Beyond Plasma Membrane Targeting: Role of the MA domain of Gag in Retroviral Genome Encapsidation

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.

Section: Discussionmentioning

confidence: 48%

Section: Discussionmentioning

confidence: 99%

Cytoplasmic Utilization of Human Immunodeficiency Virus Type 1 Genomic RNA Is Not Dependent on a Nuclear Interaction with Gag

Grewe

Hoffmann

Ohs

et al. 2012

“…The matrix (MA) domain of Gag also has several established functions in retroviral replication (11). HIV-1 MA is required for targeting of Gag to the plasma membrane of infected cells via its myristoyl moiety (12,13).…”

mentioning

confidence: 99%

Retrovirus-Specific Differences in Matrix and Nucleocapsid Protein-Nucleic Acid Interactions: Implications for Genomic RNA Packaging

Sun

Grigsby

Gorelick

et al. 2014

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro . HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

“…HIV-1 Gag and NCp7 are chaperones that remodel nucleic acids (NAs) into energetically more favorable conformations through helix destabilization and NA aggregation activities to promote annealing (21)(22)(23)(24)(25)(26). Al-though well known for its role in recruiting Gag to the PM, HIV-1 MA is also able to bind NA with mid-to low-nM affinity in vitro (20,(27)(28)(29)(30)(31)(32). This MA-RNA interaction can be abrogated by phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ]-containing liposomes (30).…”

mentioning

confidence: 99%

Mechanistic Differences between Nucleic Acid Chaperone Activities of the Gag Proteins of Rous Sarcoma Virus and Human Immunodeficiency Virus Type 1 Are Attributed to the MA Domain

Rye-McCurdy

Nadaraia-Hoke

Gudleski-O'Regan

et al. 2014

Self Cite

Host cell tRNAs are recruited for use as primers to initiate reverse transcription in retroviruses. Human immunodeficiency virus type 1 (HIV-1) uses tRNALys3 as the replication primer, whereas Rous sarcoma virus (RSV) uses tRNA Trp . The nucleic acid (NA) chaperone function of the nucleocapsid (NC) domain of HIV-1 Gag is responsible for annealing tRNA Lys3 to the genomic RNA (gRNA) primer binding site (PBS). Compared to HIV-1, little is known about the chaperone activity of RSV Gag. In this work, using purified RSV Gag containing an N-terminal His tag and a deletion of the majority of the protease domain (H6.Gag.3h), gel shift assays were used to monitor the annealing of tRNA Trp to a PBS-containing RSV RNA. Here, we show that similar to HIV-1 Gag lacking the p6 domain (Gag⌬p6), RSV H6.Gag.3h is a more efficient chaperone on a molar basis than NC; however, in contrast to the HIV-1 system, both RSV H6.Gag.3h and NC have comparable annealing rates at protein saturation. The NC domain of RSV H6.Gag.3h is required for annealing, whereas deletion of the matrix (MA) domain, which stimulates the rate of HIV-1 Gag⌬p6 annealing, has little effect on RSV H6.Gag.3h chaperone function. Competition assays confirmed that RSV MA binds inositol phosphates (IPs), but in contrast to HIV-1 Gag⌬p6, IPs do not stimulate RSV H6.Gag.3h chaperone activity unless the MA domain is replaced with HIV-1 MA. We conclude that differences in the MA domains are primarily responsible for mechanistic differences in RSV and HIV-1 Gag NA chaperone function. IMPORTANCEMounting evidence suggests that the Gag polyprotein is responsible for annealing primer tRNAs to the PBS to initiate reverse transcription in retroviruses, but only HIV-1 Gag chaperone activity has been demonstrated in vitro. Understanding RSV Gag's NA chaperone function will allow us to determine whether there is a common mechanism among retroviruses. This report shows for the first time that full-length RSV Gag lacking the protease domain is a highly efficient NA chaperone in vitro, and NC is required for this activity. In contrast to results obtained for HIV-1 Gag, due to the weak nucleic acid binding affinity of the RSV MA domain, inositol phosphates do not regulate RSV Gag-facilitated tRNA annealing despite the fact that they bind to MA. These studies provide insight into the viral regulation of tRNA primer annealing, which is a potential target for antiretroviral therapy.