Cross-Packaging of Human Immunodeficiency Virus Type 1 Vector RNA by Spleen Necrosis Virus Proteins: Construction of a New Generation of Spleen Necrosis Virus-Derived Retroviral Vectors

Abstract

The ability of the nonlentiviral retrovirus spleen necrosis virus (SNV) to cross-package the genomic RNA of the distantly related human immunodeficiency virus type 1 (HIV-1) and vice versa was analyzed. Such a model may allow us to further study HIV-1 replication and pathogenesis, as well as to develop safe gene therapy vectors. Our results suggest that SNV can cross-package HIV-1 genomic RNA but with lower efficiency than HIV-1 proteins. However, HIV-1-specific proteins were unable to cross-package SNV RNA. We also constructed SNV-based gag-pol chimeric variants by replacing the SNV integrase with the HIV-1 integrase, based on multiple sequence alignments and domain analyses. These analyses revealed that there are conserved domains in all retroviral integrase open reading frames (orf), despite the divergence in the primary sequences. The transcomplementation assays suggested that SNV proteins recognized one of the chimeric variants. This demonstrated that HIV-1 integrase is functional in the SNV gag-pol orf with a lower transduction efficiency, utilizing homologous (SNV) RNA, as well as the heterologous vector RNA of HIV-1. These findings suggest that homology in the conserved sequences of the integrase protein may not be fully competent in the replacement of protein(s) from one retrovirus to another, and there are likely several other factors involved in each of the steps related to replication, integration, and infection. However, further studies to dissect the gag-pol region will be critical for understanding the mechanisms involved in the cleavage of reverse transcriptase, RNase H, and integrase. These studies should provide further insight into the design and development of novel molecular approaches to block HIV-1 replication and to construct a new generation of SNV-based vectors.

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

“…Moreover, it is becoming evident from cross-packaging studies that switching the packaging sequences between two different retroviruses that have no primary sequence homology can maintain efficient packaging (Rizvi and Panganiban 1993;Yin and Hu 1997;White et al 1999;Parveen et al 2004;Moore et al 2007;Al Dhaheri et al 2009;Al Shamsi et al 2011). The process of gRNA packaging thus seems to involve recognition of the structure rather than the sequence of the packaging signals.…”

HIV-1 Vpr Potently Induces Programmed Cell Death in the CNSin Vivo

“…The apoptotic neurons were also observed in the cerebellar regions (25 Ϯ 3.8%), as shown in Figure 6H. The N2c envelope, although neuroinvasive (Parveen et al, 2004), and targeting the expression of Vpr to mainly neuronal cells, did not enhance the apoptotic effects of Vpr beyond the cortex. Overall, the levels of apoptosis in these studies were lower than those induced by the HIV-1 vector-expressed Vpr, suggesting that other HIV-1 proteins are also factors influencing CNS-based apoptosis in vivo.…”

“…1). Addition of N2c envelope to the SNV-based vector system was employed to determine the specific transfer of Vpr into neurons, compared with diverse cell delivery via VSV-G. Of importance, the VSV-G envelope is capable of gene delivery into most cell types, compared with N2c, which is relatively specific for neurons (Parveen et al, 2004). The qualitative and quantitative impact on the apoptosis inducing capability of Vpr, particularly focusing on potential "bystander" effects, was also studied using terminal transferase dUTP nick end labeling (TUNEL) and Annexin V assays.…”

“…Retroviral vectors, derived from SNV and its closely related avian retriculoendotheliosis virus strain (REV-A), have genus-specific relationships with mammalian type C viruses (Tsai et al, 1985). Unlike HIV-1, they are nonpathogenic in humans, and are capable of delivering exogenous genes into dividing and nondividing human cells when pseudotyped with targeted envelopes (Gautier et al, 2000;Parveen et al, 2000Parveen et al, , 2003Parveen et al, , 2004. The results were compared with Vpr expressed by HIV-1-based vectors in vitro and in vivo in similar fashion.…”

Cross- and Co-Packaging of Retroviral RNAs and Their Consequences

“…Rather, it has been shown that the packaging sequences of all known retroviruses assume a higher order structure comprising different structural motifs (reviewed in [3,5,6,8,23]). Thus, regardless of their primary sequence, these structural motifs have been strongly associated with retroviral RNA encapsidation and could explain the phenomenon of RNA cross- and co-packaging among diverse retroviruses [7,24,25,26,27,28,29,30,31]. This review summarizes the current understanding of the gRNA encapsidation determinants of a diverse group of retroviruses, elaborates on what is known about the interaction of these structural elements with their cognate structural Gag proteins, and then discusses the current understanding of cross-/co-packaging among these retroviruses.…”