The N-Terminal Basic Domain of the HIV-1 Matrix Protein Does Not Contain a Conventional Nuclear Localization Sequence But Is Required for DNA Binding and Protein Self-Association

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: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 95%

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 HIV-1 MA domain was reported previously to have two NLSs (5,21) and one NES (13), but multiple subsequent reports have shown that HIV-1 MA-GFP is excluded from the nucleus (2,10) and that it does not contain a conventional NLS (15,22). Two studies in the past year confirmed that HIV-1 Gag lacks a CRM1-dependent nuclear export signal (2,18).…”

Section: Discussionmentioning

confidence: 96%

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

Kemler

Saenz

Poeschla

2012

Lentiviral genomic RNAs are encapsidated by the viral Gag protein during virion assembly. The intracellular location of the initial Gag-RNA interaction is unknown. We previously observed feline immunodeficiency virus (FIV) Gag accumulating at the nuclear envelope during live-cell imaging, which suggested that trafficking of human immunodeficiency virus type 1 (HIV-1) and FIV Gag may differ. Here we analyzed the nucleocytoplasmic transport properties of both Gag proteins. We discovered that inhibition of the CRM1 nuclear export pathway with leptomycin B causes FIV Gag but not HIV-1 Gag to accumulate in the nucleus. Virtually all FIV Gag rapidly became intranuclear when the CRM1 export pathway was blocked, implying that most if not all FIV Gag normally undergoes nuclear cycling. In FIV-infected feline cells, some intranuclear Gag was detected in the steady state without leptomycin B treatment. When expressed individually, the FIV matrix (MA), capsid (CA), and nucleocapsid-p2 (NC-p2) domains were not capable of mediating leptomycin B-sensitive nuclear export of a fluorescent protein. In contrast, CA-NC-p2 did mediate nuclear export, with MA being dispensable. We conclude that HIV-1 and FIV Gag differ strikingly in a key intracellular trafficking property. FIV Gag is a nuclear shuttling protein that utilizes the CRM1 nuclear export pathway, while HIV-1 Gag is excluded from the nucleus. These findings expand the spectrum of lentiviral Gag behaviors and raise the possibility that FIV genome encapsidation may initiate in the nucleus.G ag is both necessary and sufficient for retrovirus particle formation and budding. The protein is translated on free polysomes and targeted to the plasma membrane, where virus particles are assembled (16). A central question is whether Gag and the viral genomic RNA (gRNA) associate at the plasma membrane or earlier during assembly and, if so, whether this occurs in the cytosol; in association with organelles, e.g., cotranslationally; or even in the nucleus, as was described previously for an alpharetrovirus (17). Human immunodeficiency virus type 1 (HIV-1) gRNAs become anchored at the plasma membrane before particle assembly is detectable, but it is not clear whether the Gag-gRNA complexes of this and other lentiviruses first form in the cytoplasm and then transit to the plasma membrane or the gRNA traffics there independently (26,27). Biochemical experiments supported the former scenario, with a monomer or low-order multimers forming on HIV-1 gRNAs and higher-order multimer formation depending on subsequent plasma membrane interactions (33).Gag is encoded by the full-length unspliced viral RNA. To circumvent the cellular checkpoint that prevents the export of intron-containing mRNA, lentiviruses express Rev, which functions as an adaptor between the cellular karyopherin CRM1/exportin-1 and a Rev response element (RRE) located in the 3= region of unspliced viral RNAs (9). However, the main cellular function of the CRM1 nuclear export pathway is the export of cellular proteins containing a...

“…This effect of the 74LR change may be accounted for by one of several mechanisms in which MA affects viral infectivity. First, although involvement of MA in nuclear import of preintegration complexes is unsubstantiated (reviewed in reference 31), several reports showed that alteration of MA residues modulates other steps of the postentry process (5,8,10,16,31,32,37,38,45). For example, substitutions of Leu20 in MA impair viral DNA synthesis early postentry (37,38).…”

Section: Vol 85 2011mentioning

confidence: 99%

Assembly and Replication of HIV-1 in T Cells with Low Levels of Phosphatidylinositol-(4,5)-Bisphosphate

Monde

Chukkapalli

Ono

2011

HIV-1 Gag assembles into virus particles predominantly at the plasma membrane (PM). Previously, we observed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ] is essential for Gag binding to the plasma membrane and virus release in HeLa cells. In the current study, we found that PI(4,5)P 2 also facilitates Gag binding to the PM and efficient virus release in T cells. Notably, serial passage of HIV-1 in an A3.01 clone that expresses polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P 2 , yielded an adapted mutant with a Leu-to-Arg change at matrix residue 74 (74LR). Virus replication in T cells expressing 5ptaseIV was accelerated by the 74LR mutation relative to replication of wild type HIV-1 (WT). This accelerated replication of the 74LR mutant was not due to improved virus release. In control T cells, the 74LR mutant releases virus less efficiently than does the WT, whereas in cells expressing 5ptaseIV, the WT and the 74LR mutant are similarly inefficient in virus release. Unexpectedly, we found that the 74LR mutation increased virus infectivity and compensated for the inefficient virus release. Altogether, these results indicate that PI(4,5)P 2 is essential for Gag-membrane binding, targeting of Gag to the PM, and efficient virus release in T cells, which in turn likely promotes efficient virus spread in T cell cultures. In T cells with low PI(4,5)P 2 levels, however, the reduced virus particle production can be compensated for by a mutation that enhances virus infectivity.