The Primary Nucleotide Sequence of the Bovine Leukemia Virus RNA Packaging Signal Can Influence Efficient RNA Packaging and Virus Replication

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The RNA packaging process for retroviruses involves a recognition event of the genome-length viral RNA by the viral Gag polyprotein precursor (PrGag), an important step in particle morphogenesis. The mechanism underlying this genome recognition event for most retroviruses is thought to involve an interaction between the nucleocapsid (NC) domain of PrGag and stable RNA secondary structures that form the RNA packaging signal. Presently, there is limited information regarding PrGag-RNA interactions involved in RNA packaging for the deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and -2, respectively). To address this, alanine-scanning mutagenesis of BLV PrGag was done with a virus-like particle (VLP) system. As predicted, mutagenesis of conserved basic residues as well as residues of the zinc finger domains in the BLV NC domain of PrGag revealed residues that led to a reduction in viral RNA packaging. Interestingly, when conserved basic residues in the BLV MA domain of PrGag were mutated to alanine or glycine, but not when mutated to another basic residue, reductions in viral RNA packaging were also observed. The ability of PrGag to be targeted to the cell membrane was not affected by these mutations in MA, indicating that PrGag membrane targeting was not associated with the reduction in RNA packaging. These observations indicate that these basic residues in the MA domain of PrGag influence RNA packaging, without influencing Gag membrane localization. It was further observed that (i) a MA/NC double mutant had a more severe RNA packaging defect than either mutant alone, and (ii) RNA packaging was not found to be associated with transient localization of Gag in the nucleus. In summary, this report provides the first direct evidence for the involvement of both the BLV MA and NC domains of PrGag in viral RNA packaging.The RNA packaging process for retroviruses involves a recognition event of the genome-length viral RNA by the viral Gag polyprotein precursor (PrGag), which acts to initiate the morphogenesis of virus particles (see reference 17 for review). The mechanism underlying this genome recognition event is poorly understood, but many biochemical and genetic analyses have revealed that this event involves the interaction between stable RNA secondary structures at the 5Ј end of the viral genome and, in many cases, amino acids in the nucleocapsid (NC) domain of PrGag (1,2,4,6,7,9,10,13,15,29,30,32,36,37).The genome recognition event is an important step in the morphogenesis of infectious retrovirus particles. This recognition event leads to the predominant packaging (encapsidation) of the genome-length viral RNA into assembling particles (38). This discrimination process, which is primarily a viral RNAprotein interaction, is known to strongly favor the full-length viral RNA over spliced viral RNAs and cellular mRNAs (38). In general, the RNA sequences necessary and sufficient for the RNA packaging process are located in a region that includes the 5Ј-...

Section: Discussionsupporting

confidence: 78%

“…Therefore, the VLPs do not have mature cores. The primary BLV RNA packaging signal, consisting of two stable RNA stemloop structures (i.e., SL1 and SL2), are located just downstream of the gag start codon (24)(25)(26). Due to the presence of the packaging signal, this RNA transcript can be packaged into VLPs through the interaction with PrGag.…”

Section: Methodsmentioning

confidence: 99%

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

Wang

Norris

Mansky

2003

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“…It has been shown that replacement of the BLV packaging signal with a similar region from either HTLV-1 or HTLV-2 leads to only a partial BLV replication defect (32,33). These data support at least some level of conserved function in deltaretroviral RNA packaging signals.…”

mentioning

confidence: 89%

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

Sun

Grigsby

Gorelick

et al. 2014

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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.

“…Because of these difficulties, information regarding the molecular details of their life cycles, including virus assembly and release, is limited. To date, most studies of deltaretrovirus assembly have been focused on BLV RNA encapsidation, BLV and HTLV-1 Gag myristylation, and the role of basic residues in MA of HTLV-1 Gag membrane localization and virus production (2,14,18,(22)(23)(24).…”

mentioning

confidence: 99%

Both the PPPY and PTAP Motifs Are Involved in Human T-Cell Leukemia Virus Type 1 Particle Release

Wang

Machesky

Mansky

2004

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In retroviruses, the late (L) domain has been defined as a conserved motif in the Gag polyprotein precursor that, when mutated, leads to the emergence of virus particles that fail to pinch off from the plasma membrane. These domains have been observed to contain the PPXY, PTAP, or YXXL motifs. The deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2, have a conserved PPPY motif in the C-terminal region of the matrix (MA) domain of Gag, while HTLV-1 also encodes a PTAP motif in MA. In this study, we analyzed the roles of the PPPY and PTAP motifs in the C terminus of MA in HTLV-1 particle release. Mutation of either motif (i.e., PPPY changed to APPY or PTAP changed to PTRP) reduced budding efficiencies. Particle buds and electron-dense regions of plasma membrane were observed by electron microscopy. When the locations of PPPY and PTAP were switched, particle release was eliminated. Intriguingly, the replacement of the PTAP motif with either the PPPY or YPDL motifs did not influence the release of virus particles, but the replacement of the PPPY motif with either PTAP or YPDL eliminated particle production. This indicates that the role that PPPY plays in HTLV-1 budding cannot be replaced with either PTAP or YPDL. A similar observation was made with the BLV PPPY motif. Finally, HTLV-1 particle release was found to be sensitive to proteasome inhibitors, implicating a role for ubiquitin in HTLV-1 budding. In summary, our observations indicate that (i) the PPPY motif plays a crucial role in virus budding and (ii) the PTAP motif plays a more subtle role in HTLV-1 particle release. Each of these motifs may play an important role in virus release from specific cell types and therefore be important in efficient virus spread and transmission.