A single amino acid substitution of the human immunodeficiency virus type 1 capsid protein affects viral sensitivity to TRIM5α

<p>Abstract</p> <p>Background</p> <p>Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously reported that efficient replication of HIV-1 in CM cells was achieved after we replaced the loop between α-helices 6 and 7 (L6/7) of the capsid protein (CA) with that of SIVmac239 in addition to the loop between α-helices 4 and 5 (L4/5) and <it>vif</it>. This virus (NL-4/5S6/7SvifS) was supposed to escape from host restriction factors cyclophilin A, CM TRIM5α, and APOBEC3G. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired.</p> <p>Results</p> <p>By long-term cultivation of human CEMss cells infected with NL-4/5S6/7SvifS, we succeeded in rescuing the impaired replicative capability of the virus in human cells. Sequence analysis of the CA region of the adapted virus revealed a G-to-E substitution at the 116th position of the CA (G116E). Introduction of this substitution into the molecular DNA clone of NL-4/5S6/7SvifS indeed improved the virus' replicative capability in human cells. Although the G116E substitution occurred during long-term cultivation of human cells infected with NL-4/5S6/7SvifS, the viruses with G116E unexpectedly became resistant to CM, but not human TRIM5α-mediated restriction. The 3-D model showed that position 116 is located in the 6^thhelix near L4/5 and L6/7 and is apparently exposed to the protein surface. The amino acid substitution at the 116^thposition caused a change in the structure of the protein surface because of the replacement of G (which has no side chain) with E (which has a long negatively charged side chain).</p> <p>Conclusions</p> <p>We succeeded in rescuing the impaired replicative capability of NL-4/5S6/7SvifS and report a mutation that improved the replicative capability of the virus. Unexpectedly, HIV-1 with this mutation became resistant to CM TRIM5α-mediated restriction.</p

Modification of a loop sequence between α-helices 6 and 7 of virus capsid (CA) protein in a human immunodeficiency virus type 1 (HIV-1) derivative that has simian immunodeficiency virus (SIVmac239) vif and CA α-helices 4 and 5 loop improves replication in cynomolgus monkey cells

<p>Abstract</p> <p>Background</p> <p>Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees but not cynomolgus or rhesus monkeys while simian immunodeficiency virus isolated from macaque (SIVmac) readily establishes infection in those monkeys. Several HIV-1 and SIVmac chimeric viruses have been constructed in order to develop an animal model for HIV-1 infection. Construction of an HIV-1 derivative which contains sequences of a SIVmac239 loop between α-helices 4 and 5 (L4/5) of capsid protein (CA) and the entire SIVmac239 <it>vif </it>gene was previously reported. Although this chimeric virus could grow in cynomolgus monkey cells, it did so much more slowly than did SIVmac. It was also reported that intrinsic TRIM5α restricts the post-entry step of HIV-1 replication in rhesus and cynomolgus monkey cells, and we previously demonstrated that a single amino acid in a loop between α-helices 6 and 7 (L6/7) of HIV type 2 (HIV-2) CA determines the susceptibility of HIV-2 to cynomolgus monkey TRIM5α.</p> <p>Results</p> <p>In the study presented here, we replaced L6/7 of HIV-1 CA in addition to L4/5 and <it>vif </it>with the corresponding segments of SIVmac. The resultant HIV-1 derivatives showed enhanced replication capability in established T cell lines as well as in CD8+ cell-depleted primary peripheral blood mononuclear cells from cynomolgus monkey. Compared with the wild type HIV-1 particles, the viral particles produced from a chimeric HIV-1 genome with those two SIVmac loops were less able to saturate the intrinsic restriction in rhesus monkey cells.</p> <p>Conclusion</p> <p>We have succeeded in making the replication of simian-tropic HIV-1 in cynomolgus monkey cells more efficient by introducing into HIV-1 the L6/7 CA loop from SIVmac. It would be of interest to determine whether HIV-1 derivatives with SIVmac CA L4/5 and L6/7 can establish infection of cynomolgus monkeys <it>in vivo</it>.</p

Replication of chicken anemia virus (CAV) requires apoptin and is complemented by VP3 of human torque teno virus (TTV)

