the amino-terminal glycine, and this myristate can either be sequestered in the globular head of MA or be exposed to interact with the plasma membrane (1). It is unclear how this myristyl switch is regulated, but we and others have hypothesized that the myristate is constitutively sequestered in 3T3 cells (27,68). As overexpression of SRp40 or SRp55 substantially enhanced Rev-dependent Gag expression well beyond the level of 4xCTE-dependent Gag expression (which produces Gag that is assembled efficiently) (Fig.…”

Section: Vol 84 2010 Sr Proteins Promote Hiv-1 Gag Translation 6751mentioning

confidence: 99%

SRp40 and SRp55 Promote the Translation of Unspliced Human Immunodeficiency Virus Type 1 RNA

Swanson

Malim

2010

Nuclear RNA processing events, such as 5 cap formation, 3 polyadenylation, and pre-mRNA splicing, mark mRNA for efficient translation. Splicing enhances translation via the deposition of the exon-junction complex and other multifunctional splicing factors, including SR proteins. All retroviruses synthesize their structural and enzymatic proteins from unspliced genomic RNAs (gRNAs) and must therefore exploit unconventional strategies to ensure their effective expression. Here, we report that specific SR proteins, particularly SRp40 and SRp55, promote human immunodeficiency virus type 1 (HIV-1) Gag translation from unspliced (introncontaining) viral RNA. This activity does not correlate with nucleocytoplasmic shuttling capacity and, in the case of SRp40, is dependent on the second RNA recognition motif and the arginine-serine (RS) domain. While SR proteins enhance Gag expression independent of RNA nuclear export pathway choice, altering the nucleotide sequence of the gag-pol coding region by codon optimization abolishes this effect. We therefore propose that SR proteins couple HIV-1 gRNA biogenesis to translational utilization.From transcription to translation to cytoplasmic mRNA degradation, the sequential phases of gene expression are coupled physically and functionally. In humans, 94% of genes have more than one exon (79) and pre-mRNA splicing plays a central role in regulating transcription, nuclear stability, 3Ј end formation, nuclear export, cytoplasmic trafficking, cytoplasmic stability, translation efficiency, and even posttranslational events, such as protein half-life (reviewed in references 56 and 75). The integration of pre-mRNA splicing with later steps in RNA metabolism can prevent the translation of deleterious RNAs in at least two ways. First, mRNAs containing functional introns are retained by the nucleus (14, 45), thereby preventing the translation of incompletely processed transcripts and the synthesis of deleterious protein products. Second, and less well understood, the coupling of splicing to the control of nuclear stability, export, and translation prevents the translation of unspliced parasitic and/or noncoding RNAs (reviewed in reference 6). Because viruses frequently depend upon the translation of unspliced viral RNAs, they need to have developed mechanisms to circumvent such barriers to gene expression.All retroviruses use a combination of spliced and unspliced RNAs to express their proteins (reviewed in references 20 and 71). The nuclear export of a full-length, unspliced, introncontaining transcript is essential for the replication of all retroviruses, since this transcript is (i) the viral genome (genomic RNA [gRNA]) that is packaged into virions, (ii) the mRNA that is translated into Gag and Gag-polymerase (Pol), the polyproteins that yield the virion structural and enzymatic proteins, and (iii) a physical scaffold that helps promote virion assembly. Human immunodeficiency virus type 1 (HIV-1) has one of the most complicated gene expression strategies of all known retroviruses, where...