Human immunodeficiency virus type 1 (HIV-1) containing human APOBEC3G (hA3G) has a reduced ability to produce viral DNA in newly infected cells. At least part of this hA3G-facilitated inhibition is due to a cytidine deamination-independent reduction in the ability to initiate reverse transcription. HIV-1 nucleocapsid (NCp7) is required both for the incorporation of hA3G into virions and for the annealing between viral RNA and tRNA 3 Lys , the primer tRNA for reverse transcription. Herein we present evidence that the interaction of hA3G with nucleocapsid is required for the inhibition of reverse transcription initiation. A tRNA 3Lys priming complex was produced in vitro by the NCp7-facilitated annealing of tRNA 3Lys to synthetic viral RNA in the absence or presence of hA3G.

Vif-negative HIV-1 produced in non-permissive human cells incorporate both APOBEC3F (hA3F) AND APOBEC3G (hA3G), and have a severely reduced ability to produce viral DNA in newly infected cells. While it has been proposed that this reduction is due to deamination of deoxycytidine in viral DNA by either hA3G or hA3F, followed by DNA degradation, recent evidence indicates that the inhibition of viral DNA production can occur independently of DNA editing by either hA3F or hA3G. We have reported that the presence of hA3G in Vif-negative HIV-1 produced from either the non-permissive cell line, H9, or from transfected 293T cells transiently or stably expressing hA3G, results in a >or=50% reduction in the ability of primer tRNA(Lys3) to initiate reverse transcription in these virions, and that this is correlated with a similar reduction in the production of early DNA transcripts in the infected cells. In this work, we show that, like hA3G, hA3F in Vif-negative virions also results in a similar reduction in the initiation of reverse transcription in HIV-1, which is correlated with the inhibition of early viral DNA synthesis in the cell, and which does not require cytidine-deamination of DNA.

Cell culture-adapted strains of Sindbis virus (SINV) initially attach to cells by the ability to interact with heparan sulfate (HS) through selective mutation for positively charged amino acid (aa) scattered in E2 glycoprotein (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72: 7357–7366, 1998). Here we have further confirmed that interaction of E2 protein with HS is crucial for cellular infection of SINV based on the reverse genetic system of XJ-160 virus, a Sindbis-like virus (SINLV). Both SINV YN87448 and SINLV XJ-160 displayed similar infectivity on BHK-21, Vero, or C6/36 cells, but XJ-160 failed to infect mouse embryonic fibroblast (MEF) cells. The molecular mechanisms underlying the selective infectivity of XJ-160 were approached by substituting the E1, E2, or both genes of XJ-160 with that of YN87448, and the chimeric virus was denominated as XJ-160/E1, XJ-160/E2, or XJ-160/E1E2, respectively. In contrast to the parental XJ-160, all chimeric viruses became infectious to wild-type MEF cells (MEF-wt). While MEF-Ext −/− cells, producing shortened HS chains, were resistant not only to XJ-160, but also to YN87448 as well as the chimeric viruses, indicating that the inability of XJ-160 to infect MEF-wt cells likely due to its incompetent discrimination of cellular HS. Treatment with heparin or HS-degrading enzyme resulted in a substantial decrease in plaque formation by YN87448, XJ-160/E2, and XJ-160/E1E2, but had marginal effect on XJ-160 and XJ-160/E1, suggesting that E2 glycoprotein from YN87448 plays a more important role than does E1 in mediating cellular HS-related cell infection. In addition, the peptide containing 145–150 aa from E2 gene of YN87448 specifically bound to heparin, while the corresponding peptide from the E2 gene of XJ-160 essentially showed no binding to heparin. As a new dataset, these results clearly confirm an essential role of E2 glycoprotein, especially the domain of 145–150 aa, in SINV cellular infection through the interaction with HS.

An infectious clone (pBR-XJ160) was constructed using the full-length cDNA of the Sindbis-like XJ-160 virus. Two nucleotide mutations, causing amino acid changes at residue 169 from Lys to Arg and at residue 173 from Thr to Ile in the nonstructural protein (nsP) 1 coding region, strongly influenced the infectivity of in vitro-synthesized RNA. We used site-directed mutagenesis to obtain clones encoding a change to Arg at residue 169 of nsP1 (pBR-169), a change to Ile at residue 173 (pBR-173), or both changes (pBR-6973). Infectivity of RNA from pBR-169 was abolished, but viral forms BR-173 and BR-6973 were obtained from pBR-173 and pBR-6973, respectively. Further, BR-173 exhibited higher propagation than BR-XJ160 in cell culture and higher neurovirulence in a suckling mouse model. BR-6973 possessed an intermediate phenotype. BR-173 and BR-6973 showed increased sensitivity to 3-deazaadenosine (3-DZA), which inhibits S-adenosylhomocysteine hydrolase. Thus, mutagenesis at residue 169 in the nsP1 region of XJ-160 is lethal, but mutation at residue 173 from Thr to Ile enhances viral infectivity and neurovirulence and suppresses the lethal effect of the mutation at residue 169. These mutations might be associated with the RNA methyltransferase (MTase) activity of nsP1.