AbstractTo test requirement for apoptin in the replication of chicken anemia virus (CAV), an apoptin-knockout clone, pCAV/Ap(−), was constructed. DNA replication was completely abolished in cells transfected with replicative form of CAV/Ap(−). A reverse mutant competent in apoptin production regained the full level of DNA replication. DNA replication and virus-like particle (VLP) production of CAV/Ap(−) was fully complemented by supplementation of the wild-type apoptin. The virus yield of a point mutant, CAV/ApT108I, was 1/40 that of the wild type, even though its DNA replication level was full. The infectious titer of CAV was fully complemented by supplementing apoptin. Progeny virus was free from reverse mutation for T108I. To localize the domain within apoptin molecule inevitable for CAV replication, apoptin-mutant expressing plasmids, pAp1, pAp2, pAp3, and pAp4, were constructed by deleting amino acids 10–36, 31–59, 59–88 and 80–112, respectively. While Ap1 and Ap2 were preferentially localized in nuclei, Ap3 and Ap4 were mainly present in cytoplasm. Although complementation capacity of Ap3 and Ap4 was 1/10 of the wild type, neither of them completely lost its activity. VP3 of TTV did fully complement the DNA replication and VLP of CAV/Ap(−). These data suggest that apoptin is inevitable not only for DNA replication but also VLP of CAV. The common feature of apoptin and TTV-VP3 presented another evidence for close relatedness of CAV and TTV

Slower Uncoating Is Associated with Impaired Replicative Capability of Simian-Tropic HIV-1

Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees, but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously generated a simian-tropic HIV-1 that replicates efficiently in CM cells. This virus encodes a capsid protein (CA) with SIVmac239-derived loops between α-helices 4 and 5 (L4/5) and between α-helices 6 and 7 (L6/7), along with the entire vif from SIVmac239 (NL-4/5S6/7SvifS). These SIVmac239-derived sequences were expected to protect the virus from HIV-1 restriction factors in monkey cells. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired. By long-term cultivation of human CEM-SS cells infected with NL-4/5S6/7SvifS, we succeeded in partially rescuing the impaired replicative capability of the virus in human cells. This adapted virus encoded a G-to-E substitution at the 116th position of the CA (NL-4/5SG116E6/7SvifS). In the work described here, we explored the mechanism by which the replicative capability of NL-4/5S6/7SvifS was impaired in human cells. Quantitative analysis (by real-time PCR) of viral DNA synthesis from infected cells revealed that NL-4/5S6/7SvifS had a major defect in nuclear entry. Mutations in CA are known to affect viral core stability and result in deleterious effects in HIV-1 infection; therefore, we measured the kinetics of uncoating of these viruses. The uncoating of NL-4/5S6/7SvifS was significantly slower than that of wild type HIV-1 (WT), whereas the uncoating of NL-4/5SG116E6/7SvifS was similar to that of WT. Our results suggested that the lower replicative capability of NL-4/5S6/7SvifS in human cells was, at least in part, due to the slower uncoating of this virus

Slower Uncoating Is Associated with Impaired Replicative Capability of Simian-Tropic HIV-1

<div><p>Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees, but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously generated a simian-tropic HIV-1 that replicates efficiently in CM cells. This virus encodes a capsid protein (CA) with SIVmac239-derived loops between α-helices 4 and 5 (L4/5) and between α-helices 6 and 7 (L6/7), along with the entire <i>vif</i> from SIVmac239 (NL-4/5S6/7SvifS). These SIVmac239-derived sequences were expected to protect the virus from HIV-1 restriction factors in monkey cells. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired. By long-term cultivation of human CEM-SS cells infected with NL-4/5S6/7SvifS, we succeeded in partially rescuing the impaired replicative capability of the virus in human cells. This adapted virus encoded a G-to-E substitution at the 116^th position of the CA (NL-4/5SG116E6/7SvifS). In the work described here, we explored the mechanism by which the replicative capability of NL-4/5S6/7SvifS was impaired in human cells. Quantitative analysis (by real-time PCR) of viral DNA synthesis from infected cells revealed that NL-4/5S6/7SvifS had a major defect in nuclear entry. Mutations in CA are known to affect viral core stability and result in deleterious effects in HIV-1 infection; therefore, we measured the kinetics of uncoating of these viruses. The uncoating of NL-4/5S6/7SvifS was significantly slower than that of wild type HIV-1 (WT), whereas the uncoating of NL-4/5SG116E6/7SvifS was similar to that of WT. Our results suggested that the lower replicative capability of NL-4/5S6/7SvifS in human cells was, at least in part, due to the slower uncoating of this virus.</p> </div

Improved capacity of a monkey-tropic HIV-1 derivative to replicate in cynomolgus monkeys with minimal modifications.

