HIV-1 Mutant Assembly, Processing and Infectivity Expresses Pol Independent of Gag.

Fu-Hsien Yu,Chin-Tien Wang,Kuo-Jung Huang

doi:10.3390/v12010054

Fu-Hsien Yu, Chin-Tien Wang + Show 1 more

Open Access

https://doi.org/10.3390/v12010054

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Abstract

The pol retrovirus gene encodes required enzymes for virus replication and maturation. Unlike HIV-1 Pol (expressed as a Gag–Pol fusion protein), foamy virus (described as an ancient retrovirus) expresses Pol without forming Gag–Pol polyproteins. We placed a “self-cleaving” 2A peptide between HIV-1 Gag and Pol. This construct, designated G2AP, is capable of producing virions with the same density as a wild-type (wt) HIV-1 particle. The 2A peptide allows for Pol to be packaged into virions independently from Gag following co-translationally cleaved from Gag. We found that G2AP exhibited only one-third the virus infectivity of the wt, likely due, at least in part, to defects in Pol packaging. Attenuated protease (PR) activity, or a reduction in Pol expression due to the placement of 2A-mediated Pol in a normal Gag–Pol frameshift context, resulted in significant increases in virus yields and/or titers. This suggests that reduced G2AP virus yields were largely due to increased PR activity associated with overexpressed Pol. Our data suggest that HIV-1 adopts a gag/pol ribosomal frameshifting mechanism to support virus assembly via the efficient modulation of Gag–Pol/Gag expression, as well as to promote viral enzyme packaging. Our results help clarify the molecular basis of HIV-1 gene expression and assembly.

Highlights

The retroviral pol gene encodes protease (PR), reverse transcriptase (RT) and integrase (IN); all essential for virus replication [1]. For orthoretroviruses such as HIV-1 and murine leukemia virus (MLV), Pol and the viral structure protein Gag are translated from the same mRNA, with Pol translated as a Gag–Pol fusion protein via ribosomal readthrough or frameshifting
Gag from separate plasmids indicate that both HIV-1 and MLV Gag–Pol lacking the Gag domain are still capable of incorporation into Gag virus-like particles (VLPs) [20,21,22]. These findings suggest the possibility of interactions involving Pol and Gag for HIV, MLV and foamy virus (FV), with an undefined Pol incorporation mechanism shared by all three retroviruses
To prevent ribosomal frameshifting at the gag/pol junction, the original slippery sequence TTT TTA within G2AP and G2AP-derived constructs was changed to CCC GCG, blocking a -1 ribosomal frameshift event [25]

Summary

Introduction

The retroviral pol gene encodes protease (PR), reverse transcriptase (RT) and integrase (IN); all essential for virus replication [1]. For orthoretroviruses such as HIV-1 and murine leukemia virus (MLV), Pol and the viral structure protein Gag are translated from the same mRNA, with Pol translated as a Gag–Pol fusion protein via ribosomal readthrough or frameshifting. Gag–Pol recruitment for assembling viral particles facilitates Gag–Pol dimerization, which is believed to trigger embedded PR domain activation [3]. Pr55gag cleavage yields four major products: matrix (p17; MA), capsid (CA, p24), nucleocapsid (p7), and C-terminal p6gag [5]. In addition to Gag cleavage products, Gag–Pol processing produces PR, RT and IN

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Conclusion