Significance: For several decades, retroviral core uncoating has been thought to occur in the cytoplasm in coordination with reverse transcription, and while some recent studies have concluded that HIV-1 uncoating occurs at the nuclear envelope during nuclear import, none have concluded that uncoating occurs in the nucleus. Here, we developed methods to study HIV-1 uncoating by direct labeling and quantification of the viral capsid protein associated with infectious viral cores that produced transcriptionally active proviruses. We find that infectious viral cores in the nuclei of infected cells are largely intact and uncoat near their integration sites just before integration. These unexpected findings fundamentally change our understanding of HIV-1 postentry replication events.

Phosphorylation of the alpha subunit of the eucaryotic translation initiation factor (eIF-2 alpha) by the double-stranded RNA-activated inhibitor (DAI) kinase correlates with inhibition of translation initiation. The importance of eIF-2 alpha phosphorylation in regulating translation was studied by expression of specific mutants of eIF-2 alpha in COS-1 cells. DNA transfection of certain plasmids could activate DAI kinase and result in poor translation of plasmid-derived mRNAs. In these cases, translation of the plasmid-derived mRNAs was improved by the presence of DAI kinase inhibitors or by the presence of a nonphosphorylatable mutant (serine to alanine) of eIF-2 alpha. The improved translation mediated by expression of the nonphosphorylatable eIF-2 alpha mutant was specific to plasmid-derived mRNA and did not affect global mRNA translation. Expression of a serine-to-aspartic acid mutant eIF-2 alpha, created to mimic the phosphorylated serine, inhibited translation of the mRNAs derived from the transfected plasmid. These results substantiate the hypothesis that DAI kinase activation reduces translation initiation through phosphorylation of eIF-2 alpha and reinforce the importance of phosphorylation of eIF-2 alpha as a way to control initiation of translation in intact cells.

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection. APOBEC3G deaminates deoxycytidines in minus strand DNA to deoxyuridines, resulting in G to A hypermutation and viral inactivation. Human immunodeficiency virus type 1 (HIV-1) virion infectivity factor counteracts the antiviral activity of APOBEC3G by inducing its proteosomal degradation and preventing virion incorporation. To elucidate the mechanism of viral suppression by APOBEC3G, we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1 deletion mutants. Virus-like particles derived from constructs in which pol, env, and most of gag were deleted still contained high levels of cytidine deaminase activity; in addition, coimmunoprecipitation of APOBEC3G and HIV-1 Gag in the presence and absence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex. Viral particles lacking HIV-1 genomic RNA which were generated from the gag-pol expression constructs pC-Help and pSYNGP packaged APOBEC3G at 30–40% of the wild-type level, indicating that interactions with viral RNA are not necessary for incorporation. In addition, viral particles produced from an nucleocapsid zinc finger mutant contained ∼1% of the viral genomic RNA but ∼30% of the cytidine deaminase activity. The reduction in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions. These results indicate that interactions with viral proteins or viral genomic RNA are not essential for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation.

Encapsidation of host restriction factor APOBEC3G (A3G) into vif -deficient human immunodeficiency virus type 1 (HIV-1) blocks virus replication at least partly by C-to-U deamination of viral minus-strand DNA, resulting in G-to-A hypermutation. A3G may also inhibit HIV-1 replication by reducing viral DNA synthesis and inducing viral DNA degradation. To gain further insight into the mechanisms of viral inhibition, we examined the metabolism of A3G-exposed viral DNA. We observed that an overall 35-fold decrease in viral infectivity was accompanied by a five- to sevenfold reduction in viral DNA synthesis. Wild-type A3G induced an additional fivefold decrease in the amount of viral DNA that was integrated into the host cell genome and similarly reduced the efficiency with which HIV-1 preintegration complexes (PICs) integrated into a target DNA in vitro. The A3G C-terminal catalytic domain was required for both of these antiviral activities. Southern blotting analysis of PICs showed that A3G reduced the efficiency and specificity of primer tRNA processing and removal, resulting in viral DNA ends that are inefficient substrates for integration and plus-strand DNA transfer. However, the decrease in plus-strand DNA transfer did not account for all of the observed decrease in viral DNA synthesis associated with A3G. These novel observations suggest that HIV-1 cDNA produced in the presence of A3G exhibits defects in primer tRNA processing, plus-strand DNA transfer, and integration.

A long-standing question in retrovirus biology is how RNA genomes are distributed among virions. In the studies presented in this report, we addressed this issue by directly examining HIV-1 RNAs in virions using a modified HIV-1 genome that contained recognition sites for BglG, an antitermination protein in the Escherichia coli bgl operon, which was coexpressed with a fragment of BglG RNA binding protein fused to a fluorescent protein. Our results demonstrate that the majority of virions (>90%) contain viral RNAs. We also coexpressed HIV-1 genomes containing binding sites for BglG or the bacteriophage MS2 coat protein along with 2 fluorescent protein-tagged RNA binding proteins. This method allows simultaneously labeling and discrimination of 2 different RNAs at single-RNA-detection sensitivity. Using this strategy, we obtained physical evidence that virions contain RNAs derived from different parental viruses (heterozygous virion) at ratios expected from a random distribution, and we found that this ratio can be altered by changing the dimerization sequences. Our studies of heterozygous virions also support a generally accepted but unproven assumption that most particles contain 1 dimer. This study provides answers to long-standing questions in HIV-1 biology and illustrates the power and sensitivity of the 2-RNA labeling method, which can also be adapted to analyze various issues of RNA biogenesis including the detection of different RNAs in live cell imaging.

We determined the in vivo forward mutation rate in a single replication cycle for spleen necrosis virus (SNV). A method was developed to clone integrated proviruses of retroviral shuttle vectors by exploiting the tight binding of the lac operator to the lac repressor protein. The vectors contained the lacZ alpha gene as a reporter of mutations. Thirty-seven of the 16,867 proviruses recovered contained five classes of mutations, including substitutions and frameshifts. Runs of 9 and 10 identical base pairs and a direct repeat of 110 base pairs were mutational hotspots. In addition, two copies of a provirus contained 15 G-to-A substitutions. Such proviruses, which we name hypermutants, may arise through the action of an error-prone polymerase and could significantly contribute to the genetic variation in retroviral populations.