Human immunodeficiency virus type 1 (HIV-1) hardly replicates in Old World monkeys. Recently, a mutant HIV-1 clone, NL-DT5R, in which a small part of gag and the entire vif gene are replaced with SIVmac239-derived ones, was shown to be able to replicate in pigtail monkeys but not in rhesus monkeys (RM). In the present study, we found that a modified monkey-tropic HIV-1 (HIV-1mt), MN4-5S, acquired the ability to replicate efficiently in cynomolgus monkeys as compared with the NL-DT5R, while neither NL-DT5R nor MN4-5S replicated in RM cells. These results suggest that multiple determinants may be involved in the restriction of HIV-1 replication in macaques, depending on the species of macaques. The new HIV-1mt clone will be useful for studying molecular mechanisms by which anti-viral host factors regulate HIV-1 replication in macaques

Measurement of the reverse transcribed products of simian-tropic HIV-1 in human cells.

<p>(A) CEM-SS cells were infected with NL-vifS, NL-4/5S6/7SvifS, or NL-4/5SG116E6/7SvifS, and DNA was extracted at 12 hr after infection and subjected to real-time PCR assays using U5/gag primers for late reverse transcription (RT), 2-LTR primers for nuclear transported viral DNA, and Alu-HIV primers for integrated DNA. Mean relative amounts of U5/gag, 2-LTR, and Alu-HIV products obtained from three independent experiments (the amount in the NL-vifS sample at 12 hr after infection is set at 1) are indicated. Mean numbers of U5/gag, 2-LTR, and Alu-HIV copies per 30 ng of total DNA of NL-vifS-infected cells were 39695, 187, and 2.17, respectively. Error bars reflect the SD of the three independent experiments. (B) CEM-SS cells were infected with NL-vifS, NL-4/5S6/7SvifS, or NL-4/5SG116E6/7SvifS, and DNA was extracted at 12, 24, 48, and 72 hr after infection and subjected to real-time PCR assays as described above. The number of viral DNA (U5/gag and 2-LTR) copies per 30 ng of total DNA and relative amount of Alu-HIV products (the amount in the NL-vifS sample at 12 hr after infection is set at 1) is indicated. Error bars reflect the SD of triplicate values of real-time PCR. Presented data are representative of three independent experiments.</p

<i>In situ</i> uncoating assay.

<p>HeLa cells were spinoculated with VSV-G-pseudotyped, S15-dTomato, GFP-Vpr -labeled NL-Nh (WT; black diamonds), NL-Nh with 4/5S6/7S CA (NL-4/5S6/7S, white squares), or NL-Nh with 4/5SG116E6/7S CA (NL-4/5G116E6/7S, gray triangles) for 2 hr at 16° C in the presence or absence of bafilomycin A (BafA). Infection was synchronized by washing off inocula and replacing with 37° <i>C</i><i>medium</i>. At the indicated time post-infection, the cells were fixed, immunostained for p24 CA (Cy-5), and imaged. The identity of the samples was blinded before counting. GFP-positive puncta then were quantified and individually examined for the presence of dTomato and Cy-5 (p24 CA) signals. The percentage of the total number of fused (dTomato-) virions that stained for p24 CA over time following fusion is shown. The 0-hr time point and BafA (+) samples represent total number of GFP-positive virions that stained positive for p24 CA. For BafA treatment, only data from the 4-hr time points on 4/5S6/7S CA (NL-4/5S6/7S+Baf, a grey square), or 4/5SG116E6/7S CA (NL-4/5G116E6/7S+Baf, a black triangle) are shown. The results shown are means and SD from three independent experiments. Actual numbers of counted dots are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072531#pone.0072531.s001" target="_blank">Table S1</a>.</p